Evaluation location: New York, United States
Brief consolidation of 2024 HudsonAlpha USDA-ARS PGRU Hemp Germplasm Collection Genotyping/Sex-determination/Phenotyping Project. Evaluate and genotype two XX and two XY individual per accession from most of the USDA-ARS Hemp Germplasm Collection, N ~ 2000 genomes. Phenotypic efforts follow established protocols in the USDA Hemp Handbook [https://www.ars.usda.gov/northeast-area/geneva-ny/plant-genetic-resources-unit-pgru/docs/hemp-descriptors/] and Brummer SAC projects. Conducted in Zone 1, in USDA-ARS Wellington Farm (1.13 ha ~ 2.8 ac), 42.535048'N 77.05422'W. Nov 2023 soil testing was most recent test. My understanding is that ENR = 94 lbs./ac. and add 100 lbs. N/Acre to the field. PGRU will share all data and workflow information using Cornell Box with all collaborators invited as editors. In early 2024, the PGRU team will generate a list of accessions for evaluation, pulled germplasm, designed field randomization, established persistent unique identifiers (PUIDs) for every potential individual, procured greenhouse and field supplies, generated plot labels, generate 50 mL tissue sampling tubes with PUID QR codes. Given germination data and seed quantity, we pulled enough seeds to generate 20 individual live plants. Some accessions could not generate all nine live plants from, but they will still be included in the trial. In April 2024, the PGRU team prepared approximately 2-acre field at the USDA-PGRU Wellington Farm in Zone 1 and applied 100 lbs./acre nitrogen before bedding. Field design will be compostable mulch with between row spacing of 7.5’. Between rows w/ landscape fabric for weed suppression. Late April, the PGRU team will fill 161 2x2x3” peat pots with ProMix BT. May 7 sowed seeds from each accession into 2x2x3” peat pots in a greenhouse using ProMix growth media. These seeds were sown in the exact order that they will be transplanted into the field. In rep one, placed a unique pot label in each peat pot. Thin and balance the seedlings in the peat pots such that all three reps have close to the same number of individuals, although the first rep will be prioritized to contain three individuals, as those accessions will be evaluated for sex first. All all flats were thinned by ZJS, TB, DJM 5/15-5/16. ZJS pulled individuals from reps 2 or 3 into rep 1 to bring the number of individuals as close to 3/rep as possible.. Any pot with multiple individuals will be randomly thinned to retain only one individual per pot. Zach conducted a seven day stand count on 5/14/24 and approximately 92% of the accessions had enough plants to proceed with transplanting and genotyping. 88% of the accessions contained at least two live plants, so there is a possibility of recovering an XX/XY pair. Plants were watered as needed in the GH, cold frames, shade house. The field design will be a complete randomized block design of 536 accessions in three replicates, with three individual plants per plot, (i.e., 536 accessions x 3 reps x 3 individuals per plot = 4,824). The true number of plants was lower due to seed quantity and viability. All germination data was recorded. A count of every plant will be made using FieldBook >10 days post sowing (5/17). Several days before transplanting (e.g., ~15-20 days post sowing), these accessions will be moved to a cold frame to harden off (Kevin). Transplanting will occur using a waterwheel at (1.5’ or 2’) spacing, 50 ppm N in tank. Last three weeks of May, DJM ran as many PACE reactions as possible to identify target plants. PGRU collected leaf tissue for DNA extraction for PACE analysis using the sex marker CSP2 in Toth dissertation. Identify two XX and two XY individual per accession (N < 1100). This number will be lower due to multiple reasons (not enough live individuals generated, “feminized”, randomly does not have either an XX or XY [ p = 1 – (2n-2)/ 2n; where n = number of individuals tested]). Data will be processed via SexDescisionTool.Rmd. Drip irrigation will be applied prior to and immediately after to reduce transplant shock. Transplanting occurred on May 23 (P1001-P3194) and May 24 (P3196-3564). The within row spacing is 3’. Fieldbook phenotyping: stand counts taken seven days after transplanting. Stand Vigor counts were taken (StandVigor_20240528, missing = no plant was transplanted, dead = plant died, 1= cotyledon, 2=small, 3 = average, 4=large, 5=very large). The results of stand count generate two slip tags for every plant in the field. Slip tags will be placed on destructive plant every plant with PUID, Accession number, Inventory Number, Inventory ID, Top Name, Plot number, Plant number, and a QR code that captures the PUID. The PUID is concat of Inventory ID, Rep Number, and Plant number. E.g. (24HA_G_33199_21UO_SD_S1004_p2 = “the 2024 Hudson Alpha trial, inventory G 33199,Plot 1004, plant 2”). Prepared 50 mL tubes of 10 g fresh, young leaf tissue, and ship overnight on dry ice. Target 10g fresh tissue. PGRU ran field walkthroughs and phenotyping at least once per week. Phenotyping efforts will mirror trials conducted in the USDA-UC-Davis SAC Trials (publication pending) and the USDA-ARS Hemp Descriptors and Phenotyping Handbook. WEEKLY: phenotypic sex, flowering date, and senescence phenotyping will occured on every individual in the trial (536x3x3). In mid Aug (~100 days after sowing) plant architecture traits were collected. Sequenced plus other key individuals were be selected for additional phenotyping. Phenotype Sex, F/M flowering date, leaf morphometrics. In mid-June, images of every plant will be taken using Field Book. CHEMICAL HARVEST taken ~35 days after terminal flowering via script every Friday to generate the chemical samples as F_FLOW_DATE + 35 days. Take three ~12 cm female inflorescence 35 days after terminal flowering, record date. Seed collection and shattering potential. Affix a seed shattering bag (Midco Global-PQ218, with a 336-um pore diameter, size 10” x 12”) to every sequenced female inflorescence with a printed label so the shattering bags can be tracked after harvest. ONE FEMALE (or Monoecious) PER Sequenced individual. DIA: stem diameter at the ground (mm). HT: plant height (cm). MCD: maximum canopy diameter (mcd) as width of plant at widest set of branches. Measure once between flowering and senescence (cm) MCDH: height evaluated at maximum canopy diameter from ground to max canopy diameter. Measure once between flowering and senescence (cm). Senescence date. Record senescence date weekly during flowering and sex phenotyping. Definition: stem starts to brown + all the fan leaves dry down. Harvest Measurements – at “Date_Seed” DATE_SEED: Take seed sample post-senescence, record date. Only sequenced plants with pistilate flowers. Try to obtain more than 5 grams of seed per plant. Chop sequenced stems at senescence. Cut at ground level with pruning shears. Trim off bottom 12 cm, save 1 m of stem, remove top. Remove branches. Make sure slip tag is on plant. Move stems and seed bags to a warm, dry place with plenty of ventilation—This will likely be the new high tunnels at Wellington. Print labels for each sample that include the "UNIQ-IND" as a QR code.