CONSERVATION AND UTILIZATION OF PLANT GENETIC RESOURCES

<br>What was accomplished under these goals? As of 2017 we are conserving 12,636 accessions of tomato, onion, radish, winter squash, cabbage, cauliflower, broccoli, other cole crops, celery, tomatillo, asparagus and other vegetables and their wild relatives. In total, 200 taxa of 151 species representing 29 genera are currently held in the seed crops repository. This provides the genetic diversity needed to develop vegetable varieties with disease and pest resistance, tolerance to abiotic stresses, varieties with improved quality and nutrition, and ensures its long-term availability. During FY 2017, seed of 110 annual (B. rapa, radish, tomato, winter squash, and buckwheat) and 52 biennial (onion and cabbage) accessions were planted for regeneration by ARS researchers at Geneva, NY and seed samples of 50 accessions (onion, winter squash, annual Brassica) were distributed to cooperators for regenerations. In addition, 10 collected short-day onion cultivars not currently in the collection are being regenerated by a cooperator. We distributed 7,942 samples of vegetable germplasm in 254 orders during calendar year 2016, and 4,247 samples in 130 orders during the first half of calendar year 2017. A large number of these samples were distributed to NE9 member states (ME, NH, VT, MA, RI, CT, NY, NJ, PA, DE, MD, WV, and DC). During 2016, 703 seed samples in 37 orders were distributed to users in the NE9 region. During 2017, through June 30, there were 508 seed samples in 23 orders distributed to the NE9 region. Delivery of germplasm to clients and stakeholders is the primary way in which the results of this unit's activities are distributed to the public. The crop Brassica rapa L., known as rape mustard, field mustard, birdsrape mustard, etc. is very diverse in its usage. It can be consumed as a raw or cooked leafy green (mizuna, Chinese cabbage, bok choy, tatsoi, choy sum, komatsuna), a spice or condiment (brown sarson, yellow sarson), a root vegetable also used as animal fodder (turnip), or a cooking oil (oilseed rape). The genetic relationships among these various types (morphotypes) is not well understood but a deeper understanding would be beneficial for crop conservation and improvement. We sequenced portions of the genomes of 333 diverse accessions of B. rapa from the NE9 and NC7 NPGS repositories that originated from the Americas, Europe, East Asia, South Asia/Middle East, and Australia and included all ten of the above mentioned morphotypes. Our results indicated that both morphotype and geography contributed to the global genetic patterns of B. rapa. We identified four distinct groups (turnip, oilseed, Chinese cabbage, and bok choy) and separated turnips by geographic origin (European vs. East Asian). These results emphasized the importance of morphotype on genetic diversity but also indicated that there exists significant differences between the same morphotypes that are related to geographic origin. These differences may have arisen through different domestication histories, breeding histories, or extensive geographic isolation. The results provided enhanced resolution to the genetic relationships of morphotypes within the species B. rapa. Understanding of these relationships is key to future breeding and scientific work in B. rapa. Winter squash (Cucurbita maxima) has traditionally been valued for its good storage properties bestowed by its thick, hard skin. Squash provides a wide range of nutrients and consumer demand has been steadily rising during recent decades. Squash contains naturally occurring cucurbatacins (tetracyclic triterpenes) that have been used in pest control and also exhibit anti-inflammatory and anticancer activities. Winter squash breeding has focused on fruit size and shape, non-bitter fruits, deep orange fruit color (high pro-vitamin A), earliness, bush habit, and insect and disease resistance. Two C. maxima core sets based on morphological (95 accessions) and geographical (95 accessions) diversity from 38 countries, (165 total accessions) were assembled and 285 plants were genotyped using GBS. Data scoring and analyses is being done in collaboration with members of the Cucurbit Coordinated Agricultural Project (CucCAP, "Leveraging applied genomics to increase disease resistance in Cucurbit crops") from Cornell University and West Virginia State University. A core set of 190 PGRU tomato accessions was genotyped using GBS. These originated from 31 countries and included six different versions of the economically valuable variety San Marzano.Based on 3,713 SNPs, there was not extensive genetic distinction among types, i.e., ornamental, processing, breeder's lines, and home gardening varieties. Many Plant Variety Protection (PVP) varieties developed by different private companies were closely related to each other, probably because they shared UC-82 in their pedigrees. Results supported the traditional view that cultivated germplasm is not a rich source of new genetic variation for the crop. However, genetically divergent accessions such as VFNT cherry, some Italian accessions, and some South American landraces could provide novel alleles for crop improvement. We also found support for a "genuine" San Marzano plus additional San Marzano accessions that did not cluster with the "genuine" accessions or with each other. For the clonal crops, a total of 451 orders for 7,160 samples were filled in 2016, including 127 orders and 2,917 samples for the NE-9 region. The number of orders (451) was a new record high two years in a row (from 2010 to 2016, the total number of orders were 318, 367, 346, 322, 383, 444, and 451, respectively). From January to June 2017, the total number of orders were 343, for 6,189 samples. We expect the trend of increasing demand for our germplasm will continue in the near future. Some of the increasing requests are attributable to the fast growing hard cider industry and growers and cider makers' desire for new cider apple varieties. In 2016, we sent out 69 Malus accessions to the Plant and Animal Genetic Resources Preservation Unit (PAGRP), USDA-ARS Fort Collins for cryo backup. We received back cryo-treated budwood of 65 accessions for viability testing by budding. The viability (budding) results showed that 8 accessions had 0% viability, 5 accessions with viability ranging from 11% to 30% (below satisfactory 40% viability standard), and the other 52 accessions had viabilities ranging from 44% to 100%. Overall, 75.36% of the accessions (52 out of 69) have acceptable viability after cryo storage treatment and there is sufficient cryo-preserved budwood for long term backup storage of these 52 accessions. For the 2017 cryo backup effort, we sent to USDA-ARS PAGRP 55 Malus and 15 Prunus accessions. The cryo back up effort for apple and tart cherry in the next 5 years is uncertain as of now due to budget constraints. The PAGRP is allocating more resources to cryo backup of other plant genetic resources that have very high critical needs, such as the citrus collection that is facing the threat of devastating greening disease (Huanglongbing-HLB). We will work with PAGRP on the apple and tart cherry cryo backup whenever resources are available. Over three years, the Vitis collection (approximately 2,500 vines) was evaluated for phenological traits, including bud break, bloom date, and fruit ripening. These traits provide information on the adaptability of an accession for a particular location, and could be used to develop broadly adapted grape cultivars. Currently, this information is being summarized for distribution. Additionally, 718 hybrid grapevines (typically cultivated grapevine crossed with wild Vitis species) were genotyped using GBS. This data will be used for future genetic studies and management of the collection. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? A total of 236 orders for 4,900 vegetable seed germplasm samples were filled for clients and stakeholders in Calendar Year 2015. In 2016, through May 1, there were 80 orders for 2,098 seed germplasm samples. A large number of these samples were distributed in the northeast. For 2015, 484 seed samples for 31 orders were distributed to users in the states that are part of NE-9. For 2016, through May 1, there were 190 seed samples for 11 orders distributed to the NE9 region. Delivery of germplasm to clients and stakeholders is the primary way in which the results of this unit's activities are distributed to the public. Seed regenerations of vegetable Brassicas, tomatoes, wild tomatoes, tomatillos, celery, artichokes, radish, onion, buckwheat, and Cucurbits in 2015 were completed for 252 accessions and 60 accessions were grown for plants and bulbs to store for seed production in 2016. For 2016, seed regenerations of 223 accessions are being completed and 40 accessions are being grown for plants and bulbs to store for seed production in 2017. In addition to standard regeneration activities, 11 short-day onion accessions were regenerated at New Mexico State University. A total of 12,605 accessions of seed-propagated vegetable crops were successfully maintained during the past year, including nine new accessions. The future availability of this germplasm is therefore assured. A set of 52 commercial varieties from PGRU's collection were profiled for cation concentrations. Cations influence fruit quality traits such as color, uniformity of ripening, hollow fruit, fruit shape, firmness, and acidity, and are also important components of human diets. Results supported a genetic basis for potassium, magnesium, calcium and sodium concentrations that was consistent across environments. Potassium and magnesium were significantly correlated with each other.No other correlations between pairs of traits were observed. Results will provide insight for development of cultivars with favorable cation profiles in terms of human health and fruit quality. The crop Brassica rapa L. is very diverse in its usage. It can be consumed as a raw or cooked leafy green (mizuna, Chinese cabbage), a spice (sarson), a root vegetable also used as animal fodder (turnip), or a cooking oil (oilseed rape). The genetic relationships among these various types is not well understood but a deeper understanding would be beneficial for crop conservation and improvement. We sequenced portions of the genomes of 333 accessions of B. rapa that originated from across the globe and included ten various morphotypes. Results supported the genetic distinction of types but also indicated differences between the same types based on geographic origins. Understanding of these relationships is key to future breeding and scientific work in B. rapa. Onion is highly consumed in the U.S.; production dollar value ranks third among vegetables. As an ancient, globally widespread crop, thousands of onion cultivars have been developed to fit diverse environments and dietary preferences. Onion is costly to breed because of its biennial habit and requirements for cross-pollination. Molecular genetic markers, which can increase the efficiency of breeding, are difficult to develop due to onion's large (16GB) genome size. We used genotyping-by-sequencing (GBS) to discover 70,000 single nucleotide polymorphism (SNP) markers in onion. After filtering for high quality and expected segregation ratios, 701 of the SNPs were mapped in an F2 population. Results indicated that GBS is a promising technology for marker development in onion. A total of 444 orders for 7,355 clonal samples were distributed to stakeholders in Year 2015. The number of order was a record high for clonal crops. For 2015, 3,278 clonal samples for 105 orders were distributed to states of NE-9. Most orders were for cuttings or dormant budwood in addition to pollen, flower, leaf, fruit, seed and sap. We maintained 5,910 Malus accessions, 1,392 Vitis accessions, and 130 Prunus accessions at PGRU. The K1 block, M. sieversii seedling block was removed in December 2015. Working with Dr. Agnello, Entomology, Cornell University, we monitored the apple black stem borer (Xylosandrus germanus) in 2016 to better manage this insect. Five publications, five meeting abstracts, and one report were published in 2015 about clonal crops. We received 14 wild Malus ioensis seed accessions from the USDA-ARS North Central Plant Introduction Station in October 2015. Chao, clonal curator, carried out an exploration from September 5-20, 2015 in Arkansas, Mississippi, Louisiana, and Alabama and collected 37 seed accessions of M. angustifolia and two seed accessions of Muscadine grape (Vitis rotundifolia). All seeds were extracted, accessioned, and stored at -200C freezer. An off cycle exploration of M. angustifolia in Arkansas, Mississippi, and Louisiana took place in August 2016. A proposal of collecting M. doumeri in Taiwan was submitted. In January 2016, we received budwood of 30 crab apples from the National Arboretum. We received one accession of M. trilobata in February 2016 from the Arnold Arboretum and one seed accession of M. trilobata in April 2016 from Turkey. We received one seed accession of M. coronaria from Ohio State University in February, 2016. We received 'Campfield' M. hybrid from B. O'Gorman, East Sandwich, MA also in February, 2016. We collected over 33,000 additional open pollinated seeds from the wild M. sieversii seedling block in 2015. We sent 69 Malus accessions in January 26, 2016 to NCGRP, Fort Collins, CO for cyro storage. We also shipped a large quantity of budwood of 8 tart cherry and 11 Malus to NCGRP in January 20, 2016 for cryo experiment. Fruit of selected M. orientalis, new Spanish cider apples, and other Malus accessions were evaluated in fall 2016 for their hard cider potential with cooperation with C. Gerling, Food Science, and G. Peck, Horticulture, Cornell University. GBS-derived SNP markers from 1,251 progenies for 7 F1s from 'Royal Gala' x 7 M. sieversii revealed about 476,000 putative SNPs. The number of useful SNP markers for initial screening per F1 calls ranges from 3,023 to 5,489 SNPs. Both male and female linkage maps were constructed using JoinMap 4.1through an iterative marker screening workflow adopted from HetMappS. Although additional marker curation could enhance the marker density, the number of SNP markers for the linkage maps of the four largest F1s were 821 (GMAL4591), 753 (GMAL4596),748 (GMAL4590) and 738 (GMAL4593), giving an average marker density per linkage group for about 2.1cM/marker. Additionally, a linkage map with 646 markers based on RoseBreed SNP Chip was also constructed (GMAL4593). The close agreement in the marker order and linkage group size between GBS and SNP Chip derived maps indicate the robustness of the adopted iterative marker screening workflow. Marker screening for the remaining F1s continues.We will also use the GBS data to investigate other questions of the Malus collection. Other traits such as phenology and growth traits are also being evaluated. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? A total of 266 orders for 6,232 vegetable seed germplasm samples were filled for clients and stakeholders in Calendar Year 2014. In 2015, through July 1, there were 133 orders for 3,983 seed germplasm samples. A large number of these samples were distributed in the northeast. For 2014, 381 seed samples for 40 orders were distributed to users in the states that are part of NE-9. For 2015, through July 1, there were 163 seed samples for 17 orders distributed to the NE9 region. Delivery of germplasm to clients and stakeholders is the primary way in which the results of this unit's activities are distributed to the public. Seed regenerations of vegetable Brassicas, tomatoes, wild tomatoes, tomatillos, celery, artichokes, radish, onion, buckwheat, and Cucurbits in 2014 were completed for 312 accessions and 88 accessions were grown for plants and bulbs to store for seed production in 2015. For 2015, seed regenerations of 252 accessions are being completed and 60 accessions are being grown for plants and bulbs to store for seed production in 2016. In addition to standard regeneration activities, 15 short-day onion accessions were regenerated at New Mexico State University. A total of 12,600 accessions of seed-propagated vegetable crops were successfully maintained during the past year, including nine new accessions. The future availability of this germplasm is therefore assured. We are in the process of completing studies that i)characterized a diverse set of 50 historic USA tomato varieties for fruit quality (antioxidants, sugars, acids, Vitamin C and color), and size and morphological (size and shape) variables using replicated trials across two environments, ii) evaluated the reproducibility of assays for organic acids, carbohydrates and antioxidants in cryopreserved samples of homogenized fruit, iii) estimated effects due to genotype, environment and their interaction on fruit quality and morphological variables, iv) provided insight into which traits distinguished the varieties and as to whether these traits were interrelated, correlated or predictive of each other, and v) discovered which traits are tractable for crop improvement based on their heritability, relationships and ease of assay. A core set of 190 PGRU tomato accessions was genotyped using the Genotyping By Sequencing (GBS) tool. These originated from 31 countries and included six different versions of the economically valuable variety San Marzano. A total of 3,713 mapped, high quality SNPs were discovered. We are currently in the process of analyzing genotypic data. Data will be used to address questions regarding the amount and global distribution of tomato genetic diversity. A core set of 152 radish accessions was assembled based on geographical origin and representation in the PGRU collection (weighted sampling). A total of 35 countries were represented. Five plants per accession were genotyped using GBS. Two squash core sets of 95 accessions each, one plant per accession, were genotyped using GBS. The core sets are based on morphological diversity and geographical diversity. In collaboration with M. Gore (Cornell University) and C. Pires (University of Missouri, Columbia) PGRU's Brassica rapa collection of 268 accessions along with 100 accessions from the North Central Regional PI station were genotyped using GBS. A field trial of 360 accessions of Brassica rapa was conducted in Geneva, NY in the 2015 season. Phenotypic data collected from this experiment will be used for GWAS. A total of 388 orders for 6,128 samples of fruit clonal germplasm were filled for clients and stakeholders in Calendar Year 2014. A large number of these samples were distributed in the northeast. For 2014, 2,719 clonal samples for 86 orders were distributed to users in the states that are part of NE-9. From July 1, 2014 to June 30, 2015, we had 105 orders and we distributed 3,268 samples to the NE-9 states. Most distribution of the fruit clonal collection in NE-9 states was for dormant budwood and cuttings in addition to pollen, leaf, fruit, and seed samples. We maintain 6,631 Malus accessions, 1,392 Vitis accessions, and 130 Prunus accessions at Geneva. We will collect additional "Botany of Desire seeds" in the fall of 2015 before the removal of the K1 M. sieversii seedling block. M. sieversii is a wild progenitor of cultivated apples. From the original 1,300+ M. sieversii seedlings, we have selected and propagated 178 seedlings for inclusion into the permanent collection. We also have 79 wild M. sieversii seedling selections from Dr. J. Luby, University of Minnesota, in our permanent collection. Overall, we have 299 accessions of M. sieversii in our permanent collection. We still maintain 56,865 M. sieversii seeds from the original exploration trips plus an additional 47,978 seeds from specific crosses among M. sieversii accessions. We are also working on selecting representative seedlings from 36 half-sib families of wild Vitis species in the W2 block based on genetic profiles (GBS genotyping), powdery mildew resistance, gender, and overall vine health. The goal is to re-propagate the representative seedlings to the main Vitis collection block (I1 block) and eventual removal of the W2 block for new Malus plantings. We received one seed accession of M. orientalis from Georgia in October 2014 through cooperative exploration; 22 Malus accessions from APHIS Quarantine Center in March 2015 that included two M. sieversii accessions from Kazakhstan from the 1996 exploration; 'Seker Elmasi' and 'Turkish' from Turkey; 'Clara', 'Limon Montes', 'Verdialona', 'Marialena' cider apples from Northern Spain; 'M23', 'M24', 'Jongrines', 'Barnick Beauty', 'Schoner VonNord Hausen', 'Jersey Black', 'Ralls Janet' from the United Kingdom; 'Shukar' from Pakistan; 'Poorhouse', 'Babbit', 'Cloud' from Tasmania, Australia; '14/5' from Israel; and 'GE-061', 'Champawski Apple' from Georgia. Currently we have 47 accessions that are in "pre-quarantine" release status at different stages of propagation. All 47 have not been fully-released from the APHIS quarantine process yet. We received two accessions of Ampelopsis brevipedunculata (Porcelain vine, a wild relative of Vitis from Asia) from Dr. Bruce Reisch, Cornell University. We collected an additional 22,549 "Botany of Desire Seeds" (open pollinated seeds of M. sieversii seedlings) in the fall of 2014, which now totals 76,353 seeds in cold storage. We sent 11 Malus accessions and 8 Prunus accessions on January 12 and February 9, 2015 to the National Center for Genetic Resources Preservation (NCGRP), Fort Collins, CO for a cryo-backup protocol experiment in the winter of 2014/2015. The viability testing of the cryo-treated buds is ongoing. To provide critical information to stakeholders and clients for better utilizing these germplasm, we finished the third year recording of phenology traits of all permanent Malus accessions in the spring of 2015. Fruit samples of apples and tart cherries were collected in 2014 and are being analyzed for profiles of 30 secondary metabolites using high-pressure liquid chromatography (HPLC). Fruit weight, color and titratable acidity for apple and tart cherry samples were also collected. GBS genotyping of seven F1 populations from 'Gala' x M. sieversii was completed and genetic maps for all 7 F1 populations will be created as a community resource. We added 252 floral photos of Malus and berry photos of Vitis to the GRIN database in 2015. No Malus fruit photos were taken in 2015 due to poor fruit set and severe hail damage. A plant exploration for collection of wild M. angustifolia germplasm in the Southern U.S. took place in September 2015. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? A total of 229 orders for 4,786 vegetable seed germplasm samples were filled for clients and stakeholders in calendar year 2013. In 2014, through August 1, there were 192 orders for 3,388 seed germplasm samples. A large number of these samples were distributed in the northeast. For 2013, 461 seed samples for 51 orders were distributed to users in the states that are part of NE-9. For 2014, through August 1, there were 351 seed samples for 33 orders distributed to the NE9 region. Delivery of germplasm to clients and stakeholders is the primary way in which the results of this unit’s activities are distributed to the public. Seed regenerations of vegetable Brassicas, tomatoes, wild tomatoes, tomatillos, celery, artichokes, radish, onion, buckwheat, and Cucurbits in 2013 were completed for 307 accessions and 88 accessions were grown for plants and bulbs to store for seed production in 2014. For 2014, seed regenerations of 312 accessions are being completed and 65 accessions are being grown for plants and bulbs to store for seed production in 2015. In addition to standard regeneration activities, 25 short-day onion accessions were regenerated at New Mexico State University. A total of 12,666 accessions of seed-propagated vegetable crops were successfully maintained during the past year, including 29 new accessions of B. rapa, 22 new accessions of radish, and 33 accessions of buckwheat, all from China. The future availability of this germplasm is therefore assured. Understanding the genetic and environmental components of health beneficial and nutritional compounds of tomato will aid in development of superior cultivars. We are in the process of analyzing a study that i)characterized a diverse set of 52 historic U.S.A. tomato varieties for fruit quality (antioxidants, sugars, acids, Vitamin C and color), and size and morphological (size and shape) variables using replicated trials across two environments, ii) evaluated the reproducibility of assays for organic acids, carbohydrates and antioxidants in cryopreserved samples of homogenized fruit, iii) estimated effects due to genotype, environment and their interaction on fruit quality and morphological variables, iv) provided insight into which traits distinguished the varieties and as to whether these traits were interrelated, correlated or predictive of each other, and v) discovered which traits are tractable for crop improvement based on their heritability, relationships and ease of assay. Genotyping by sequencing data have been analyzed for tomatillo (Physalis philadelphica) (190 samples). Geographic Information Systems (GIS) data from Genetic Resources Information Network (GRIN) and from the International Rice Research Institute (IRRI) project on geospatial references was verified using web and PC-based software tools. We found that multiple plants sampled per accession were closely related to each other, but there was no apparent pattern related to state in Mexico, latitude or isolation-by-distance. Average proportion of heterozygous sites was halved in nine inbred lines developed at PGRU relative to open-pollinated accessions (0.04 versus 0.08). Tests of population differentiation showed that 29% of pairs of accessions were significantly differentiated from each other. The genetic characterization of these accessions can help end users choose germplasm to support increased US production of fresh and processed tomatillo products such as taco and enchilada sauces. We assembled a set of 95 onion (Allium cepa) accessions as a PGRU core collection. These originated from 28 countries or regions. High quality DNA was isolated from one plant per accession and these have been assayed in replicate at Cornell University Institute for Genomic Diversity. The resultant SNP markers are being mapped to a draft onion transcriptome. This will result in thousands of high-quality markers for crop improvement and germplasm management. A total of 297 orders for 5,795 samples of fruit clonal germplasm were filled for clients and stakeholders in Calendar Year 2013. In 2014, through August 1, there were 273 orders for 3,343 samples of fruit clonal germplasm. A large number of these samples were distributed in the northeast. For 2013, 2,996 clonal samples for 87 orders were distributed to users in the states that are part of NE-9. For 2014, through August 1, there were 690 clonal samples for 66 orders distributed to the NE9 region. Most of the samples of the fruit clonal collection were for DNA and leaf samples for DNA extraction, but multiple samples of cuttings, pollen and seeds of wild species were distributed as well. We maintain 6,717 Malus accessions, 1,432 Vitis accessions, and 130 Prunus accessions at Geneva. Fourteen new Malus accessions were added to the collection, including nine new foreign Malus accessions from the APHIS Quarantine Center and five seed accessions of M. sylvestris from Albania. We collected additional “Botany of Desire Seedsâ€