Duration: 10/01/2003 to 09/30/2009

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

In response to the negative impacts of biotic and abiotic variables on crop production, a broad genetic base is critical for U. S. agriculture in the development of new cultivars or the improvement of existing ones. The wide array of plant genetic resources (germplasm) that are maintained as part of the W-6 Regional Research Project, also known as the Western Regional Plant Introduction Station (WRPIS), provide stakeholders the genetic materials to achieve crop development. For W-6 these resources include forage and turf grasses, beans, cool season food legumes (pea, lentil, chickpea, fava bean, lupine, etc.), lettuce, safflower, onion relatives, and forage legume crop species to name just a few. Other important taxa relate to new crops, ornamental species, and medicinal plants. Availability of this germplasm is critical to researchers in the Western Region who represent both public and private sectors. Ready availability of the most diverse collections of these genes is best maintained through the existing infrastructure of the USDA, ARS, National Plant Germplasm System (NPGS) and the close association with the other germplasm related Regional Research Projects, which include the North Central (NC-7), Northeastern (NE-9) and Southern (S-9), as well as the other 22 special clonal and seed germplasm repositories. These genetic resources are readily recognized as important and crucial in the agricultural production system as water, air, soil, and minerals.

The conservation and utilization of these genetic resources ultimately affect our quality of life. This project safeguards and promotes utilization of a wide array of plant genetic resources for grower, processor and consumer stakeholders. This project, one of the four initial Regional Plant Introduction Stations established in the 1950s, is a vital component of the USDA, NPGS. The WRPIS is responsible for 15.4% of the total accessions and 23.5% of the total taxa in the NPGS. The size of the collection has grown from 53,000 accessions to over 70,000 accessions in the past ten years (Attachment: Growth in WRPIS Germplasm Collections). Project research, information documentation and germplasm conservation relate directly to all aspects of the USDA, ARS National Program Action Plan.

Specifically, the use of plant germplasm by researchers in the Western Region, particularly scientists associated with the SAES Universities, is significant. Plant germplasm is utilized in the region to support crop development, help to sustain small farm agriculture, preserve endangered species, and even repatriate crops to centers of diversity. It is also used to develop new U.S. crops, and encourage international trading diplomacy through exchange of plant germplasm. Over the past seven years public and private sector plant researchers from the Western Region request approximately 26% of the germplasm distributed by the whole NPGS, and ranges between around 26,000 to over 61,000 accessions per year (Attachment: Germplasm Use in the Western Region, Table 1.). Of this material, W-6 directly provides 5,000 to 7,000 accessions each year (Attachment: Germplasm Use in the Western Region, Table 2.) to researchers in the Western Region.

Much of the literature dealing with germplasm conservation and evaluation is specific (23,51,52,56), indicating that each crop must be tested before general rules for maintenance, preservation, and evaluation can be applied. There are, however, some general rules that are effective in germplasm management. Marshall and Brown (40), argue that relatively common alleles merit priority during regeneration. They point out that for sample size stored in collections, and for sample size distributed, the objective should be to assure (0.95 probability) that at least one copy of each allele occurring at a frequency of >0.05 is obtained and maintained. To meet this requirement quantitative genetic analysis and statistical predictions are used to determine regeneration populations based primarily on the pollination biology of the respective species. Theoretical population sizes, however, must sometimes be modified to a lesser number of plants due to practical concerns and functional realities. This may present the risk of losing alleles at very low frequencies, but is the best compromise when considering the limitations of resources at the WRPIS. Regeneration protocols for any given taxa are under constant refinement. There are few published guidelines on the regeneration of the wild relatives of crop species (33,41), and none for many of the "new crop" species.

Use of molecular techniques in germplasm management has provided a more exacting way of comparing genetic diversity in various aspects of the maintenance and evaluation programs. The advantages and disadvantages of such methods were covered in detail by Bretting and Widrlechner (3). They stressed that genetic marker data should be considered a complement to, not a replacement of, managerial experience with germplasm. The use of molecular techniques in the development of core subsets is well described (3,4,37). Utilization of these cores, especially in large collections, continues to be examined to evaluate its utility in improving the efficiency of germplasm management (24,29,62,67).

The WRPIS research on disease and insect resistance has been an important part of the evaluation program in the past (8-13,31,32,35,38,49). This type of research will be continued to better characterize important traits of use to breeders and geneticists.

Biotic pressures and stresses (i.e. insect and disease) encountered in the course of germplasm maintenance efforts play critical roles in the ultimate success of the NPGS, and particularly the WRPIS, to provide the best quality and ample distribution quantity of germplasm accessions. Sound hypothesis testing research, conducted to assess etiologies of diseases and positive or negative interactions between arthropods and hosts, is critical to the success of the overall mission of the management unit.

The range of plant taxa (over 2,400 species) maintained at the WRPIS includes almost the entire spectrum of pollination biologies found in the plant kingdom. Understanding the specificity and mechanics of the various pollination systems with which we are confronted is critical to the success of conservation of plant biodiversity. Similarly, understanding the relationships between insect vectors and the potential array of plant pathogens (viral, bacterial, fungal) or endophytes or beneficial microbes that can infect germplasm increase plots is essential to the efficient and economic maintenance of the collections.

Adequate characterization of each germplasm accession and subsequent
documentation of those data into the GRIN database will provide the necessary information to the germplasm user community to efficiently utilize the wide range of genes available. This necessarily entails the inception of or development of plant character descriptors that is a standard among U.S., or even international, plant scientists.

Molecular characterization of accessions in any given plant germplasm collection will provide an additional and valuable tool for collection management. These data will be important in both measuring genetic diversity among accessions, as well as help to identify duplicates within the collections. Research, and the subsequent information generated, will facilitate the identification of universal marker sets for future characterizations of new accessions added to the collections, for gene mapping activities, and construction of consensus diversity tables.

Because of the diversity of environments and needs in the Western Region, and the diversity of research interests and expertise available, it is both efficient and economical that a multi-disciplinary effort continue to evolve in order to utilize the talents of all interested researchers in the region. It is also critical that these valuable germplasm resources be made available, kept in superior quality condition and subsequently provided expeditiously to researchers.

Related, Current and Previous Work

At Pullman, W-6 has a team of ten scientists who interact and collaborate to achieve mission goals. This is the largest scientific team at one germplasm site in the NPGS. In the course of conducting germplasm conservation, each curator interacts with the Research Plant Pathologist, Research Entomologist, and Research Agronomist. Collaboratively they conduct both applied research to solve practical and imminent problems, and at times collaborate on basic research. Since the National Temperate Forage Legume Genetic Resources Unit (NTFLGRU), Prosser, WA and the National Arid Land Plant Genetic Resources Unit (NALPGRU), Parlier, CA are part of the WRPIS management unit, collaborative efforts are routine between all three sites. Additionally, although they are not recipients of W6 funds, the NPGS sites in the Western Region (Riverside, Davis, Corvallis, Hilo, Palmer, Aberdeen) participate in the W6 annual meetings and some collaborative projects. The need for a plant germplasm acquisition and preservation system for the United States is adequately documented in numerous reports (14,43,44,48,61). These papers describe the components of the U.S. system, including this Plant Introduction Station. The only duplication of these collections deemed necessary by the NPGS is the long-term base collection storage at the National Center for Genetic Resource Preservation (NRGRP), Ft. Collins, Colorado. These back-up samples are stored at either -18 C or under liquid nitrogen (LN, vapor phase). At the WRPIS, original samples and multiple samples for regeneration are stored at -18 C separate from the distribution samples. The active collection, including germplasm for distribution, is stored at 4 C, 28%RH.

The WRPIS station maintains over 2400 plant species. The project documents, conserves and distributes economically important and diverse germplasm, with associated information, and encourages their use in research and crop development. Activities benefit U. S. agriculture by addressing issues of genetic erosion and genetic vulnerability in crops represented in the WRPIS collections. Accessions in each germplasm collection are characterized and freely available for scientific research and evaluation worldwide. New sources of pest-resistant germplasm help to reduce U.S. dependency on pesticides. New fungal endophytes for turf and forage grass improvement are expected as well. In addition to germplasm, information in publications and the data in GRIN and other public databases are valuable products. There is a wide and diverse customer base for the germplasm that the WRPIS distributes each year. The user community for germplasm and the associated information primarily includes universities and colleges, commercial seed companies (i.e. Seminis, Pioneer Hybrid, Syngenta Seeds, Novartis, etc.) plant and animal scientists at international research centers (i.e. CIAT, ICRISAT, ICARDA, AVRDC, IPGRI, etc.), other national programs in many countries, production specialists, farmers, and occasionally home gardeners. The most direct customers are public and private breeders trying to improve or develop crop cultivars, and university researchers conducting basic research. Curators and scientists at the WRPIS interact with national Crop Germplasm Committees (CGC), which are composed of researchers from the public and private sectors. Due to the breadth of the plant diversity at the Pullman station the WRPIS curators actively represent our collections on 11 of the 40 CGCs (28%).

Within the Western Region there is a wide range of research activity utilizing NPGS plant germplasm. The WRPIS distributes between 19,000 to 23,000 accessions each year to researchers worldwide. Every state in the region conducts plant research utilizing NPGS germplasm. For example, Dr Martin at Montana St. Univ. has winter and spring wheat breeding projects using lines with resistance to Russian wheat Aphid (PI372129 and PI 262605) from National Small Grains Collection. Genera and species related to hexaploid wheat obtained from National Small Grains Collection has been used extensively to study the evolution of hexaploid wheat. At Utah St. Univ., germplasm obtained from the W-6 Western Regional Plant Introduction Station (WRPIS) has been a source of genetic material instrumental in the development and release of arid- semiarid range grass cultivars Hycrest, CD-II, Douglas, RoadCrest, and Vavilov wheatgrass and Bozoisky-Select and Tetra-1 Russian wildrye. In addition, source plant material for a tall fescue, orchardgrass, and meadow bromegrass breeding programs originated from WRPIS. Also from Utah St. Univ., cultivars of meadow brome, Altai wildrye and Russian wildyre, which have utilized plant materials from WRPIS, will be released within the next several years. Other uses included the private sector for plant breeding, the home gardener (hobby interests), seed bank survey's, and academic studies on pathogen resistance. Numerous research projects at the University of Arizona have utilized germplasm from the National Plant Germplasm System. These include completed research that dealt with measurement of differences in water requirements for germination in Digitaria californica, a grass native to the Sonoran Desert. Ongoing research using NPGS germplasm at the University of Arizona involves such things as quantification of variation phenotypic plasticity in the aggressive invasive grass, Bromus tectorum and response to low winter temperatures in guayule, Parthenium argentatum. Germplasm from numerous sites in the NPGS has been used in several research programs associated with the University of Idaho. In the area of plant breeding and cultivar development germplasm has been utilized in cereals, beans, rapeseed, mustard and potato to develop new cultivars with improved biotic stress resistance, abiotic stress tolerance/resistance and improved or modified end-use quality. Included in this research are Dr. Zemetras efforts to develop molecular markers to assist in the transfer of any desired traits identified into adapted germplasm. Associated with research in cultivar development have been programs in entomology and plant pathology that used germplasm to study the mechanisms of resistance/tolerance to various insects and diseases. With the advent of transgenic crops studies have also been done to determine the potential for genes to move from cultivated crops such as rapeseed and wheat into wild relatives such as field mustard and jointed goatgrass. Faculty at Oregon St. Univ. use the PI system extensively. Additional users in the state include state and federal researchers as well as private seed companies and private individuals. Grass breeders use the system as a source of standard cultivars for PVP evaluations, and as a source of genetic diversity for use in turf grass breeding. SunSeeds has used PI material in their vegetable breeding programs, including cucumber accessions resistant to gummy stem blight. Large numbers of accessions have been requested for evaluation of disease susceptibility; large evaluation projects include white mold in runner bean (J. Myers, OSU Horticulture), and eastern filbert blight in hazelnut (S. Mehlenbacher, OSU Horticulture). PI material is commonly requested for use in taxonomic and genetic diversity studies, which in recent years have emphasized a molecular approach. Recent studies include a taxonomic study of Trifolium (A. Liston, OSU Botany) and birdsfoot trefoil (J. Steiner, USDA Forage Lab), and a genetic diversity study in sunflower using microsatellite markers (S. Knapp, OSU Crop & Soil Science). Users also have made trips to collect plant germplasm. Recent acquisitions include strawberry and blackberry (Chad Finn, USDA-HCRL) and hazelnut (S. Mehlenbacher, OSU Horticulture). Dr. Brick at Colorado State Univ. has accessed the W6 Phaseolus collection extensively and has been a long standing participant in W6. Medicago germplasm from W6 us used by Dr. Groose at Wyoming St. Univ. to develop better forage crops.

A broad CRIS search of just the major taxa and the relevant areas of research being conducted by our SYs on those taxa produced 1,250 projects. This represents the stakeholders and customers of the germplasm provided by this project. When the search is refined to include a given taxon and 'germplasm conservation', in almost all cases the WRPIS is the only ARS project responsible for acquisition, maintenance, distribution and genetic resource research in the CRIS system. A search for Phaseolus, our largest single genus collection, germplasm yielded 32 CRIS projects. Excluding our CRIS, and that of the National Center for Genetic Resource Preservation (used to be National Seed Storage Laboratory and is the site for long term back up of the NPGS collections) the remaining CRIS projects are all associated with the Multi-state Regional Project W-150, which utilizes plant germplasm from our station as well as other Phaseolus germplasm repositories outside of the U.S. A search on the cool season food legume taxa (primarily Cicer, Lens, Pisum and Vicia) indicated 24 breeding programs, but only the WRPIS as the genetic resource conservation project. Similarly, for the grass, safflower, wild onion relatives, and lettuce collections, The WRPIS is the sole CRIS project explicitly responsible for developing and maintaining the wide array of genetic resources utilized by U.S. breeders for crop improvement or new crop development. The species assigned to the WRPIS are not duplicated at any other NPGS active collection site.

A search of the databases for the WRPIS entomology research components yielded 73 active records for research on Allium ampeloprasum, but none of these involved insect pollination for seed production. A search of 'Pisum' and 'Cicer' records produced a total of 67 records, but none indicated active research in the U.S. on evaluation of genebank accessions or selections from unadapted germplasm for resistance to seed Bruchidae or pod-infesting Lepidoptera. One project at the University of Idaho (S.D. Eigenbrode in the Department of Plant, Soil and Entomological Sciences) is examining surface wax variation in peas for pea aphid management and one project (5348-21000-007-00D) by the USDA-ARS Grain Legume Genetics Research Unit (F.J. Muehlbauer and K.E. McPhee at Pullman, Washington) mentions that insect pests reduce seed yield and quality of cool season food legumes, however, the focus of this latter project is disease resistance. A search of active Neotyphodium fungal endophyte research in the U.S. recovered 50 records, most addressing animal toxicity research, synthesis of alkaloids present in endophyte-infected forage grasses, and the genetics and biochemistry of endophytes and their association with grass hosts. Two projects are investigating endophyte-mediated resistance of grasses to pests: one (TEN00173 by K.D. Gwinn at the University of Tennessee) is assessing herbivore resistance to endophyte-produced compounds, using Drosophila melanogaster as the study insect; and one (OHO00882-SS by P. Grewal at the Ohio State University) is exploring the nature of interactions between fungal endophytes of turfgrasses, entomopathogenic nematodes, and insect (below ground white grubs, black cutworm, fall armyworm) and nematode herbivores. This search indicated no overlap between the WRPIS research objective of determining extent and nature of multiple cereal aphid species and Hessian fly resistance in endophyte-infected and endophyte-free wild barley and tall fescue germplasm accessions and other U.S. research programs studying endophyte-insect interactions. This search revealed the absence of research in the U.S. to assess survival of Neotyphodium coenophialum in tall fescue seed during the course of seed production and regeneration in the field.

Searches were conducted at the USDA/CRIS web site, and there were no records recovered for two searches: 'Selenophoma OR Pseudoseptoria' and 'Neotyphodium AND Hordeum'. Nineteen records were recovered for 'Cladosporium' and these projects focused on individual diseases caused by Cladosporium species on peach, pecan, mums, spinach and tomato. Only one project (Sedlacek and Weston at Kentucky State) mentioned Cladosporium in the context of grass family hosts (as a contaminant of stored corn). None of the 19 projects focused on phylogeny of Cladosporium or cladosporium-like taxa, nor on distinguishing between such taxa. A search of 'Alternaria' produced 138 records, but 'Alternaria AND grass' only seven, one of which was the CRIS at the WRPIS. The other six were not pertinent to identification of graminicolous Alternaria species. The majority of the 138 projects focused on or at least pertinent to Alternaria were devoted to fruits, vegetables or ornamentals attacked by Alternaria species, although one looked at Alternaria on sorghum. Three projects (Peever at Washington State; Roberts at USDA Tree Fruit Lab, Wenatchee WA; and Pryor at Arizona State) involved a strong taxonomy/phylogenetic component, but the emphasis of the first two is on Alternaria spp. attacking citrus and tree fruits respectively, and the latter on the species attacking fruits and vegetables. (Peever is a collaborator with the WRPIS project; Roberts and Pryor have both been reviewers on Alternaria-related manuscripts produced by the WRPIS lab.) A search of 'Fusarium and garlic' produced 5 records exclusive of the WRPIS CRIS. None of the five concerned the Fusarium species responsible for the new disease of garlic under investigation in our lab.

Objectives

1. Acquire and conserve cool season food and forage legume, turf and forage grass, vegetable, ornamental and medicinal plant germplasm of over 2,400 species in 268 genera.
   
2. Characterize and evaluate germplasm by using morphological characteristics and molecular marker technology to enhance conservation management, increase utilization of the germplasm collections, and to incorporate the resulting genetic data into publicly accessible databases
   
3. Evaluate interactions of key associated pathogens, and/or symbionts to improve management and utilization of plant germplasm collections. Conduct research on selected germplasm collections for response to, or relationship with, close organismal associates such as microorganisms, pathogens, and saprophytes.
   
4. Evaluate interactions of key associated insects to improve management and utilization of plant germplasm collections. Conduct research on selected germplasm collections for response to, or relationship with, significant insect pests and disease vectors.
   
5. Evaluate and improve seed regeneration protocols and methodologies to both maximize efficiency and quality of germplasm, and to preserve selected beneficial plant microbes and insect-pollinated out-crossing species.
   
6. Within the Western Region, throughout the U.S., and internationally, encourage the use of a broad diversity of germplasm to reduce crop genetic vulnerability. Through different avenues of technology transfer in the form of plant germplasm propagules (seed/clones), research publications and other associated information to scientists world wide, provide resources to scientists world wide for future crop development.
   

Methods

1. The major collections at the WRPIS are the grasses, safflower, Phaseolus , lettuce, wild onion relatives, cool season food legumes, beets and an array of medicinal, ornamental, and wild legume species. All curators work closely with the appropriate Crop Germplasm Committees (CGC). The curators are responsible for entering accessions into the PI system. New germplasm is obtained through exchanges with University breeders, foreign national programs, international centers and from NCGRP as part of the CSR process. Often, W6 participating state representatives provide new germplasm to the W6 and other NPGS collections. Every effort is made to obtain complete passport documentation and enter it into GRIN. GIS technology and morphological and molecular characterization are used to identify genetic gaps in the collections. To fill acquisition strategies are devised. Regeneration protocols that are effective in preserving the genetic integrity, and diversity of collection(s) are developed. Efforts are made to ensure pest and disease free seed production. Collaborate with CGC members to the curators characterize. The entomologist and plant pathologist assist by screening germplasm for pest resistance. Curators send seed samples to the NCGRP, Fort Collins, CO for backup. Determination of seed viability in the distribution seed lots is critical. We will emphasize germination testing. The seed manager will coordinate this work. The curators will collaborate with scientists to incorporate research results into germplasm increase methodologies. The primary focus of development will be on controlled pollination of the out-crossing taxa in order to provide effective, efficient and economically feasible increase of accessions. Greenhouse increases of beans will continue with use of pot culture. Pot culture facilitates the virus testing on an individual plant basis. Pest control will continue to be based on parasites and predators. The entomologist and plant pathologist will examine new germplasm entering this collection. Regeneration plots will be monitored to ensure production of pest and pathogen-free seeds. We will continue to expand our virus-testing lab for seed borne virus assays. 2a. Reducing genetic drift in heterogenetic accessions: Heywood (26) derived the equation to quantify the proportional reduction in effective population size (Ne) associated with variation in potential fecundity. Under those conditions genetic drift is equal to the traditional Wright-Fisher binomial sampling model (78). The reductions in effective population size for three sampling methods will be examined in field plots at Central Ferry and Pullman, WA locations with accessions of Lolium perenne, Festuca pratensis, and Pseudoroegneria spicata. Cutting and rubbing are the traditional sampling methods, but direct comparisons of how all these procedures affect Ne has never been completed. Cut method: Each plant will be cut at seed maturity and placed in a separate bag. Seeds will be cleaned and variation in seed per plant and mean seed number determined. Ne/Nc will be determined. Rub method: Each plant will be rubbed one or more times as needed and seeds from each plant kept separate. Ne/Nc will be determined. Inflorescence method: Two inflorescences from each plant will be harvested and kept separate. Variation between inflorescences can be calculated to predict the optimum needed to maximize Ne. Ne/Nc will be determined. There will be 4 reps/location, with 3 treatments of 10 plants each in a split block design and analyzed over locations. All components contributing to variation in seeds per plant will be assessed and compared. Data will be analyzed to predict the number of inflorescences needed from each plant to maximize Ne. This will be repeated for two years. 2b. Genotyping grass and alfalfa germplasm collections for diversity analysis and duplication: We will use 3 alfalfa accessions, each with a duplicate of cultivar names, and will be comparing original and increased seed. There will be different sampling methods and marker systems used to determine the most effective sampling/marker system combination for genotyping alfalfa. This will be done on the three increased seed populations for each. DNA extractions will be completed using known methods (42). For the three increase seed populations, DNA from 98 plants per accession will be extracted to develop baseline information on marker systems. For each original seed population, 48 plants per accession will be extracted for each of the original seed populations. For bulk samples there will be both leaf bulking followed by DNA extraction, and bulking of DNA from individual plant extractions. We will use four maker systems (PCR based) to compare different sampling methods. We will choose the most efficient system to examine the entire set of duplicates for each cultivar. The criteria for efficiency will be ability to distinguish populations with the least input in terms of supplies and labor. 1. Chloroplast primers: Two primer pairs from the chloroplast genome in alfalfa were developed by Skinner (63) for use to describe the levels of genetic relatedness among alfalfa populations. 2. AFLP (Amplified fragment length polymorphisms): This marker system is now being widely used for genotyping work in forage grasses and legumes (36,39,50,72). It is highly reproducible and usually provides numerous loci. However, the markers are generally dominant and therefore calculation of factors related to heterozygosity is not possible. 3. RAPD (Random amplified polymorphic DNA): RAPDs are relatively easy and inexpensive, but may be less reproducible. 4. SSR (Simple sequence repeats): SSRs have the reliability that AFLPs provides, and they also have the advantage of being co-dominant. This allows more extensive data analysis (73). SSR markers are available for alfalfa (15,16). After scoring, genetic distances will be calculated for each sampling and marker system using Sokal Binary Distance Coefficient (55). Multivariate techniques such as cluster analysis and principal components will be used to visualize distances among accessions (80). Comparisons will be made among accessions and sampling method to determine if statistical separation is possible. Factors for evaluation will include fall dormancy, forage production, forage quality factors, winter survival, and observations on disease and insect infestations. Average Euclidean distance will be used to develop a distance matrix for each population. This matrix will be correlated with matrices based on the molecular data to determine if molecular and agronomic data correspond. For both molecular and agronomic data sets, multivariate statistical techniques will be used to visualize distances among accessions (80). 2c. Characterization and enhancement of Kentucky bluegrass (KBG: The experiment will consist of 10 entries; 8 are PIs and 2 are check cvs. (Kenblue and Midnight). Kenblue is a common type with high yield, and Midnight has high turf quality. The selected accessions represent a range of responses to residue treatments. Seeds of each accession will be germinated, grown in under greenhouse conditions and transplanted to the field. Heading date, anthesis date, physiological maturity date, seed yield and yield components, vegetative spread, plant height, leaf length and leaf width will be measured on individual plants. Ratings of leaf habit, abundance and color will be made. Leaf tissue will be gathered and DNA extracted from one replication/entry. AFLP analysis will be done. The agronomic and molecular variation within and among entries will be assessed along with seed production. Distance matrices will be calculated and comparison of the extent of agronomic and molecular variation will be compared. Seed production data from individual plants will be used to select individual plants from each accession and within each block for high and low panicle number. The selected material will be increased in seed increase nurseries to obtain sufficient seed to carry out future genotype by environment studies on yield and turf quality of selections. 2d. Application of markers to elucidate allelic diversity across legume germplasm: The work will focus on the plant model species Medicago truncatula for SSRs, SNPs and ESTs for microarray, Genotyping will be conducted using single sequences repeats (SSR) from M. truncatula, C. arietinum(28,59,76,77), L.culinaris (22), and P. sativum (5). DNA has been isolated from the pea and chickpea core accessions (42). Single nucleotide polymorphisms are available for M. truncatula and P. sativum (68,81). We will apply the method developed for M. truncatula to develop universal PCR-based marker sets (17,34). To develop PCR primer sets that will amplify a corresponding locus from the genomes of multiple species, we will analyze the unigene EST set (69,70). Putative orthologus loci of mapping parents and nodal accessions will be identified using five legume taxa. STMS genetic diversity experiments will focus on markers linked to economic traits. We will identify sequence tagged microsatellite (STMS) markers closely linked to the Fusarium wilt race 2 resistance gene (Fnw). Recombinant inbred line populations will be evaluated for disease reaction. By converting the markers to co-dominant markers (36,58,79), we will also expand their utility for genetic diversity assessment (45). SNP analyses of genetic diversity of legume procedures will initially be developed for peer. Methodologies will be compared to determine efficacy, through-put and cost-effectiveness. Two models will be pursued for use of microarrays to assess allelic diversity of plant germplasm collections: cDNA arrays (57) and whole genome DNA arrays (30). Microarray allelic diversity scans will use the available unigene set (70). The mRNA will be isolated using a commercial kit. Accessions will be hybridized in sets of 16 using a hybridization station following manufacturers directions and scanned using an array reader. Diversity values for each locus will be calculated using the genetic diversity index (73). Genetic similarity will be calculated by Jaccard's coefficient. A dendrogram will be generated from the Sequential, Agglomerative, Hierarchical, and Nested (SAHN) clustering method using the unweighted pair-group method, arithmetic average (UPGMA) by NTSYS (54,65). To obtain the multidimensional scaling, a distance matrix will be used (53). The ABSOLUTE option will be used in the MDS procedure (60). Distance matrices giving the relatedness of individual accessions within collections will be developed and used to identify duplication and unique accessions. 3: Evaluate interactions of key associated pathogens, and/or symbionts to improve management and utilization of plant germplasm collections: Taxa of fungal pathogens and other important fungi associated with seeds will be identified and characterized by traditional morpho-taxonomic methods (21,27) and/or molecular genetic techniques focusing on ITS (20), beta-tubulin or endo-PG sequences (46), and/or RAPD (47). Sequences from 18S will be utilized (2) with regard to phylogenetics of Cladosporium and cladosporium-like taxa. Taxa of primary interest are currently Alternaria , Cladosporium, Neotyphodium, and Selenophoma (64). Koch's postulates will be applied to suspected new diseases and positive results published for new pathogens or new diseases associated with seeds. Selected portions of the garlic germplasm will be screened for resistance to a new Fusarium bulb rot by plantings in infested and un-infested soil. Methods exist for assessing the reaction of Allium species to infection by soil-borne fusaria (1,66,71). Surveys of grass accessions for infection by endophytes and defining patterns of colonization by endophytes in host tissues will continue using traditional staining techniques (74), histology and electron microscopy (19,80) and/or molecular techniques (18). The Neotypodium endophyte of wild barley has been characterized extensively by the WRPIS scientists (8,19,75). This system will remain a primary investigative focus. 4. Evaluate interactions of key associated insects to improve management and utilization of plant germplasm collections. Pea seed weevil resistance in wild pea: Study will be conducted near Pullman, WA, where the weevil occurs in high numbers and where a previous study was conducted (25). Seed of P. fulvum and interspecific crosses must be scarified and germinated as per established procedures (33). In spring of each year plants will be grown in a greenhouse. Potted plants of the susceptible pea, Alaska 81, parent and resistant P. fulvum parent will be randomly interspersed among the F3 plants. After 7-10 days, weevil eggs will be counted on all pods/plant to measure weevil oviposition rates. Mature pods will be collected and returned to a laboratory to be scored for weevil damage (25). Pod-boring Lepidoptera resistance in wild annual Cicer spp.: This study is to quantify mortality and development of larvae of Helicoverpa punctigera on 22 accessions of annual wild Cicer spp., and to compare findings with larval performance on a susceptible chickpea cultivar. The study will use 70 accessions (22 PIs) in four Cicer spieces. We will obtain larvae for this study from Perth, Western Australia. Dr. Clement will evaluate the germplasm in Perth. In the first experiment 3-5 plants per accession will be infested with five 1-2 instar larvae. Larval development and survival will be observed 7-10 d. A second experiment will infest plants with 3-4 instar larvae, but only one per plant. Five plants per entry will be infested with 3-4 instar larvae and larval development and mortality recorded for 7-10 days. Leaf and pod damage will be estimated. Additionally, a detached branch section with foliage and a developing pod from a glasshouse-grown plant will be placed into a petri dish. Five first and second instar larvae selected from the colony will be placed on the plant material in each petri dish. After 24 and 48 h the numbers of live larvae, and their position (stem, foliage, pod, off plant material) will be recorded. Experiments will be conducted in a temperature and light controlled environmental chambers. The above experiments will later be expanded to evaluate a larger number of plants (n = 10) of selected resistant accessions. Grass-endophyte-insect interactions: Endophyte-infected and endophyte-free wild barley source plants of four perennial Horeaum accessions will provide replicate clones for each experiment at a rate of 25 adult apterous aphids/clone. Before aphids are added, the endophyte status of each replicate will be confirmed (8). Live aphids will be recorded after 10 d. For Hessian fly experiments, each replicate clone will be exposed to ovipositing females for 12 hours, after which eggs will be counted and live adults removed. Each Hessian fly experiment will last 24 days, at which time the numbers of live second instar larvae and puparia per replicate will be recorded. Replicate clones of each wild barley accession will be obtained from parent plants at WRPIS. There will be 24 separate experiments involving wild barely-endophyte-insect interactions. The presence of N. coenophialum in tall fescue adversely affects the survival of bird cherry-oat aphid: Endophyte-infected plants from two Tunisian tall fescue accessions were susceptible to this aphid in an earlier study (7). To test that the differential response of bird cherry-oat aphid to infected plants from North Africa reflects the presence of diverse Neotyphodium genotypes in this germplasm, we will conduct experiments with 23 Neotyphodium-infected tall fescue accessions from Tunisia and Morocco (7). Tall fescue source plants (1.5- 2 yrs-old) of each treatment are maintained in a glasshouse. The infection status of each replicate clone will be confirmed by a PCR method using tub-2 primers (18). After 6 wk. each clone will be pruned to two vigorously growing tillers. Ten of the most clones per treatment will be selected for experiments. Experimental clones (each with 25 aphids) will be placed in environmental chambers for each 10-day experiment. Three separate experiments will be conducted, each with 8 or 9. Data on number of live aphids at the close of each experiment will be analyzed using ANOVA procedures. 5. Evaluate and improve seed regeneration protocols and methodologies: Caged insect pollination of leeks, A. ampeloprasum: In July 2003 the PI line for the study will be grown in glasshouse in September the young plants will be transplanted to field (75 per cage). In June 2004 - Place cages over plants (28 cages). Add flies to each cage (except controls) each week during pollination period. In August, stop adding flies and count number of plants that produced viable umbels and thin to uniform number of viable plants and umbels per cage. In September and October, harvest umbels as they mature in each cage. Weigh seed harvested from each cage and perform seed germination tests (100 seeds per cage). This procedure will be repeated for the second year of the study, commencing with sowing of seed in July 2004 and placement of new plants in the field in late-September 2005. Treatments will be 100, 250, and 500 pupae of Musca domestica and Calliphora vicina, as well as the no-insect control (4 reps/trt, RCB design). Feral insects that may appear in control cages will be captured to maintain the no-insect status of these cages. Commercial suppliers of flies will be used. ANOVA procedures will be used to analyze data. Preserving viable endophytes in tall fescue: A PCR method using tub-2 primers specific for detection of diverse and viable Neotyphodium endophytes in tall fescue will determine the endophyte status of plants, tillers from infected plants, and individual seeds from infected tillers. Research in 2003 and 2004 will address: 1) the accuracy of the PCR method to detect endophyte in individual seed. This is necessary to determine if accession infection rates after seed-regeneration can be generated using seed from field plants; 2) the ratio of infected to uninfected tillers on an infected plant. Such information is unknown for infected plants of wild and unadapted accessions of tall fescue; 3) the ratio of infected to uninfected seed produced by an infected tiller on an infected plant. This information is required to design a protocol for sampling seed of regeneration plants in field plots. Seed sampling protocols will be developed. Three infected accessions will be selected and seed sown to obtain 80 healthy plants of each accession for transplanting to field plots. The endophyte status of each plant will be established prior to their being moved to the field. 6. Within the Western Region, throughout the U.S., and internationally, encourage the use of a broad diversity of germplasm to reduce crop genetic vulnerability: Germplasm related data on Plant Introduction accessions in the NPGS reside in the Germplasm Resource Information Network (GRIN) data base. All data designated as available to the public is accessible via the Internet. GRIN is the primary repository for passport, evaluation and characterization data related to WRPIS germplasm. Germplasm is received by the WRPIS as material transferred from other NPGS sites, from NCGRP, through exchanges with other national genetic resource conservation programs around the world, from public and private sector breeding programs, from donations by U. S. citizens, and from plant exploration expeditions. Passport data is assembled at Pullman and loaded into GRIN. Research and evaluation data not yet in GRIN is maintained and distributed by the respective scientists in the unit. Information and results from research projects is transferred to the user community via peer-reviewed publications, posters, abstracts and presentations at professional and commodity meetings. These publications and presentations lead to personal interaction with scientists from around the world. As part of the germplasm maintenance program, protocols for each step of the process of 'seed-to-seed' conservation are documented in an operations manual which is a dynamic reference that is continually being developed and updated. These protocols are routinely requested by other genetic resource conservation organizations. Available germplasm is distributed to researchers around the world. Seed orders are processed within 7-10 days if the request is routine. An important aspect of this technology transfer is the interaction of the germplasm user with the respective curator to best refine the germplasm request to meet the needs of the scientists.

Measurement of Progress and Results

Outputs

• The most important output will be the continued provision of quality germplasm of the species maintained at this site and deliver to researchers worldwide. From the utilization of this germplasm both basic and applied research will result. This includes new cultivars, production of genetic maps, analyses of diversity, new medicinal plants, ornamentals, etc. and restoration or re-patriotization of germplasm to seed banks in countries from whence the original germplasm came.
• Research information will be applied to regeneration programs to provide cost effective methodology to maximize effective population size during regeneration of heterogenetic species and accessions. The results may also be applied to germplasm collecting to promote representative field sampling.
• Results from molecular genetic research will provide the basic information needed to expand the molecular characterization program for germplasm collections in a cost effective manner. It will also be useful to other scientists interested in the interaction between marker systems and DNA sampling, especially in heterogenetic crops. As characterization data is collected it will be made available to the public on the Germplasm Resources Information Network (GRIN).
• An enhanced understanding of turf quality and yield will provide an understanding if, and to what extent, yield can be improved without detrimental effects to turf quality. This should assist the plant breeding community in their cultivar development efforts. This will enhance the potential for utilization of the Kentucky bluegrass collection. Informative AFLP markers will be identified and the correspondence between molecular and agronomic variation both among and within accessions will be determined.
• Genetic research on important food legumes will result in the identification of unique germplasm in each food legume taxa readily available for basic research and applied plant breeding programs. Novel methods of genetic diversity assessment will be tested and implemented.
• Output 6: Successful completion of entomology research will identify good sources of insect-resistant pea and chickpea germplasm for use by breeding programs, and will further advance our knowledge about the presence of diverse fungal endophytes in tall fescue and wild barley germplasm and their potential use for developing novel forage- and cereal-endophyte associations for pest resistance. Output 7: Criteria for identification (descriptive keys, DNA sequences etc) of important taxa of seed-associated fungi will be published in peer-reviewed journals, as will discovery of new diseases or new disease agents. Control measures, including assessment of resistance, will be similarly published in collaboration with plant pathologists, curators and/or breeders. Plant and microbial germplasm of benefit to breeders and pathologists will be identified, preserved and distributed to bone fide researchers.

Outcomes or Projected Impacts

• The most important outcome will be the continued utilization of this germplasm for both basic and applied research. This includes new cultivars, production of genetic maps, analyses of diversity, new medicinal plants, ornamentals, etc. and restoration of germplasm to seed banks in countries from whence the original germplasm came. Research information applied to regeneration programs will provide cost effective methodologies. Successful completion of the leek-fly pollination study will result in an effective seed regeneration system for Allium germplasm at the WRPIS.
• Results from molecular genetic research will provide the basic information needed to expand the molecular characterization program for germplasm collections in a cost effective manner. It will also be useful to other scientists interested in the interaction between marker systems and DNA sampling, especially in heterogenetic crops. As characterization data is collected it will be made available to the public on the Germplasm Resources Information Network (GRIN). Genetic research on important food legumes will result in the identification of unique germplasm in each food legume taxa readily available for basic research and applied plant breeding programs. Novel methods of genetic diversity assessment will be tested and implemented.
• An enhanced understanding of turf quality and yield will assist the plant breeding community in their cultivar development efforts. This will enhance the potential for utilization of the Kentucky bluegrass collection. Informative AFLP markers will be identified and the correspondence between molecular and agronomic variation both among and within accessions will be determined.
• Successful completion of entomology research will identify good sources of insect-resistant pea and chickpea germplasm for use by breeding programs, and will further advance our knowledge about the presence of diverse fungal endophytes in tall fescue and wild barley germplasm and their potential use for developing novel forage- and cereal-endophyte associations for pest resistance. The endophyte preservation study will determine if viable endophyte is retained under current tall fescue seed regeneration protocols, and will pinpoint the need for modifications if there is significant loss of endophyte viability after seed regeneration.
• Proper identification of important taxa of seed-associated fungi will provide a basis for development of control measures, including assessment of resistance. Plant and microbial germplasm of benefit to breeders and pathologists will be identified, preserved and distributed to bone fide researchers.

Milestones

(2003): With regard to the collections, based on the current of new accession introductions, the WRPIS total collection could increase to over 76,000 accessions over the next five years. Our current level of available seed will be increased in increments of 1-2% each year, and germplasm backed up at NCGRP will be increased from 73% to 85%. Research activity includes: First year for cage insect pollination of leeks: Pea weevil resistance in P.fulvum x P. sativum, field evaluation of F3s: Compile data on pod-boring Lepidoptera resistance in wild Cicer: Extract DNA from plants to genotype grass and alfalfa accs.: Establish Kentucky Bluegrass plots and collect agronomic and molecular data: Determine endophyte infection frequencies of three fescue accessions in field plots: Artificial inoculation of wild barley for endophyte surveys: Conduct Hessian fly/wild barley/endophyte experiments: Identification of graminicolous Selenophoma: Determine etiology of Fusarium proliferatum bulb rot of garlic: Taxonomy & identification of Alternaria on grass family hosts, and conduct PCR based studies: Complete STMS genotyping for M. truncatula: Complete map Pisum genes Fnw, Fw, Fwf : Identify Pisum SNPs and do allelic diversity analysis: Identify biochemical pathways for allelic diversity analysis. Manuscript preparation for completed work.

(2004): Continue cage insect pollination study in leeks: Begin field evaluation of F3 plants for pea weevil resistance: Continue collaboration on pod-boring Lepidoptera resistance in wild annual Cicer with Australians: cooperators continue research: Complete interaction of DNA sampling and marker system to genotype grass and alfalfa study, and publish: Survey grass seed accessions for Neotyphodium: Finish data collection and make yield selections of Kentucky Bluegrass: Analyze Hessian fly/endophyte data in fescue: complete Bird cherry-oat aphid and rose grass aphid experiments: RAPDs and sequence analysis of graminicolous Selenophoma: Screen garlic accessions for resistance to F. proliferatum. List of 500+ names in Cladosporium.:: Complete STMS genotyping for M. truncatula: Complete map Pisum genes Fnw, Fw, Fwf: Identify Pisum SNPs and identify biochemical pathways for allelic diversity analysis. Manuscript preparation for completed work.

(2005): Complete or continue the above studies. Manuscript preparation and submittal for completed work. Based on results of the above model crops, begin new research on PCR based analysis and evaluation of collections, beginning with core sub-sets. Get assembled data entered into the GRIN system. Provided additional resources, initiate tissue culture/cryogenic preservation of clonal germplasm assigned to WRPIS.

(2006): Incorporate results of controlled pollination experiments into regeneration protocols. Continue evaluations of collections and/or core sub-sets. Enter data into GRIN. Manuscript preparation and submittal for completed work. Begin large scale evaluations for duplicates in the collections. Continue to address disease problems related to germplasm conservation.

(2007): And 2008. Continue evaluations of collections and/or core sub-sets. Enter data into GRIN. Manuscript preparation and submittal for completed work. Begin large scale evaluations for duplicates in the collections. Continue to address disease problems related to germplasm conservation: Complete STMS genotyping of Lens and Vicia faba core collections: Complete screening Pisum Core collection using SNPs: Identify SNPs for cross taxa genotyping: Prepare chips for allelic diversity analysis using microarrays.

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Germplasm related data on accessions in the NPGS reside in the Germplasm Resource Information Network (GRIN) database, which are available to the public, and accessible from anywhere in the world via the Internet. The WRPIS utilizes GRIN in an interactive way as the primary repository for passport, evaluation and characterization data. Germplasm is received by the WRPIS as material transferred from other NPGS sites, from NCGRP, through exchanges with other national genetic resource conservation programs around the world, from public (universities primarily) and private sector breeding programs, from donations by U. S. citizens, and from plant exploration expeditions. Passport data is assembled at Pullman and loaded into GRIN. Information and results from research projects is transferred to the user community via peer-reviewed publications, posters, abstracts and presentations at professional and commodity meetings. These publications and presentations lead to personal interaction with scientists from around the world, and subsequent additional technology transfer occurs.

Protocols for each step of the process of 'seed-to-seed' conservation are documented in an operations manual, which is a dynamic reference that is continually being developed and updated. These protocols are routinely requested by other genetic resource conservation organizations within the US and from other countries and international conservation institutes. We provide this technology on a regular basis.

As part of the mandate of the CRIS, available germplasm is distributed to researchers around the world. Seed orders are are processed within 7-10 days if the request is routine. An important aspect of this technology transfer is the interaction of the germplasm user with the respective curator to best refine the germplasm request to meet the needs of the scientists.

Organization/Governance

The recommended Standard Governance for multi-state research activities includes the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative. Over the next five years we will use internal benchmarks and accountability systems to assess progress and then determine future needs. In addition to the input from the W6 Technical Advisory Committee, we will use the ARS, National Program 301 review process, input from the CGCs, and if appropriate, suggestions from and external review.

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