Duration: 10/01/2003 to 09/30/2008

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

STATEMENT OF ISSUE:


America's abundant and inexpensive supply of food and fiber is based on a productive and progressive agricultural system. The foundation for this productivity has been based on scientific knowledge and exploitation of useful genetic diversity for developing new, higher quality cultivars that can resist pests, diseases, and environmental stresses. Genetic uniformity of modern cultivars results in a noteworthy lack of genetic diversity with the potential attendant susceptibility to new pest and abiotic stresses. The genes that are needed to provide a continued flow of new varieties that produce higher yields with better quality, and better withstand pests, diseases, and abiotic stresses can only come from diverse plant germplasm.


JUSTIFICATION:


Biological diversity in plants benefits human welfare directly, as various species are used to satisfy basic human needs, and indirectly, as diversity supports many processes essential to human survival and progress (Office of Technology Assessment, 1987). Without an assured source of food we would not be free to engage in those activities which are associated with the quality of life to which we have become accustomed (Wilkes, 1983). However, plant genetic resources native to the United States are few. The tremendous progress made by the agricultural sector of the United States has been founded almost exclusively on plant genetic resources imported from areas outside the U.S. Recent international treaties and the adoption of restrictive laws governing germplasm access in many countries have seriously complicated the acquisition and exchange of germplasm. Since the Convention on Biological Diversity (CBD) entered into force in 1993, the free and open access to genetic resources from other countries has largely become a thing of the past. These restrictions make it imperative that the U.S. maintain and secure existing national germplasm collections to provide the genetic basis of future crop improvement.


Seed collections at the Plant Genetic Resources Unit (PGRU) located at Geneva, NY include 11,737 accessions representing approximately 162 species in 32 genera. Major holdings include tomato (Lycopersicon), onion (Allium), Crucifers (Brassica), celery (Apium), winter squash (Cucurbita), radish (Raphanus), other vegetables, and buckwheat (Fagopyrum). The clonal collections contain 52 species of apple (Malus), 23 species of grape (Vitis), and 11 species of cherry (Prunus) for a total of 5205 accessions. The National Germplasm Repository (NGR) and the Northeast Regional Plant Introduction Station (NERPIS) comprise PGRU. Both the seed and clonal crops for which Geneva has responsibility are important components of agriculture in the northeast. Many northeastern State Agricultural Experiment Stations (SAESs) have research and extension responsibilities for these valuable commodities. The vegetable crops maintained at Geneva account for about 45% of the value of U.S. production and the fruit crops account for 53% of the value of production of fruit trees and vines.


The Regional Research Project NE-9 serves as a unique component of a national effort to enhance crop improvement by providing accessibility to genetic resources of selected horticultural and agronomic crops and by conducting problem-oriented research to establish higher quality collections for ultimate conservation and utilization. As a component of a national germplasm system, PGRU serves as a convenient conduit to and from the Northeast for all types of germplasm and genetic resources information. Over the years, NE-9 has contributed greatly, either directly or indirectly, to the producer and consumer as evidenced by increased regional and domestic agricultural products and productivity. These contributions have been tangible, as in the form of germplasm, and intangible, as in the form of increased knowledge. Both service and research activities have been conducted concurrently. Contributions of plant genetic resource conservation and utilization efforts will affect future studies of plant biology as well as those of commodity improvement. The overall effort is an endeavor involving multiple disciplines, locations, and interests. Because of these complexities (biological, operational, and logistical in nature), the cooperating organizations are best served by a regional approach.


Research at PGRU focuses on activities that are complementary to its conservation responsibilities to increase the effectiveness by which the PGRU performs its conservation and utilization mission. Primary disciplines associated with the research include plant genetics and crop improvement. This groundwork also may serve as a springboard for future germplasm enhancement within state experiment stations of the Northeast and the United States as a whole. Typical applications of molecular markers to conservation and management of the germplasm collections involve solving problems in maintenance and genetic characterization of collections; determining genetic diversity to identify genetic gaps and duplicates in collections; and establishing and validating core subsets. Screening germplasm collections for resistance to common diseases is performed in cooperation with Cornell University and USDA pathologists.


If we are to move towards greater utilization of plant genetic resources, research targeted at all five objectives (p. 7-8) of this project proposal must be integrated. Plant genetic resources acquisition, conservation, and use have had a major, positive effect on the improvement of field and horticultural crops. Throughout history, plant scientists have shown flexibility in utilizing new resources and tools that contribute to progress in crop improvement. As biotechnology programs in the northeastern U.S. have grown, plant genetic resource conservation has become more critical. Molecular biologists must have this reservoir of genes available if they are to transfer useful genes to plants that breeders can then exploit. Continuing progress in improving the performance of crop plants while simultaneously improving our understanding of plant biology will be accomplished by the integration of new technologies with the broadest possible array of genetic resources.

Related, Current and Previous Work

The Regional Research Project NE-9 partially supports and complements service and research activities encompassed within the scope of the responsibilities at Geneva. Similar regional research projects, i.e., North Central (NC-7), Southern (S-9), and Western (W-6), associated with other components of the NPGS are well-known. Despite similarity among missions at locations, unique regional interests as well as crop responsibilities determine the specific efforts of a location. The major crops supported by the Regional Research Project NE-9 are onion, vegetable Crucifers (cabbage, broccoli, cauliflower, kale, etc.), winter squash, tomato, apple, tart cherry, and cold-hardy grape. Responsibility for United States national germplasm collections of these crops lies solely at Geneva. The data in Appendix A, Tables 1-4 provide a measure of the economic importance of these crops on a worldwide and domestic basis. (Tables begin on p. 20)

Most crops of commercial significance in the northeastern U.S. are of exotic origin. NE-9 support has enabled the United States Department of Agriculture, Agricultural Research Service, other federal agencies, the Agricultural Experiment Stations of the 12 northeastern states and the District of Columbia, and other cooperators, to engage in a coordinated effort to conserve and utilize plant genetic resources. The continued demand for plant genetic resources underscores the importance of maintaining collections which preserve the vast reservoirs of genes needed to fulfill the plant and agricultural research activities so critical to the economic well-being of the northeastern U.S. and other regions of the United States. With the actual or potential loss of some of our most effective herbicides, fungicides, and insecticides, the need for novel sources of resistance or tolerance genes will increase. Furthermore, changing landscapes, urbanization, irrigation, and other habitat changes increase the need for plants having tolerances to more stressful environments.

Acquisition:

In a survey by the General Accounting Office (1997), all 40 Crop Germplasm Committees (CGCs) stated that acquisition of additional germplasm is a moderately to extremely important activity and about one-half of the CGCs reported that wild and weedy relatives are under-represented in germplasm collections. We agree that judicious additions to all of PGRU's collections are needed. Because of changes in the international political climate as well as ramifications of the Convention on Biological Diversity, continued plant exploration and exchange is needed. PGRU in cooperation with Animal and Plant Health Inspection Service (APHIS) also needs to take an active role in developing improved quarantine procedures for both import and export of germplasm. This was an area that the GAO Survey (1997) also highlighted as needing additional attention.

Significant accomplishments in the period 1998-2003 include:

• 467 new accessions added to the germplasm collections (Appendix A, Table 5)
  
• Collection conducted in southern Mexico for tomatillo (Physalis philadelphica) which added 107 accessions to the previous collection of 18 accessions for this ethnically important crop
  
• Started transfer of tomato accessions from the National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, CO to Geneva, with 200 accessions out of 1400 transferred.
  
• We received elite tetraploid as well as intraspecific cherry hybrids that contain novel disease resistant genes from a plant exchange trip in 1998 to four institutes in Russia and 20 accessions of 5 Malus species from China donated by the genebank in Dresden Germany.
  
• We collected 28 accessions of wild Malus orientalis from the Caucasus Mountains, 63 accessions of M. orientalis and 7 elite local apple cultivars were collected in 1999 in Turkey, and 10 accessions of M. orientalis were collected in Armenia in 2002.
  
• We produced 7 new accessions (seed populations of "Gala" X scab resistant M. sieversii clones) that are being used to study the genetics of apple scab resistance.
  
• Establishment of the research evaluation quarantine block for evaluation of grape germplasm at Geneva allowing postponement of sanitation of the grapevines until the potential utility is determined; 25 accessions were introduced in the previous 5 years.
  

Maintenance, regeneration, and characterization

It is no surprise that accessions having little or no characterization, evaluation, or passport data tend not to be ordered for use by cooperators. Furthermore, accessions lacking adequate numbers of seeds cannot be securely backed up at the NCGRP. For seed accessions, both secure backup and characterization are dependent upon regeneration activities. Plans for regeneration and NCGRP backup of the major seed collections are in place, as are plans for further evaluation studies of clonal crops. Regeneration of our cucurbit collection could be made more efficient by the acquisition of larger pollination cages, which would obviate the need for labor-intensive hand pollinations. More attention should be paid to Geneva's minor seed crops, such as buckwheat (an important crop in Japan and a crop for which we have a breeder / researcher at Geneva, Thomas Bjorkman), artichoke, and radish. These three crops are particularly in need of regeneration. Core subsets need to be established for some of the major seed crops, winter squash, vegetable Crucifers and Allium onion. The entire apple collection of approximately 2600 accessions must be repropagated on fire blight tolerant rootstock EMLA7.


Significant accomplishments in the period 1998-2003 include:

• Core collections for cultivated tomato, apple (Forsline, 1996), and grape have been established using strategies as discussed by Brown (1989) and Kresovich et al. (1995).
  
• Increased availability of seed crops from 60% to 76% accomplished from 1998 to 2003 through 1500 regenerations.
  
• Established a system for routine monitoring of seed viability for the vegetable crops to ensure that high quality seed is always available.
  
• Developed minimal descriptor lists of priority descriptors in consultation with the relevant CGCs for tomato, cabbage, bulb and bunching onion, apple, grape, and cherry.
  
• Completed characterization of 250 tomato, 400 cabbage, 600 onion, 900 apple, 800 grape, and 39 cherry accessions, including digital images of product and foliage for all vegetable characterizations and 700 apple, and 800 grape accessions.
  
• Developed system for renting of bee hives for pollination that frees up one technician position for other regeneration, characterization, and distribution activities
  
• Establishment of a SCA with the New Mexico State University in Las Cruces for regeneration of short-day onions.
  
• Barcode labels on all plants in the orchards and vineyards have been installed, reducing typographical errors and making data collection more reliable and efficient.
  
• Cryopreservation of apple dormant buds has been successful with no decline in ability to repropagate such buds after 12 years of storage; over 89% of apple and 60% of cherry accessions are now in cryopreservation at the USDA-ARS, NCGRP.
  
• Repropagation of the apple to EMLA 7 rootstock for superior control of fire blight is 70% completed.
  
• 2000 seedlings from Russia, Turkey and Europe have been screened for resistant reactions to apple scab, fire blight, and cedar-apple rust. Kazak apple seedlings continue to be evaluated in 20 worldwide institutes.
  
• A book was completed which is a translation of Kazakh apple studies along with the work of collection and evaluation of apple from Kazakhstan (Forsline et al., 2003)
  

Documentation

The Genetic Resources Information Network (GRIN) National Database is the focal point for PGRU germplasm information documentation. Accessions, inventory, order, and characterization data are continually updated to provide the most current PGRU information possible. A major effort was also started at PGRU to produce digital images of all crops at various stages of development to show their characteristics which are then uploaded to the GRIN National Database where they may be accessed via the Internet.

Significant accomplishments in the period 1998-2003 include:

• Digital images for 400 cabbage accessions and 220 tomato accessions have been uploaded into GRIN for online access. 1050 images of apple and grape and 1200 onion images have been prepared for uploading to GRIN
  
• 100 seed weight information has been collected for eventual loading into the database.
  
• Cucurbita and Raphanus inventory has been loaded into the database, Vitis and Malus morphological characteristics such as fruit size, shape, weight, and flower-sex have been updated.
  
• The PGRU website is undergoing a major revision and is expected to be completed by the middle of 2003.
  
• Workstations are being replaced with newer technology systems that include operating system (Windows XP) and application software upgrades.
  

Distribution

Distribution of germplasm is primarily through requests that are received through web access to GRIN. More rapid movement of germplasm through the quarantine system needs to be encouraged by PGRU, for both seed and clonal crops. PGRU, in collaboration with the Plant Exchange Office, must be prepared with a reasoned response to actual or potential restrictions to germplasm importation and exportation that may result from the Convention on Biological Diversity.

Significant accomplishments in the period 1998-2003 include:

• Malus and Vitis catalogs for ordering accessions are accessible via the Internet
  
• order processing has been automated, and the information is immediately placed in GRIN.
  
• During 1998-2002 11,638 seed accessions were distributed by PGRU (Appendix A, Table 6)
  
• During 1998-2002 14,091 clonal accessions were distributed by PGRU (Appendix A, Table 7)
  
• There were 397 orders for 6,612 accessions sent to recipients in the Northeast region (Appendix A, Table 8)
  
• DNA of apples and grapes is being distributed as part of the normal order process; PGRU is the first germplasm unit in the country to do this.
  

Research

To complement current conservation efforts, problem-oriented research has been conducted that facilitates improvements in germplasm acquisition, maintenance, and characterization. Research focusing to increase the effectiveness and efficiency of the management of PGRU's germplasm is an essential component in achieving the unit's service goals (References cited in Appendix E). The primary goal of the research has been to describe intra- and interspecific genomic variation and to define genetic relationships among individuals, populations, and species. Gene bank curators have used the results to establish and maintain useful and representative crop collections and core subsets based on the integrated information representing DNA sequences, genes, genotypes, and phenotypes. Such information has also been used to determine collection strategies for wild foreign germplasm in the field, and in designating domestic populations of wild species for in situ preservation.

The personnel associated with the Regional Research Project NE-9 cooperate and participate in activities associated with 11 relevant Crop Germplasm Committees (CGCs) of the NPGS. These activities include planning and participating in plant germplasm exploration/exchange proposals and conducting germplasm evaluation studies. In addition, research carried out by current and former members of the Regional Technical Advisory Committee for NE-9 has interfaced productively with the goals of the NE-9 project at PGRU (see Appendix D for a list of relevant publications).

Significant accomplishments in the period 1998-2003 include:

• Evaluation of the grape collection for disease and abiotic stress resistance/tolerance has been initiated collaboratively in: 1) powdery mildew (B. Reisch, unpublished); 2) crown gall (Sule et al., 1994); root knot nematode (Boyden and Cousins, 2003); and cold hardiness (Pool et al., 1990) with sources for resistances/tolerances found in the germplasm.
  
• Collaborative projects with Cornell University, North Carolina State University, the University of Arkansas, and the University of Minnesota has led to sources of resistance to fire blight, apple scab, cedar apple rust, sooty blotch, flyspeck, bitter rot, black rot, white rot as well as resistance to an array of arthropod pests as noted in the following publications: Aldwinckle et al. (2002); Luby et al. (2001); Aldwinckle et al. (1999); Momol et al. (1999); Williamson and Sutton (2000); and Yan and Correl (1993).
  
• Evaluation of the apple core collection and wild seedling populations for resistance to abiotic stresses along with other desirable traits (antioxidant levels in fruit) has begun. Publications from these collaborative projects include Stushnoff et al. (2002), Miller and Zhang (2002), and Luby et al. (2001).
  
• Resistances to twig brown rot and cherry leafspot have been found in material collected in 1998 (Forline et. al., 1998).
  
• Resistance to white mold has been found in cauliflower (Griffiths and Robertson 2002).
  
• We have demonstrated that storage in liquid nitrogen (LN) is reliable and can be used for backing up a germplasm collection (Forsline et al., 1998). At present, the majority of apple and tart cherry are backed up at NCGRP (Towill and Forsline 1999, Towill et al., 2002).
  
• Curators in gene banks now are able to type or "fingerprint" plants and accessions held in their collections. Currently simple sequence repeat DNAs (SSRs) (Litt and Luty, 1989; Edwards et al. 1991; Lamboy and Alpha, 1998) and DNA sequences (Single Nucleotide Polymorphisms or SNPs, Gibson and Muse, 2002) are being used at PGRU for genotype and species identification.
  
• The apple core collection has been refined through fingerprinting (Hokanson et al. 1998, 2001). Vitis species from the grape core collection have also been fingerprinted using SSR markers (Lamboy and Alpha, 1998).
  
• SSR markers were used to reveal genetic identities, genetic diversity and relationships in the apple core subset. Genetic diversity in 24 wild populations of the grape species V. rupestris was estimated using SSRs. This species has been a critical source of disease resistance genes for cultivars and our study was aimed at enhancing its in situ preservation.
  
• Morphologic, molecular, taxonomic, and census data of V. rupestris were used to establish the first NPGS in situ conservation sites for American wild relatives of a crop.
  
• New major equipment for high-throughput assaying of molecular genetic markers was purchased and protocols were optimized. A molecular marker data base for long-term storage and easy access to and manipulation of data is under development.
  
• Eliminating unintentional duplication in germplasm collections will allow more resources to be used in essential activities. Results using SSRs show that identically named accessions of tomato sometimes differed genotypically. Additional markers and phenotypic data will be used to develop rigorous protocols for identifying and eliminating unwanted duplicates.
  
• Discovering and describing underutilized genetic variation in the collection will be valuable to stakeholders who are searching for novel alleles for continued crop improvement. Using SSRs we found heirloom tomato accessions to be slightly more diverse than the modern types.
  
• We adapted an assay to estimate tomato fruit quality (lycopene content) in high-throughput mode and designed molecular markers for several candidate genes in the lycopene biosynthetic pathway. These will be used to study the genetics of tomato nutraceutical quality through characterizing germplasm.
  
• Broccoli and cauliflower are different botanical varieties of the same species (Brassica oleracea) with very different phenotypic traits. In collaboration with Thomas Bjorkman from Cornell University. Horticultural Sciences, we tested the correlation between phenotype (broccoli versus cauliflower) and genotype at a locus that is a candidate for conferring cauliflower phenotype in B. oleracea. Results showed that the correlation was highly significant but genotype at this locus did not absolutely predict phenotype.
  

Objectives

1. Acquire, maintain/regenerate, characterize, document, and distribute plant genetic resources for use in the Northeast, the United States, and the World while ensuring the identity of each accession as to species (or hybrid) and cultivar.
   
2. Determine the basis for and the extent of genetic variations, the geographic distribution of cultivated species, and their taxonomic relationships with closely related species and to determine the genetic mechanisms controlling the inheritance of important traits.
   
3. Characterize/evaluate accessions for specific traits, including high-priority traits such as nutritional content of fruits and vegetables.
   
4. Combine genes from diverse sources into germplasm more useful to plant breeders and to breed, release, maintain, and evaluate improved germplasm and cultivars.
   
5. Note: Objectives 3-5 require the cooperation of collaborators. Forging the links between PGRU and reliable and productive cooperators should be viewed as part of these objectives.
   

Methods

The Regional Research Project NE-9 will continue to serve as a conduit for movement of valuable plant genetic resources from worldwide origins to the northeastern states as well as the entire United States (see Appendix A, Tables 6-8). As such, the objectives and the procedures of the project are organized as a continuum. Objectives 1 and 2 will be performed primarily by personnel of PGRU, whereas latter objectives (3-5) will be in collaboration with cooperators associated with NE-9 activities. PGRU is relatively well equipped with facilities and equipment for conduct of its service and research activities (Appendix F). 1. Acquire, maintain/regenerate, characterize, document, and distribute plant genetic resources for use in the Northeast, the United States, and the World while ensuring the identity of each accession as to species (or hybrid) and cultivar. Acquisition Germplasm is acquired through transfer from other domestic repositories, breeder's collections (foreign and domestic), foreign repositories, private individuals, or by means of both foreign and domestic exploration and collection trips. Any foreign acquisitions will be brought in via the National Plant Germplasm Quarantine Office in Beltsville or via APHIS import permits. Depending on the particular crop, seed importation may be subject to quarantine restrictions; clonal accessions always are. Impetus for foreign germplasm explorations comes from the respective CGCs. The recognition that windows for exploration into certain regions of the world open and close, sometimes unpredictably, catalyzes explorations into specific areas of the world during opportune times. All foreign explorations will comply with the Convention on Biological Diversity (CBD) and national access laws in host countries. National authorities will be contacted for permission for access to germplasm under terms acceptable to both the host country and USDA. The National Germplasm Resources Laboratory (NGRL) will assist with requests for access and determination of appropriate benefit sharing. The NPGS will not claim ownership or seek intellectual property rights over the germplasm collected. The NPGS cannot accept germplasm with intellectual property rights. Small non-monetary benefit sharing projects that contribute to conservation of plant genetic resources in the host country may be associated with foreign explorations. Such projects are often funded as a component of plant exploration proposals supported by the USDA-ARS Plant Exploration Program. In Vitis we have some excellent opportunities to expand the collection across broad ranges of wild grape in North America. Presently, we have minimal representation of about 12 North American species. Most of the wild types presently on inventory were collected in the 1960s (Barrett et al., 1969). With the addition of a support scientist to this project, and with technical input by the Grape GGC, we plan to make additional collections across North America. A second collection emphasis with grapes will be on the little known Chinese species. Some reports indicate upward of 50 Vitis species are present in China. With vegetables the emphasis will be on collection of onion from Central Asia (center of domestication) and with tomatillo from Guatemala. Germplasm will be collected using optimum sampling strategies (Allard, 1970; Hawkes, 1975; Marshall and Brown, 1975; Sykes, 1975; Zagaja, 1970). Maintenance / Regeneration Regeneration of a seed accession is done when germination falls below 60% or the number of seed available for distribution falls below 5,000. Seed regenerations are conducted using the appropriate pollination techniques and pollinators. Bumble bee, honey bee, solitary bee, fly, or hand pollination is done, as appropriate, in the greenhouse, screenhouse, field, or pollination cages. After extraction of seed from fruits, seed are left at 1 o C and 20% relative humidity until equilibrated. These are then stored under optimal conditions in a freezer room at -20o C, in heat-sealed, moisture-proof, foil-lined bags. In addition, duplicate seed of many accessions are backed up in cold storage or cryopreservation at the NCGRP. Eventually all seed accessions will be backed up in this fashion. During the next five years, PGRU plans to regenerate an average of 300 tomato, 100 Brassica, 100 onion, 25 cucurbits, and 30 Raphanus (radish) annually. Some of these regenerations will be carried out at the USDA-ARS regeneration site in Parlier, California, which enables PGRU to regenerate a greater number of total accessions. An SCA will be used to regenerate short-day onions at New Mexico State University. In the case of apples, each cultivar was formerly maintained as two plants, one grown on seedling rootstock and one on dwarfing rootstock (EMLA 9). We have been able to better manage the field collections of apple with phased repropagation (70% complete) to EMLA 7 a semi-dwarf, fire-blight resistant rootstock that limits the vigorous growth that induces shoot blight. Backup collections of apple and tart cherry are cryogenically-stored as dormant buds at the NCGRP. We have successfully stored 2,100 accessions (Towill et al., 2002) of apple (89 % of the clonal collection) and 49 tart cherry (60%) of the collection. We will also collaborate with NCGRP to store broad arrays of alleles of wild species as seeds using controlled pollination of seedling populations to produce additional seed for long-term preservation to supplement original seed that may be in short supply. Research to develop protocols to preserve grape will continue in collaboration with the National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado. Clonal accessions can be re-identified any year during the growing season, usually when flowers are present. Seed accessions can be re-identified only during regeneration of those accessions, which may occur infrequently. Comparison of plant features to those which are characteristic of an accession assists in determining whether the accession is true-to-type. Records of digital images will assist in this project. International and domestic experts are sometimes consulted about the identity of specific accessions. New technologies allow use of DNA fingerprints to determine the identity of an accession. Simple sequence repeat DNAs (SSRs) have been particularly useful in this regard, and research in this area will continue at PGRU. In the time period, 2003-2008, plans are to completely fingerprint the apple, grape, and tart cherry collections using SSRs. For the tomato collection, identically named accessions will be evaluated phenotypically by grow-outs in the field and genotypically by using molecular markers. These data will be used to help eliminate unwanted duplicates. Characterization Characterization of PGRU's major crops, apple, grape, vegetable Brassica, tomato, winter squash, and Allium, is carried out for characteristics designated by the respective CGCs. These generally include vegetative characters of leaves, stems, and flowers, and fruit characteristics, such as size, shape, color, flavor, sugar content, etc. Evaluations of traits that are difficult and expensive to measure, e.g., nutritional quality of fruits and vegetables, are done in collaboration with appropriate specialists or experts. Characters are assessed according to international standards, if they exist. Characterization of seed crops will mostly be done during regeneration, i.e., when we have living plants to examine, so that characterization schedule depends upon regeneration schedule for a particular crop. Since the tomato collection is almost completely characterized, emphasis during the next five years will be on the other crops that are planned for regeneration: Crucifers, onion, cucurbits, and radish. Plans are to complete characterization of the apple, tart cherry and grape collections using a minimal descriptor list during the next two years. Digital imaging of fruit samples using a grid background for reference will be completed for all accessions. In addition we will document digital images for inflorescences as well as tree and vine forms and other plant parts. Documentation While PGRU still maintains local databases for all PGRU collections, the GRIN National Database is established as the major source of germplasm information. The applications supporting all maintenance processing have been rewritten from a text based system to a Windows based system, thus improving the throughput of the system. A major effort was also started at PGRU to produce digital images of all crops at various stages of development to show their characteristics. They are then uploaded to the GRIN National Database where they may be accessed via the Internet. Distribution PGRU distributes germplasm in four different forms: seeds, dormant cuttings, pollen, and green cuttings. Generally, private overnight delivery services (UPS, FedEx, etc.) are used for shipment; no special handling is necessary for seeds (coin envelopes), pollen (microfuge tubes or vials), or DNA (microfuge tubes). Record keeping for order processing is completely automated. We expect orders for distribution of DNA will increase over the next five years. 2. Determine the basis for and the extent of genetic variations, the geographic distribution of cultivated species, and their taxonomic relationships with closely related species and to determine the genetic mechanisms controlling the inheritance of important traits. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) will be used for 1) partitioning genetic variability between plants, populations, species, and geographic regions, 2) determining relationships among accessions, and 3) fingerprinting cultivars. We also utilize the expertise of taxonomists in specific crops. All Vitis and Malus populations sampled during germplasm acquisition trips during 2003-2008 will be analyzed using SSRs. At least 50 individuals per population from populations spread across the geographic range of the species will be sampled. Samples will be identified to the proper species and interspecific relationships will be analyzed using cluster analysis and principal component analysis. SNPs in select chloroplast and nuclear DNA will be used to construct phylogenies for Vitis and Malus. Because the primers chosen for the Vitis phylogeny are "universal" and may be useful in Malus, marker development is being pursued concurrently within the two genera. For the vegetable crops, we will apply SNPs and/or SSRs to two plants from each of approximately half of 5,000+ tomato accessions during the next five users. High priority has been placed on genotyping the tomato core collection, followed by subsets from L. esculentums geographical range. We are also testing polymorphic Brassica oleracea markers for their usefulness in distinguishing among botanical varieties within this species. Molecular markers will be used to aid in identification of duplicates in the tomato collection. Development of molecular markers (SNPs) for candidate genes is ongoing. These are genes that have been cloned and sequenced and have provided evidence that they can potentially influence a trait of interest. High-priority traits we are currently working on are lycopene content in tomato, heading morphology in Brassica oleracea, anthocyanin and cold-tolerance in grapes, and dwarfing and disease resistance in apples. To increase efficiency in use of our molecular genetic data we will deposit it into a database to be developed and linked to supplemental information such as that stored in GRIN. Among the 192 taxa in the seed collection, Lycopersicon and Brassica are of highest priority for molecular genetic characterization because this is the germplasm that we receive the most requests for. We will use molecular genetic data generated from genome projects in tomato and Brassica to design nuclear and organellar molecular genetic markers for germplasm characterization. These will be used to identify gaps in collections, reduce redundancy, optimize regeneration protocols to prevent contamination and genetic drift, describe heterotic groups, and define and verify core subsets. In the context of plant genetic resources conservation, phylogenies are useful in assessing units for in situ or ex-situ conservation. The purposes of constructing a molecular phylogeny of Vitis (with an emphasis on wild North American species) in conjunction with a survey of intraspecific variation are: i) taxonomy, the identification of species and delineation of species boundaries. Vitis species are often difficult to identify because few discrete morphological characters are available (e.g. Moore, in press), and because many Vitis species freely hybridize, ii) construction of an evolutionary framework within which traits of interest can be studied, most commercially important cultivars come from a narrow genetic base and this evolutionary framework facilitates the rational choice of new genetic material, and iii) identification of units for conservation to fill gaps in the collections. 3. Characterize and evaluate plant genetic resources for specific desirable traits. Other evaluations are carried out by cooperators and researchers who have the specialized knowledge, equipment, and technology to judge attributes such as disease and insect resistance, phytonutrient concentrations, and tolerance to environmental stresses or extremes. Particular areas of evaluation emphasis in recent years include screening for disease resistances, cold hardiness, leaf hairiness, and anti-oxidant concentrations. Many of these evaluations will be funded through the CGCs using crop evaluation proposals funded by the NPGS. Recently PGRU hired three breeders (apple rootstock and grape rootstock and scions). They will assist us to acquire and evaluate exotic germplasm for potential utilization in breeding. SCAs have been funded to: 1) evaluate apple germplasm for disease resistance (Aldwinckle); 2) identify diverse levels of antioxidants in the apple collection (Stushnoff); 3) identify resistance to cherry leaf spot from the gene pool of interspecific hybrid populations collected in Russia in 1998 (Iezzoni); 4) identify resistance of grape to crown gall (Burr); 5) evaluate enhanced M. sieversii germplasm for apple scab resistance genes (Luby, Aldwinckle, Gardiner and Bus); and 6) evaluate grape core collection for resistance to powdery mildew and Phomopsis viticola (Reisch and Wilcox). In another collaborative Northeast Region project, J. Goffreda of Rutgers University is evaluating over 1,000 seedlings of M. sieversii from Kazakhstan for disease resistance and horticultural traits (Forsline et al., 2003). 4. Combine genes from diverse sources into germplasm more useful to plant breeders and to breed, release, maintain, and evaluate improved germplasm and cultivars. Three scientists recently joined PGRU with the following objectives: 1) Evaluate and breed new dwarfing and semi-dwarfing apple rootstock varieties resistant to biotic and abiotic stresses through conventional and molecular genetic means; 2) Genetically improve wine and table grape scions through the molecular genetic and genomic characterization of disease resistance, cold hardiness, and fruit quality; and 3) Breed, evaluate, and introduce improved grape rootstocks with resistance to biotic and abiotic stresses. All these objectives will be met by utilizing the germplasm collections at PGRU as sources of diversity for recombination with adapted high value breeding lines and varieties. Over eight races of apple scab (Venturia inaequalis) have been identified and breeders have been unable to develop durable sources of apple scab resistant cultivars. In collaborative work with Cornell University, the University of Minnesota and Horticultural Research, Palmerston, North, New Zealand, we have identified sources of resistance in the Central Asian germplasm that has potential as new parental material in breeding programs. In spring 2002, PGRU made crosses of scab resistant selections of Malus sieversii and a susceptible cultivar, Gala. Seven distinct populations were produced. Initial screening results have shown remarkable segregation for resistance. Using genetic markers, these populations and a broad spectrum of Malus sieversii from the original collections made in Kazakhstan will be characterized for the different scab resistant genes.

Measurement of Progress and Results

Outputs

• Increased representation of grape, apple, Physalis, Brassica, onion, wild and weedy relatives, tomato introgression populations, modern cultivars of vegetables and buckwheat. Seed increase of wild apple M. sieversii populations and selected wild apple clo
• Representative, secure genetic resources collections. Ongoing repropagation of apple and regeneration of vegetable germplasm. Cryopreserved apple and tart cherry accessions. Storage and backup at NCGRP of high-quality vegetable seed. Research and improved
• An expanding database of passport, characterization and evaluation data made available through GRIN to improve efficiency of use of germplasm. Digital images and characterization of apple, grape, cherry, tomato, onion and cabbage.
• Molecular markers (SNPs, SSRs) and genotypic data for accession identification and fingerprinting, phylogenetics, population genetics, genetic mapping and marker-assisted selection of apple, grape, tomato and Brassica.
• Continued refinement of core subsets for major crops.
• Evaluation and genetic studies of quality traits. These include nutritional components such as antioxidants in apples, carotenoids in tomato and anthocyanins in grapes; disease resistance to apple fire blight, apple scab, and grape nematodes; cold tolerance in grapes and dwarfing in apples.
• Development and release of improved apple rootstocks and improved grape rootstocks and scions.

Outcomes or Projected Impacts

• Genetic base of crops will be broadened to reduce the dangers of genetic vulnerability. Wild species and introgression lines are very promising sources of favorable new alleles and are in increasingly high-demand for crop improvement and research.
• Valuable germplasm is readily available at PGRU and back-ups are secured at NCGRP.
• Available genetic diversity in the collections will be more efficiently utilized and conserved.
• Critical tools for germplasm conservation, characterization and improvement, in the form of molecular markers and genotypic data for high-priority species and quality traits, will be publicly available.
• Molecular genotyping will improve existing core subsets and result in more efficient characterization and management of our collections.
• Discovery of genes for increased disease and pest resistance will decrease reliance on pesticides and reduce growers costs and negative environmental impacts. Discovering genetic bases and novel alleles for improved nutrient levels will lead to more nutritious food.
• Productivity, profitability and viability of agriculture in the northeast will be ensured.

Milestones

(2004): Ongoing regeneration, storage and backup of germplasm; develop molecular markers in tomato, grape, apple; ongoing acquisition of modern vegetable cultivars and outreach training in seed-production; ongoing characterization, digital imaging and evaluation of vegetable and clonal germplasm

(2005): Complete cryo of tart cherry, genotype tomato core subset; collect wild grape

(2006): High-quality tomato stored at PGRU; complete upload of backlog of data on GRIN; genotype wild tomato taxa; complete Physalis collection, acquire tomato introgression lines; complete apple repropagation and cryo; collect wild grape and apple

(2007): Genotype landraces of tomato; complete tomato backup at NCGRP, define protocol for grape cryo; fingerprint grape and apple; seed increase and clone selection of M. sieversii; collect wild grape

(2008): High-quality onion stored at PGRU, collect onions Central Asia, genotype N. American tomato collection, complete characterization of grap

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Projected Participation

View Appendix E: Participation

Outreach Plan

Scientists are responsive to requests for information, advice, or assistance from colleagues in ARS, SAES, industry and foreign groups. Scientists initiate and/or participates in program-related activities which support ARS operations and goals, and which contribute meaningfully to the accomplishment of the ARS mission, strategic objectives, and National Programs of which the Management Unit is a part. Included in this are: 1) Lectures and demonstrations to visitors at PGRU; 2) Education modules for study by secondary and college students; and 3) Participation in press releases for the popular media including radio, television, and other avenues.


A Reimbursable Cooperative Agreement with Cornell University will allow outreach activities with small scale seed producers and organic farmers. This project will provide training and facilities for seed processing for organic farmers and small-scale seed producers interested in seed production of heirloom and new publicly bred varieties of PGRU. 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. This work will be expanded with Native Americans through contacts recently developed with Non Governmental Organizations.

Organization/Governance

The NE-9 project lacks a well-defined end point since there is a continual and ever-increasing need for plant genetic resources. It also follows that the organization and technical aspects of the project outlines will change only in points of emphasis from year to year.


Regional Research Project NE-9 can be effective only through federal, state, and private cooperation. The federal agency ARS, through acquisition, maintenance, characterization, documentation, and distribution activities, will make plant genetic resources available for evaluation and utilization research. ARS will provide support, staff, facilities, equipment, and specialized technical assistance at both the regional and national levels. The SAESs provide facilities, some staff, equipment, utilities, and local assistance.


The NE-9 Regional Technical Advisory Committee will provide technical guidance in this effort. This committee is composed of an Administrative Advisor, Regional Coordinator, plus technical representatives invited to participate from each of the northeastern SAESs plus the District of Columbia. ARS representatives from the National Program Staff, the National Germplasm Resources Laboratory, the National Seed Storage Lab, and the National Plant Germplasm Quarantine Office are also included on the committee as ex officio members. The names, affiliations, and areas of specialization of these individuals are presented subsequently (Appendices B and C).


Other committees contribute to the planning and management and are active participants in the NPGS. These include:


1. The ARS Plant Germplasm Operations Committee evaluates and recommends foreign/domestic exploration proposals, and assists the NPGS, ARS National Program Staff and other officials with plans needed to manage the NPGS.


2. CGCs have been established for about 40 crops (or crop groups) to help advise the NPGS with regard to genetic vulnerability, gaps in current collections, operational procedures, evaluation needs, and current enhancement and utilization research associated with their specific commodity.

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Attachments

Land Grant Participating States/Institutions

CT, DE, MA, ME, NH, NJ, NY, PA

Non Land Grant Participating States/Institutions

National Germplasm Resources Laboratory, USDA-ARS, USDA, ARS