Details for: G 33599, Cannabis sativa L., '----'

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Regulatory testing compliance protocols are determined in accordance with the USDA Hemp Descriptor Standards – Secondary Metabolites , which establish the relevant analytical and classification criteria for cannabinoid and secondary metabolite evaluation.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.

Field trials were conducted in Zone 1 at the USDA-ARS Wellington Farm (42.535048°N, 77.05422°W) on approximately 1.13 ha (2.8 acres). The most recent soil test was completed in November 2023. Estimated ENR was 94 lbs./acre, and 100 lbs. nitrogen/acre was applied prior to bedding.

Experimental Design and Germplasm Preparation

In early 2024, the PGRU team generated the accession list for evaluation, pulled germplasm, designed field randomization plans, established persistent unique identifiers (PUIDs) for all potential individuals, procured greenhouse and field supplies, generated plot labels, and prepared 50 mL tissue collection tubes containing PUID QR codes.

Based on available germination data and seed quantities, enough seed was pulled to target 20 live plants per accession when possible. Some accessions produced fewer viable individuals but were still included in the trial.

In April 2024, the field was prepared using compostable mulch with 7.5 ft row spacing and landscape fabric between rows for weed suppression. Approximately 161 trays of 2 × 2 × 3 inch peat pots were filled with ProMix BT media.

On May 7, 2024, seeds from each accession were sown into peat pots in greenhouse conditions using ProMix growth media. Seed sowing order matched final transplant order in the field. Unique pot labels were assigned to all pots in replicate one.

Seedlings were thinned and balanced among replicates to maintain similar plant counts across replications, while prioritizing three individuals in replicate one for early sex evaluation. Flats were thinned between May 15–16 by project personnel, and individuals from replicates two and three were transferred into replicate one when necessary. Pots containing multiple seedlings were randomly thinned to one individual per pot.

A seven-day stand count conducted on May 14, 2024 indicated that approximately 92% of accessions had sufficient plants to proceed with transplanting and genotyping. Approximately 88% of accessions contained at least two live plants, enabling the potential recovery of XX/XY pairs.

Field Design and Transplanting

The field experiment followed a complete randomized block design consisting of 536 accessions with three replicates and three individual plants per plot (maximum planned total = 4,824 plants). Actual plant numbers were reduced due to variation in seed quantity and viability. Germination data were recorded for all accessions using FieldBook.

Prior to transplanting, seedlings were hardened in cold frames approximately 15–20 days after sowing. Transplanting was conducted using a waterwheel transplanter at 3 ft within-row spacing with 50 ppm nitrogen in the transplant tank solution. Drip irrigation was applied immediately before and after transplanting to reduce transplant shock.

Field transplanting occurred on May 23, 2024 (P1001–P3194) and May 24, 2024 (P3196–P3564).

Genotyping and Sex Determination

During the final three weeks of May 2024, PACE reactions were conducted to identify target XX and XY individuals using the CSP2 sex marker described in the Toth dissertation. Leaf tissue was collected for DNA extraction and PACE analysis.

The target sampling scheme aimed to identify two XX and two XY individuals per accession (N < 1100 accessions successfully represented). Final numbers were reduced due to insufficient viable plants, feminized accessions, or stochastic absence of XX or XY individuals among tested plants.

Sex determination data were processed using the SexDecisionTool.Rmd workflow.

Field Phenotyping

Stand counts were collected seven days after transplanting. Stand vigor ratings were recorded using the following scale:

• Missing = no plant transplanted
• Dead = plant died
• 1 = cotyledon stage
• 2 = small
• 3 = average
• 4 = large
• 5 = very large

Each plant received a slip tag containing the PUID, accession number, inventory number, inventory ID, plot number, plant number, and QR code. The PUID format consisted of Inventory ID + Rep Number + Plant Number (e.g., 24HA_G_33199_21UO_SD_S1004_p2).

Leaf tissue samples (~10 g fresh tissue) were collected into 50 mL tubes on dry ice.

Field walkthroughs and phenotyping evaluations were conducted at least weekly throughout the growing season. Phenotyping protocols mirrored reflected the USDA Hemp Descriptors Handbook.

Weekly phenotyping included:

• Phenotypic sex determination
• Flowering date
• Senescence scoring

Approximately 100 days after sowing, plant architecture traits were recorded, including:

• Plant height (HT)
• Maximum canopy diameter (MCD)
• Height at maximum canopy diameter (MCDH)

In mid-June, images of all plants were collected using the Field Book platform.

Chemical and Reproductive Phenotyping

Chemical harvest sampling occurred approximately 35 days after terminal female flowering. Sampling dates were generated programmatically using the formula:

F_FLOW_DATE + 35 days

For each sampled female inflorescence, three ~12 cm floral samples were collected and dated. One female or monoecious inflorescence per sequenced individual was retained for seed collection and shattering evaluation.

Seed shattering bags (Midco Global-PQ218; 336 µm pore diameter; 10 × 12 inches) were attached to sequenced female inflorescences and labeled for tracking after harvest.

Cannabinoid analysis was performed by Dr. Korey Brownstein (USDA-ARS NCAUR) using a UPLC protocol described in the USDA Hemp Descriptors Handbook: Cannabinoid Analysis Protocol .

Senescence and Harvest Procedures

Senescence dates were recorded weekly during flowering and sex phenotyping. Senescence was defined as stem browning accompanied by fan leaf dry-down.

Following senescence, seed samples were collected from sequenced plants with pistillate flowers. The target collection amount was greater than 5 g seed per plant.

At harvest, sequenced stems were cut at ground level using pruning shears. The lower 12 cm of stem tissue was removed, approximately 1 m of stem was retained, and upper branches were removed. All harvested material retained its corresponding slip tag.

Stem and seed samples were transferred to a warm, dry, ventilated storage area, likely within the Wellington high tunnels. Sample labels containing the “UNIQ-IND” identifier and QR code were generated for all harvested materials.