2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The major objectives of this CRIS are the acquisition, documentation, maintenance, evaluation/characterization, and distribution of seed propagated crop plants and their wild and weedy relatives. The primary crops involved are tomato, the cole crops, onion, radish, and winter squash. Crops of secondary importance include asparagus, celery, buckwheat and tomatillo. We are meeting these objectives by such activities as (list is not exhaustive). 1)conducting explorations for foreign germplasm, making exchanges of germplasm with other genebanks and with crop improvement institutes both public and private,. 2)developing and carrying out our improved methods for seed production and storage, and for improving its initial viability,. 3)updating, error-checking, and consolidating our germplasm database documentation,. 4)participating in Crop Germplasm Committees for these crops,. 5)promoting germplasm evaluation studies and conducting our own characterization projects,. 6)applying molecular genetic technology to conservation and management of the germplasm collections to solve problems in - i) acquisition, maintenance, and genetic characterization of collections; ii) determining levels of genetic diversity and iii) developing or modifying molecular genetic marker technology for particular crops as needed,. 7)improving the efficiency and timeliness of our germplasm seed distribution procedures,. 8)promoting the utilization of the germplasm through development of contacts and linkages with breeders, researchers and Non-Governmental Organizations (NGOs) such as organic farmer groups and seed savers associations,. 9)a Specific Cooperative Agreement with the Tomato Genetics Research Center (TGRC) supports conservation and distribution of wild species from the TGRC collection, 10) a Specific Cooperative Agreement with New Mexico State University for rescue of short-day onion accessions, and 11) two Reimbursable Cooperative Agreements with Cornell University support efforts to train organic farmers and small seed producers in production of seed of heirloom and new public improved varieties of vegetables, provide backup support for trialing of vegetables under organic conditions, and provide backup and germplasm for participatory plant breeding projects for vegetables adaptable to organic conditions. The continuing development of new improved vegetable cultivars to meet increasing pressures from insect, fungal, and bacterial pests, to satisfy the nutritional and health requirements of the American consumer, to increase productivity in existing environments, and to continue production in the face of environmental stresses requires a reliable source of plant genes. The development of organic agriculture in the US has led to the demand for alternative sources of germplasm that the NPGS genebanks have a comparative advantage in fulfilling. Healthy, viable germplasm is the source of these genes that are critical to the long-term sustainability of American agriculture. With increased international emphasis on national sovereignty of germplasm limiting the free flow of germplasm, proper management and distribution of genetic resources already in the United States becomes more and more critical. This Project falls under Component 1 (Genetic Resource Management) of National Program 301 (Plant, Microbial, and Insect Genetic Resources, Genomics, and Genetic Improvement). The National Plant Germplasm System (NPGS) is a multi-component system of the United State Department of Agriculture's Agricultural Research Service within NP 301 that has responsibility for the conservation and utilization of plant genetic resources. The NPGS delivers both germplasm and associated knowledge to the stakeholder community. This project deals with all five problem areas of component 1 (Safeguarding Threatened Genetic Resources and Associated Information; Maintaining Genetic Resources and Associated Information Efficiently and Effectively; Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; and Technology Transfer of Genetic Resources and Associated Information). The project will also contribute to genome characterization in Component 2. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Provide adequate storage and backup of germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Distribute germplasm of vegetable crops and buckwheat to stakeholders in the United States and worldwide. This is a service activity that will continue throughout the life of the project. Regenerate germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Acquire germplasm of vegetable crops and buckwheat; specifically acquire germplasm of tomatillo from Mexico. This is a service activity that will continue throughout the life of the project. Perform SNP (single nucleotide polymorphism) prediction from NCBI L. esculentum EST database and verification by resequencing two or three tomato lines. Optimize genotyping markers for two flower developmental pathway genes in broccoli and cauliflower, genotype 3 to 30 plants for each of 40 PGRU accessions. Collect characterization data and digital images for germplasm of vegetable crops and buckwheat and upload these for use by stakeholders on GRIN. This is a service activity that will continue throughout the life of the project. Promote awareness of the value of NPGS with NGOs by providing training in small scale seed production of heirlooms and modern public varieties. This is supported by a RCA. Year 2 (FY 2005) Provide adequate storage and backup of germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Distribute germplasm of vegetable crops and buckwheat to stakeholders in the United States and worldwide. This is a service activity that will continue throughout the life of the project. Regenerate germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Acquire germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Genetic diversity study of one plant from each of 31 tomato lines at 48 genes comparing marker types (EST, COSII, carotenoid loci). Optimize genotyping markers for two glucosinolate pathway genes in broccoli and cauliflower, genotype 3 to 30 plants for each of 40 PGRU accessions. Complete a manuscript on SNP prediction from NCBI EST database and verification in two or three tomato lines. Continue development of a PGRU molecular marker database. Collect characterization data and digital images for germplasm of vegetable crops and buckwheat and upload these for use by stakeholders on GRIN. This is a service activity that will continue throughout the life of the project. Promote awareness of the value of NPGS with NGOs by providing training in small scale seed production of heirlooms and modern public varieties, provide backup support for trialing of varieties under organic conditions, and provide backup support and germplasm for participatory plant breeding projects for vegetables adapted to organic growing conditions. This is supported by two RCA agreements. Year 3 (FY 2006) Provide adequate storage and backup of germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Distribute germplasm of vegetable crops and buckwheat to stakeholders in the United States and worldwide. This is a service activity that will continue throughout the life of the project. Regenerate germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Acquire germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Describe partitioning of tomato genetic diversity within and among plants, accessions, geographical regions, and time periods by sequencing 50 mapped genes in 61 total plants. Design primers for 100 mapped genes in B. oleracea. Complete a manuscript on genetic diversity of 31 tomato lines at 48 genes comparing marker types (EST, COSII, carotenoid loci). Continue development of a PGRU molecular marker database. Collect characterization data and digital images for germplasm of vegetable crops and buckwheat and upload these for use by stakeholders on GRIN. This is a service activity that will continue throughout the life of the project. Promote awareness of the value of NPGS with NGOs by providing training in small scale seed production of heirlooms and modern public varieties, provide backup support for trialing of varieties under organic conditions, and provide backup support and germplasm for participatory plant breeding projects for vegetables adapted to organic growing conditions. This is supported by an RCA agreement. Year 4 (FY 2007) Provide adequate storage and backup of germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Distribute germplasm of vegetable crops and buckwheat to stakeholders in the United States and worldwide. This is a service activity that will continue throughout the life of the project. Regenerate germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Acquire germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Estimate linkage drag in domesticated tomato by sequencing alleles in two plants each of six wild species at the same 50 mapped genes sequenced in L esculentum in FY2003. Optimize sequencing in B. oleracea for 50 mapped genes using inbred line BI87503. Complete a manuscript on partitioning of tomato genetic diversity within and among plants, accessions, geographical regions, and time periods. Continue development of a PGRU molecular marker database. Collect characterization data and digital images for germplasm of vegetable crops and buckwheat and upload these for use by stakeholders on GRIN. This is a service activity that will continue throughout the life of the project. Promote awareness of the value of NPGS with NGOs by providing training in small scale seed production of heirlooms and modern public varieties, provide backup support for trialing of varieties under organic conditions, and provide backup support and germplasm for participatory plant breeding projects for vegetables adapted to organic growing conditions. This is supported by an RCA agreement. Year 5 (FY 2008) Provide adequate storage and backup of germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Distribute germplasm of vegetable crops and buckwheat to stakeholders in the United States and worldwide. This is a service activity that will continue throughout the life of the project. Regenerate germplasm of vegetable crops and buckwheat; specifically acquire germplasm of cultivated onion from Central Asia. This is a service activity that will continue throughout the life of the project. Acquire germplasm of vegetable crops and buckwheat. This is a service activity that will continue throughout the life of the project. Study linkage disequilibrium and population structure in L. peruvianum by sequencing 50 mapped genes within and among 3 to 9 accessions. Test robustness of PCR and DNA sequencing of 50 mapped genes on B. oleracea doubled-haploid variety Nedcha and one progeny line of Nedcha x BI87503. Complete a manuscript on linkage drag in domesticated tomato. Continue development of a PGRU molecular marker database. Collect characterization data and digital images for germplasm of vegetable crops and buckwheat and upload these for use by stakeholders on GRIN. This is a service activity that will continue throughout the life of the project. Promote awareness of the value of NPGS with NGOs by providing training in small scale seed production of heirlooms and modern public varieties, provide backup support for trialing of varieties under organic conditions, and provide backup support and germplasm for participatory plant breeding projects for vegetables adapted to organic growing conditions. This is supported by an RCA agreement. 4a.What was the single most significant accomplishment this past year? Vegetable breeders and other researchers worldwide need a ready source of diverse germplasm. At Geneva, approximately 11,500 accessions of tomato, onion, radish, winter squash, cabbage, cauliflower, broccoli, other cole crops, celery, tomatillo, asparagus and other vegetables were maintained and 173 new accessions were acquired including 111 accessions of tomatillo from Mexico and 43 accessions of collards from the southern United States. During FY 2005 approximately 406 accessions were regenerated; additionally, 177 biennial crop accessions were grown to produce plants for use in seed production in 2006; and 2837 seed lots of 2124 accessions were distributed in 206 orders (163 domestic and 43 foreign). This germplasm is available for use by qualified researchers and other bona vide users worldwide. 4b.List other significant accomplishments, if any. Short-day onion accessions cannot be regenerated in Geneva, NY. A SCA with New Mexico State University was extended for regeneration of these accessions. In FY2005 this resulted in regeneration of 43 short-day onions that were in danger of being lost (25 for seed and 19 for bulb production for seed production in 2006). This resulted in these accessions being made available for distribution to short-day onion breeders. We empirically tested for predicted SNPs in 85 of 764 EST markers by sequencing two or three tomato lines. In doing so we discovered more polymorphism (62 SNPs and 12 small insertion/deletions in 21 genes) than had been previously reported in domesticated tomato (Labate and Baldo, accepted in Molecular Breeding). All polymorphisms we verify will be useful in saturating intraspecific genetic maps of cultivated tomato. We also found evidence for highly diverged alleles (1.6 to 13 fold more variable) at five genes within L. esculentum, and have hypothesized that these are cryptic wild species alleles that represent linkage drag. Recognizing linkage drag within domesticated tomato will enable breeders to study its effects on agronomic quality during introgression and backcrossing. We collected data to compare polymorphism across a genetically diverse panel of 30 PGRU L. esculentum accessions and breeding line TA496 (provided by S. Tanksley) at 11 carotenoid, 11 COSII, 10 EST markers where a predicted SNP had previously been verified, 11 EST markers where a predicted SNP had not been verified, and five EST markers that we hypothesized to be cryptic wild species alleles within L. esculentum. The purpose is to improve SNP prediction methods from ESTs and to estimate ascertainment bias in EST markers with predicted SNPs by comparing them to other markers (carotenoid and COSII). Improved SNP prediction methods and increased understanding of ascertainment bias will be useful for genetic diversity studies for all crops, especially those that are relatively low in genetic variation. We have worked on developing diagnostic tools to distinguish between broccoli and cauliflower seeds and seedlings by genotyping several-hundred plants from PGRU broccoli and cauliflower accessions for flowering-developmental and glucosinolate-biosynthetic pathway markers. We found evidence to support published claims that alleles at BoCAL, AP1-a, ALK, and ELONG are correlated with a cauliflower phenotype in B. oleracea but the correlations were weak and not predictive in PGRU accessions. Paralogous loci may have confounded our results. Continued improvement of such tools will allow curators and breeders to predict phenotype without growing a plant to maturity. There is need for heirloom and new publicly bred germplasm for use by organic farmers. A RCA with Cornell University provides support to PGRU for training of organic farmers and small seed producers in small-scale seed production of heirloom and new publicly improved vegetable varieties, whose production has been limited because of lack of seed. In FY2004 demonstrations, training workshops, and community seed days were conducted for over 700 participants. This has increased the availability of heirloom and new publicly bred germplasm for use by small and organic farmers. 4c.List any significant activities that support special target populations. Workshops, demonstrations and community seed days were held at the USDA-ARS, PGRU in Geneva, NY 23 August, 2005; at the Connecticut Agricultural Experimental Station in Hamden, CT on October 7, 2004; at Peacework Organic Farm in Newark, NY on October 22, 2004, at Gorzynski's Ornery Farm in Cochecton Center, NY on November 8, 2004, at the On-Farm Seed Production Workshop at the NOFA-NY Annual Winter Conference in Syracuse, NY on January 28, 2005; at the NOFA-MA Summer Conference in Amherst, MA on August 13, 2005; at Harvest Home Organics in Moravia, NY on September 7, 2005 and at the Common Ground Fair in Unity, ME on September 2 and 24, 2005. These workshops served small scale farmer and organic grower target populations. They provided essential training in small-scale seed production to growers and organic-farmers interested in incorporating seed production into their existing farm systems. These workshops also provided a means to display the mobile seed processing unit. This unit consists of various seed processing and threshing devices used in wet and dry seed processing. During the community seed day workshops held in Geneva, Newark, Moravia, and Cochecton Center, NY; and in Hamden, CT organic farmers and small-scale seed producers were trained and given the opportunity to clean their seed using the equipment provided in the mobile seed processing unit. These activities were supported by the RCA with Cornell University where the PGRU has the lead role in this project in the extension of small-scale seed production of heirloom vegetable germplasm and new public open-pollinated varieties by organic farmers and small-scale seed producers. The PSI (Public Seed Initiative) website continues to be a major source of information for organic farmers with over 5,000 hits since its creation. This website supplies detailed information on upcoming events, pepper genomics, seed production, participatory breeding, on-farm commercial variety trials, and other related projects throughout the country: www.plbr.cornell.edu/psi. The OSP (Organic Seed Partnership) website contains information about the new goals and direction of the new OSP project. This website will contain all the detail that the PSI website has plus information about nationwide cooperators, updated news and events around the country, and streaming video detailing on-farm vegetable breeding and seed production: www.organicseedpartnership.org. To complete the PSI project, a final PSI CD has been created. This CD combines all annual reports for participatory breeding, on-farm commercial variety trialing and seed production. The CD shows detailed instruction for seed production and breeding of several common vegetable crops. 4d.Progress report. 1910-21000-016-02R-This report serves to document research conducted under a Reimbursable Cooperative Agreement between ARS and Cornell University. Workshops. Workshops on seed saving, seed production and/or the economics of seed production were conducted at the USDA-ARS, PGRU in Geneva, NY on 23 August 2005; at the PSI On-Farm Seed Production Workshop at the NOFA-NY Annual Winter Conference in Syracuse, NY on January 28, 2005; the NOFA-MA Summer Conference in Amherst, Massachusetts on August 13, 2005, and the Common Ground Fair in Unity, Maine September 23 - 24, 2005. These training workshops were attended by over 800 people. Demonstrations: Displays and demonstrations of the mobile seed processing unit and/or PSI outreach material were held at the NOFA-MA Summer Conference in Amherst, MA on August 13, 2005; and at the Common Ground Fair in Unity, ME on September 24 and 25, 2004. Community Seed Days. The mobile seed processing unit was set up at several locations for wet and dry seed processing during weeklong community seed days. The community seed days were held in Geneva, Newark, Moravia and Cochecton Center, NY; and in Hamden, CT. 1910-21000-016-03S-This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Texas Agricultural Extension Service. Cucurbit Yellow Stunting Disorder Virus is a whitefly-transmitted crinivirus which infects watermelon and other cucurbits. It was first reported in the U.S. in south Texas during 1999. It is efficiently vectored by the silverleaf whitefly and has caused serious reductions in yield and quality throughout the Mediterranean basin and some regions of Mexico. It can be severe in south and west Texas when whitefly populations are moderate to large. Primary symptoms are cholrosis of older leaves and green chlorotic spots, reduced vigor and poor fruit quality- primarily low sugars. Resistance has been identified in melon (Cucumis melo L.) but not in watermelon (Citrullus lanatus). Procedures: The core collection of the Citrullus spp. Germplasm was acquired from the southern regional plant introduction station in Georgia during 2003. Six seeds per accession were planted in sunshine germination mix in 72 cell styrofoam trays inside the greenhouse. Twenty mature melon plants infected with CYSDV and infested with silverleaf whiteflies were growing in pots inside the greenhouse. CYSDV infection was verified by RT-PCR using total RNA extracts and primers developed by Dr. Bryce Falk. Typical leaf symptoms were also evident. All watermelon seedlings were exposed to these viruliferous whiteflies for 20 days post-germination. During early April, all seedlings were then transplanted to field plots at the Texas Agricultural Experiment Station at Weslaco. The CYSDV infected melon plants were planted interspersed throughout the field. Subsurface drip irrigation, chemical weed control and fertigation with ammonium nitrate were employed. Additionally, fungicides were applied to prevent downy and powdery mildews, but no insecticides were applied. Watermelons typically evince leaf symptoms later than melons at Weslaco. After 6 weeks, few symptoms were observable, though cool temperatures reduced overall vine growth. April weather was unusually cloudy and wet, often impeding the application of sufficient fungicides. As a result, severe infestation by gummy stem blight (Didymella bryoniae) began to occur. By mid-May when the weather finally returned to the typical dry and sunny conditions, more than half of all the accessions had been killed by gummy stem blight and downy mildew. In addition, CYSDV symptoms were still not widespread in the plots, likely due to the destruction of the whitefly population by the rains. Fungicides were applied to stop the downy mildew and gummy stem blight with the hope of no further rains. The dry conditions prevailed for two weeks and the whiteflies rebounded by early June. Ratings for CYSDV symptoms were taken during this period, with most remaining accessions demonstrating symptoms. Unfortunately, by the second week of June, the rains returned and the station received 10 inches over the next two weeks. This flooded the plots and killed 90% of the remaining plants. In early July we took a final rating and also noted the apparent resistance of the remaining accessions to gummy stem blight and flooding. As a whole, the C. colocynthis accessions showed the least disease, including CYSDV and low whitefly infestations. Many of the C. lanatus citroides accessions also demonstrated less gummy stem blight and downy mildew infection. The following accessions are grouped according to CYSDV symptom expression, but many accessions had died from gummy stem blight before good CYSDV development occurred. No CYSDV symptoms and apparent DM+GSB resistance: PI's 386018, 248774, 482259, 482261, 482276, 482302, 500354, 482335, 482338, 485580, 596656 Very slight CYSDV symptoms on oldest leaves: GRIF 14201, PI's 195927, 244017, 244019, 254428, 254744, 271778, 296341, 295842, 271770, 271771, 368526, 379243, 386019, 386021, 386024, 386025, 386026, 482286, 482331, 629107 Obvious CYSDV symptoms on foliage: PI’s 388770, 525082, 537277, 542616, 549161, 295848, 482318, 482343, 600896, 600951, 629102, 629103, 632751, 632753, 179881, 244018, 270562, 271767, 271769, 271773, 271775, 271779, 295850, 299378, 296337, 296334, 296335, 296337, 296339, 296342, 296343, 482273, 482277, 482282, 500343 Recommendations: Currently, leaves of the accessions with slight yellowing are being tested for CYSDV using PCR primers. These and the acessions which exhibited no symptoms will be grown in the Fall greenhouse and infested with viruliferous whiteflies to verify resistance and exlude the possibility of escapes. All accessions, which died in the field before expressing CYSDV symptoms could also be re-screened in the greenhouse to prevent weather related catastrophes, but space limitations would make this a very time consuming and laborious process. Field screening were attempted again during the Spring 2005. 1910-21000-016-04S-This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Brigham Young Univeristy. Foot rot of tomato caused by Fusarium solani was first reported on the Queensland Coast of Australia in 1975. Symptoms include brown to reddish-brown lesions (about 1 to 2.5 cm long) of the cortex of the tap or main lateral roots with a discoloration of the stele extending 2 to 10 cm from the lesion. Although plants usually do not die, yields are often reduced. In California, the disease was first reported in 1991. Once confined to a few fields, the disease has spread significantly and continues to do so. Based on our 2002 survey work, the incidence of foot rot in individual fields in Yolo County has been as high as 45%. Today, the disease is now found in most of the Sacramento Valley (Sutter, Yolo, Sacramento, and Solano counties) and has moved into the San Joaquin Valley (San Joaquin, Stanislaus, and Merced counties). The only realistic long-term solution for managing the disease is through breeding for disease resistance. Genetic variability to susceptibility to Fusarium so/ani, the cause of tomato foot rot, exists within the wild germplasm collection. Because this disease is spreading in California and no control methods currently exists, resistance offers the best possible solution; resistance in wild tomato germplasm needs investigation for potential incorporation into commercial tomato varieties. To the best of our knowledge, there has been no systematic screening for resistance in North American breeding lines or wild tomato relatives. Germplasm screened included 103 accessions in the collection at the University of California, C. M. Rick Tomato Genetics Resource Center. Germplasm screened included core accessions in the collection at the University of California, C. M. Rick Tomato Genetics Resource Center. Several accessions possessed apparent resistance to the fungus; these represent possible targets for tomato breeders working on the development of resistance in tomato. This research could lead to tomato cultivars that are resistant to Fusarium foot rot, resulting in sound and economical disease control. 1910-21000-016-05S-This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of California. Germplasm Acquisitions: 25 new stocks were acquired, including additional Solanum lycopersicoides introgression lines in tomato (Lycopersicon esculentum), a marker stock for chromosome 11 (genes up, j and wv-3), and a population of Lycopersicon peruvianum from Peru. The current total of number of active accessions is 3,617. Germplasm Maintenance: A total of 2,257 cultures were grown for various purposes, of which 1,544 were for seed increase, and 240 for seed germination tests. Seed of over 182 wild accessions were regenerated, including relatively large numbers of Lycopersicon cheesmanii, Lycopersicon chilense, and Lycopersicon hirsutum accessions. As allowed by harvests, large seed lots were submitted to the NCGRP for backup storage. Germplasm Evaluation: Progeny tests were conducted on 53 segregating monogenic mutants, including recessive lethals, male-steriles, etc. Seed samples of 36 accessions of L. esculentum originally received from commercial sources were tested for the presence of transgenes containing NPT2 or 35S (all were negative). Distribution and Utilization: Approximately 4,500 seed samples, representing over 1,450 unique accessions, were sent in response to over 414 requests from 300 colleagues in 40 countries; an additional 43 requests were for information only. Information provided by recipients indicates TGRC stocks were used for research on a wide variety of topics. Over 83 publications mentioning use of TGRC stocks were identified in our annual survey of the scientific literature. Documentation of the Collection: A large number of mutant stocks were photographed and digital images were uploaded to the TGRC database and website. New accessions were defined with appropriate genetic descriptors and/or collection site information. Various improvements were made to our database and website. Our annual stock list, this year covering wild species stocks, was published in the Tomato Genetics Coop. Report. 1910-21000-016-06S-This report serves to document research conducted under a specific cooperative agreement between ARS and New Mexico State University. The objective of this cooperative research project was to regenerate accessions of the short-day Allium cepa collection maintained at the Plant Genetic Resources Unit (PGRU) of Geneva, NY. These accessions had sub-standard viability and low seed supply and had to be regenerated or they would be lost from the germplasm collection. Short-day onions could not be regenerated at Geneva, NY because of day length requirements to initiate bulbing. In September of 2003, the vegetable curator at Geneva, NY identified 28 short-day onion accessions that urgently required regeneration and sent them to New Mexico State University for regeneration. Seed of 16 short-day onion accessions were shipped from Geneva, NY to Las Cruces, NM. The seeds from these accessions and from 4 additional accessions on hand with the NMSU Onion Breeding program were sown in Metro Mix 510 in plastic flats. Four accessions failed to germinate any plants. Plants from 8 accessions were transplanted into a field in order to produce bulbs. Since the remaining 8 accessions had so few plants, plants from those accessions were transplanted into Metro Mix 510 in plastic 1 gallon nursery pots. Seeds from 8 additional accessions on hand with the NMSU Onion Breeding program were sown directly into a field in order to produce bulbs of these accessions. Bulbs from 28 accessions were harvested and placed in storage until September 2004. Bulbs from 3 accessions did not survive during storage. Bulbs from each separate remaining accession were placed in a seed production field. Bulbs were arranged such that individual caging structures were constructed over the bulbs and allowed for seed production without cross contamination. Once accessions begin to flower, the plants were covered with a frame structure and netting. Honeybees and blue bottle flies were introduced to the cage structure once flowers started to open. The pollination vectors were allowed to pollinate the flowers for 6 weeks. After the pollinators were removed, the plants remained in the cages for seed set. Once open capsules were visible, umbels were harvested from each cage and were kept separate by accession. The umbels were allowed to dry for 4 weeks. Once the umbels are completely dry, they will be crushed and the seed will be extracted and cleaned. We expect that seed from all accessions will be cleaned and delivered to PGRU within two months of the date of this report. In September of 2004, the vegetable curator at Geneva, NY identified 30 short-day onion accessions that urgently required regeneration and sent them to New Mexico State University for regeneration. Seed of these onion accessions were shipped from Geneva, NY to Las Cruces, NM. The seeds from these accessions were sown in Metro Mix 510 in plastic flats. Twenty accessions failed to germinate any plants. In November of 2004, the vegetable curator at Geneva, NY identified 16 additional short-day onion accessions that urgently required regeneration and sent them to New Mexico State University for regeneration. There were six accessions that had been previously regenerated and one accession that was currently being regenerated. The seeds from these accessions were set aside and were not planted. The seeds from the remaining accessions were sown in Metro Mix 510 in plastic flats. Seeds from the remaining 9 accessions germinated. Plants from a total of 19 accessions were transplanted into a field to produce bulbs. Once these plants mature, bulbs will be harvested and stored until September 2005. After this time, the bulbs will be handled similarly to the manner described previously for seed production. 1910-21000-016-09S-This report serves to document research conducted under a specific cooperative agreement between ARS and Cornell University. A total of 3459 accessions were received from USDA NC-7 and USDA NE-9 collections representing 185 crucifer species and sub-species. Seeds were sown in 18 cell Styrofoam trays and inoculated with 4 isolates of Xanthomonas campestris pv. campestris (Xcc) race 4 in order to induce black rot infection. Symptomless accessions were identified, and due to the sheer number of accessions involved only those lines with less than 50% infected planted were selected for race 1 inoculation. Of the 3459 accessions, 579 were re-tested with Xcc race 1, including most of the B. juncea accessions. 135 accessions are currentluy being re-tested to confirm resistance and identify the species and accessions that may be used by breeders for incorporation of resistance into Brassica vegetables. The 135 accessions do not include the B. juncea accessions that have been determined to be resistant to races 1 and 4 in most cases. The 135 accessions currently being tested with race 1 and race 4 represent the following species: B. carinata (9), B. fruticulosa (1), B. napus (47), B. nigra (11), B. oleracea (4), B. rapa (41), Camelina sativa (1), Crambe abbyssinica (2), Erucs sativa (2), Erucastrum virgatum (1), Erysimum diffusum (1), Hersperis matronalis (2), Lepidium heterophyllum (1), L. sativum (2), Lepidium (1), Sinapsis abla (5), S. arvensis (4). The full data from this work will be sent to the curators of NC-7 and NE-9, and the recommended accessions for black rot resistance will be published in HortScience. 1910-21000-016-10R-This report serves to document research conducted under a specific cooperative agreement between ARS and Cornell University. A total of 3459 accessions were received from USDA NC-7 and USDA NE-9 collections representing 185 crucifer species and sub-species. Seeds were sown in 18 cell Styrofoam trays and inoculated with 4 isolates of Xanthomonas campestris pv. campestris (Xcc) race 4 in order to induce black rot infection. Symptomless accessions were identified, and due to the sheer number of accessions involved only those lines with less than 50% infected planted were selected for race 1 inoculation. Of the 3459 accessions, 579 were re-tested with Xcc race 1, including most of the B. juncea accessions. 135 accessions are currentluy being re-tested to confirm resistance and identify the species and accessions that may be used by breeders for incorporation of resistance into Brassica vegetables. The 135 accessions do not include the B. juncea accessions that have been determined to be resistant to races 1 and 4 in most cases. The 135 accessions currently being tested with race 1 and race 4 represent the following species: B. carinata (9), B. fruticulosa (1), B. napus (47), B. nigra (11), B. oleracea (4), B. rapa (41), Camelina sativa (1), Crambe abbyssinica (2), Erucs sativa (2), Erucastrum virgatum (1), Erysimum diffusum (1), Hersperis matronalis (2), Lepidium heterophyllum (1), L. sativum (2), Lepidium (1), Sinapsis abla (5), S. arvensis (4). The full data from this work will be sent to the curators of NC-7 and NE-9, and the recommended accessions for black rot resistance will be published in HortScience. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. All major accomplishments of this project deal with all five problem areas of component 1 of National Program 301 (Plant, Microbial, and Insect Genetic Resources, Genomics, and Genetic Improvement): Safeguarding Threatened Genetic Resources and Associated Information; Maintaining Genetic Resources and Associated Information Efficiently and Effectively; Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; and Technology Transfer of Genetic Resources and Associated Information. The project will also contribute to genome characterization in Component 2. At Geneva, approximately 11,500 accessions of tomato, onion, radish, winter squash, cabbage, cauliflower, broccoli, other cole crops, celery, tomatillo, asparagus and other vegetables were maintained and 179 new accessions were acquired including 111 accessions of tomatillo from Mexico, 43 accessions of collards from the southern United States, and 6 accessions of onion from Central Asia. This activity is a continuation of previous project plans over the past 50 years. During the first two years of the project approximately 789 accessions were regenerated and 6248 seed lots were distributed in 464 orders (387 domestic and 77 foreign). Component 1: Safeguarding Threatened Genetic Resources and Associated Information; Maintaining Genetic Resources and Associated Information Efficiently and Effectively. A three year SCA with New Mexico State University was initiated for regeneration of short-day onion accessions. In FY2004 and FY 2005 this resulted in regeneration of 28 and 25 short-day onions, respectively, that were in danger of being lost. Component 1: Safeguarding Threatened Genetic Resources and Associated Information; Maintaining Genetic Resources and Associated Information Efficiently and Effectively. We developed a software tool to efficiently predict SNPs in 764 tomato genes based on public ESTs. Based on our current results, we estimate that these genes contain between 600 and 1,000 SNPs. These results will enable breeders and researchers to efficiently target existing genetic variation in cultivated tomato. Unlike other commonly used markers, our predicted expressed-gene markers are likely to be associated with functional variation. Component 1: Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; Component 2: Genome Characterization. We empirically tested for predicted SNPs in 85 of the 764 EST markers by sequencing two or three tomato lines. In doing so we discovered more polymorphism (62 SNPs and 12 small insertion/deletions in 21 genes) than had been previously reported in domesticated tomato (Labate and Baldo, in Molecular Breeding). All polymorphisms we verify will be useful in saturating intraspecific genetic maps of cultivated tomato. We also found evidence for highly diverged alleles (1.6 to 13 fold more variable) at five genes within L. esculentum, and have hypothesized that these are cryptic wild species alleles that represent linkage drag. Recognizing linkage drag within domesticated tomato will enable breeders to study its effects on agronomic quality during introgression and backcrossing. Component 1: Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; Component 2: Genome Characterization. We collected data to compare polymorphism across a genetically diverse panel of 30 PGRU L. esculentum accessions and breeding line TA496 (provided by S. Tanksley) at 11 carotenoid, 11 COSII, 10 EST markers where a predicted SNP had previously been verified, 11 EST markers where a predicted SNP had not been verified, and five EST markers that we hypothesized to be cryptic wild species alleles within L. esculentum. The purpose is to improve SNP prediction methods from ESTs and to estimate ascertainment bias in EST markers with predicted SNPs by comparing them to other markers (carotenoid and COSII). Improved SNP prediction methods and increased understanding of ascertainment bias will be useful for genetic diversity studies for all crops, especially those that are relatively low in genetic variation. Component 1: Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; Component 2: Genome Characterization. We have contributed to developing diagnostic tools to distinguish between broccoli and cauliflower seeds and seedlings by genotyping several-hundred plants from PGRU broccoli and cauliflower accessions for flowering-developmental and glucosinolate-biosynthetic pathway markers. We found evidence to support published claims that alleles at BoCAL, AP1-a, ALK, and ELONG are correlated with a cauliflower phenotype in B. oleracea but the correlations were weak and not predictive in PGRU accessions. Paralogous loci may have confounded our results. Continued improvement of such tools will allow curators and breeders to predict phenotype without growing a plant to maturity. Component 1: Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; Component 2: Genome Characterization. We tested two types of molecular markers (SSRs and SBAPs) to estimate diversity in C. maxima accessions and optimized assay conditions. These markers will be used to study geographic variation in this species and to genotype our collection to better characterize it for end-users. Component 1: Documenting and Characterizing Genetic Resources, Expanding Germplasm Evaluations and Characterizations; Component 2: Genome Characterization. There is need for heirloom and new publicly bred germplasm for use by organic farmers. Two RCA agreements with Cornell University support to PGRU for training of organic farmers and small seed producers in small-scale seed production of heirloom and new publicly improved vegetable varieties, whose production has been limited because of lack of seed. In FY2005 demonstrations, training workshops, and community seed days were conducted for over 750 participants. This has increased the availability of heirloom and new publicly bred germplasm for use by small and organic farmers. The PSI website continues to be a major source of information for organic farmers with over 5,000 hits since its creation. This website supplies detailed information on upcoming events, pepper genomics, seed production, participatory breeding, on-farm commercial variety trials, and other related projects throughout the country: www.plbr.cornell.edu/psi. The OSP website contains information about the new goals and direction of the new OSP project. This website will contain all the detail that the PSI website has plus information about nationwide cooperators, updated news and events around the country, and streaming video detailing on-farm vegetable breeding and seed production: www.organicseed partnership.org. To complete the PSI project, a final PSI CD has been created. This CD combines all annual reports for participatory breeding, on-farm commercial variety trialing and seed production. The CD shows detailed instruction for seed production and breeding of several common vegetable crops. Component 1: Technology Transfer of Genetic Resources and Associated Information. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A total of 11,982 seed samples were distributed in the past five year project in 698 orders (521 domestic and 177 foreign). The first year of this project there were 3411 seed lots were distributed in 224 orders (258 domestic and 34 foreign) and in the second year (through August 15) this resulted in 2837 seed lots distributed in 206 orders (163 domestic and 43 foreign) . Records of all our collections are publicly available via the GRIN (Genetic Resource Information Network) database that is accessible via the Internet. Developed and maintained a web site providing information on personnel, research projects, and protocols in the PGRU seed crops molecular lab. The site includes an educational section on DNA fingerprinting. The technology is transferred via the internet to anyone who has internet access. The constraints to adopting the provided technologies are the equipment and reagents necessary to perform the protocols. Crop Bioinformatics Research web site - developed and maintained a web site providing information on research projects, and computational platforms for PGRU Bioinformatics activities. The technology is transferred via the internet to anyone who has internet access. Demos were offered on laboratory robotics, DNA extraction, and DNA fingerprinting at PGRU Open House and Bring Your Child to Work Day. Basic principles of DNA isolation using common household chemicals and literature on some of the applications of DNA fingerprinting (e.g., forensics, conservation biology) were transferred. The technology was transferred through a demonstration to a total of approximately 100 laypersons and several science teachers. Anyone who purchases about $5 of common household chemicals from a supermarket can apply the technology at home. One undergraduate employee was trained in molecular biology laboratory techniques. The technology was transferred through intensive hands-on experience. The experience can be carried with the individual and transferred to future work and educational environments. A summer undergraduate student was trained in Perl and BioPerl for design and implementation of marker-identification and data-mining software to aid in genetic marker development. As with the laboratory experience, the bioinformatics training can be carried with the individual and transferred to future work and educational environments. Workshops, demonstrations and community seed days were held at the USDA-ARS, PGRU in Geneva, NY 23 August 2005 at the Connecticut Agricultural Experimental Station in Hamden, CT on October 7, 2004; at Peacework Organic Farm in Newark, NY on October 22, 2004, at Gorzynski's Ornery Farm in Cochecton Center, NY on November 8, 2004, at the On-Farm Seed Production Workshop at the NOFA-NY Annual Winter Conference in Syracuse, NY on January 28, 2005; at the NOFA-MA Summer Conference in Amherst, MA on August 13, 2005; at Harvest Home Organics in Moravia, NY on September 7, 2005 and at the Common Ground Fair in Unity, ME on September 2 and 24, 2005. These workshops served small farmer and organic grower target populations. During the community seed day workshops held in Geneva, Newark, Moravia, and Cochecton Center, NY; and in Hamden, CT organic farmers and small-scale seed producers were trained and given the opportunity to clean their seed using the equipment provided in the mobile seed processing unit. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Baldo, A.M., L.D. Robertson, and J.A. Labate. 2005. Discovery of highly polymorphic genes in tomato cultivars. In Conference Program, Intelligent Systems for Molecular Biology 2005, Detroit, MI. Baldo, A.M., D. Huntley, L.D. Robertson, and J.A. Labate. 2004. High-throughput SNP prediction in tomatoes based on ESTs. p. 14 In Program Abstracts, Tomato Breeders Roundtable, Annapolis, MD. Baldo, A.M., J. Labate., and L.D. Robertson. 2004. Prediction of single nucleotide polymorphisms in domestic tomato: How useful is EST sequence diversity? In Conference Program, Intelligent Systems for Molecular Biology 2004, Glasgow, Scotland. Cramer, C.S. and L.D. Robertson. 2004. Seed Regeneration of Short-Day Onion Accessions in the U. S. collection. Poster at National Allium Research Conference, 9-10 December 2004, Grand Junction, CO. Robertson, L.D. 2005. Apium Collection of the PGRU at Geneva, New York: July 2005. Report to the Leafy Vegetable Crop Germplasm Committee. Las Vegas, Nevada. July 2005. Oral Presentation and Written Report. Robertson, L.D. 2005. Allium Collection of the PGRU at Geneva, New York: July 2005. Report to the Root and Bulb Crop Germplasm Committee. Providence, Rhode Island. July 2005. Oral Presentation and Written Report. Robertson, L.D. 2005. Brassica Collection of the PGRU at Geneva, New York: October 2003. Report to the Crucifer Crop Germplasm Committee. Providence, Rhode Island. October, 2003. Oral Presentation and Written Report. Robertson, L.D. 2004. Tomato Collection of the PGRU at Geneva, New York: October 2004. Report to the Root and Bulb Crop Germplasm Committee. Annapolis, Maryland. October 2004. Oral Presentation and Written Report. Public Seed Initiative Website: www.plbr.cornell.edu/psi. Organic Seed Partnership Website: www.organicseedpartnership.org. Review Publications Baldo, A.M., Huntley, D., Robertson, L.D., Labate, J.A. 2005. High-throughput snp prediction in tomatoes based on ests. Tomato Breeders Roundtable Proceedings. p. 7 In Final Abstracts Guide, Plant and Animal Genome XIII, San Diego, Ca. Robertson, L.D., Baldo, A.M., Sheffer, S.M., Labate, J.A. 2005. Use of sequence-based polymorphisms for studing genetic diversity of winter squash. Meeting Abstract. p. 153 In Final Abstracts Guide, Plant & Animal Genome XII, San Diego, CA. Baldo, A.M., Robertson, L.D., Labate, J.A. 2005. Highly polymorphic genes in cultivated tomato. Hortscience. 40:999. Labate, J.A., Robertson, L.D., Bjorkman, T. 2004. Utility of bocal-a and boap1-a genotypes in identifying broccoli and cauliflower accessions. Hortscience. 39:773.