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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections


2018 Annual Report

Genebanks have an urgent mandate to increase efficiency and number of germplasm forms and species within collections. This mandate will be achieved through technologies that prolong germplasm shelf-life, tools that nondestructively detect early changes in viability and genetic integrity, and methods that quantify and compare wild and collected diversity. Through time, genebanked materials must stay fit-for-purpose. PGPRU’s goals are to provide state-of-art genebanking methods that address current needs of genebank operators to manage collections cost-effectively and future needs of users for access to well characterized, diverse collections of living germplasm. To meet this challenge, PGPRU will perform research in the next 5 years that will: OBJECTIVE 1: DETECT GAPS AND REDUNDANCIES IN GENEBANK COLLECTIONS Develop and validate methods that integrate statistical genetics and spatial analyses to detect gaps and redundancies in genebank collections, and to estimate and compare the genetic diversity among genebank collections and in situ populations, especially for crop landraces and wild relatives. • Subobjective 1a. Partition genetic diversity within NPGS collections into subsets and provide metrics to relate genetic distance among accessions. • Subobjective 1b. Confirm whether geospatial and climatic data can identify collection gaps or ecotypes. OBJECTIVE 2: IMPROVE INITIAL AND LONG-TERM SURVIVAL OF STORED GERMPLASM Devise or refine tools that enhance the long-term viability of stored germplasm, including clonal propagules, and provide the means for curators to assess and predict the response of germplasm to conventional and cryogenic storage treatments. • Subobjective 2a: Develop methods to recover vigorous plants from shoot tips. • Subobjective 2b: Quantify variation of shoot tip response to established preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2c: Quantify variation of dormant bud response to preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2d: Quantify variation of seed response to desiccation and cooling. • Subobjective 2e: Quantify interactions between temperature, moisture and seed longevity. OBJECTIVE 3: EVALUATE CHANGES IN QUALITY AND GENETIC IDENTITY Design metrics for monitoring and validating biological quality (viability, health, etc.) of stored and regenerated plant germplasm and assess genetic integrity of germplasm and the genetic shifts that occur during germplasm management. • Subobjective 3a: Develop new tools to measure initial vigor and detect aging. • Subobjective 3b: Assess risks of genetic change during genebanking. OBJECTIVE 4: LOCATE MASKED DESIRABLE GENES IN CROP WILD RELATIVES Develop and apply genome annotation methods to evaluate collections of crop land races and wild relatives for genetic diversity in key agricultural traits, so as to enable more effective germplasm curation and to improve access to that diversity for marker-assisted breeding.

The Plant Germplasm Preservation Research Unit (PGPRU) has the unique mission of troubleshooting plant genebanking methods to solve the most critical problems of genetic resource collections: keeping germplasm alive, healthy and representative of the source population; describing collection composition; and ensuring stored germplasm meets the needs of diverse users. This 5 year plan describes PGPRU’s strategy to apply creative, multidisciplinary approaches that balance the special requirements of diverse living germplasm (seeds, pollen and explants) with the practical needs of curators and users. Research will provide tools that compare genetic diversity of collections with species diversity extant in the wild; broaden the array and longevity of propagules in storage; assess germplasm health with minimum sample depletion; and account for genebanking effects on the biological and genetic integrity of the sample. Our research efforts will be vital to the overall goal of creating relevant scientific collections to understand, protect and use plant diversity in a changing world. PGPRU will approach the task of improving genebanking as the challenge of achieving apparently contradictory goals, such as maximizing genetic diversity while minimizing collection size; standardizing preservation treatments for diverse propagules that respond differently; monitoring for signs of deterioration during early storage when few changes are known to occur; maintaining genetic heterogeneity in an agricultural context where quality and uniformity are highly valued; and finding specific alleles of interest that may be masked by the genetic background. In a real sense, these contradictions underscore the complex mission of genebanking. PGPRU will remain at the forefront of plant repository biology and will continue to play a global role in technology transfer for plant genebank management.

Progress Report
This project terminated on June 12, 2018. The new project “Innovative Strategies and Methods for Improving the Management, Availability, and Utility of Plant Genetic Resource Collections” was certified in spring 2018 after review and a “no revision” score. There are no milestones reported for the new report. Milestones for the ending project have been added. During the previous 5-year project, we saw substantial advances in the use of genomic tools to structure plant genetic resource collections and to predict diversity that is underrepresented in important collections. Much of this work focuses on wild relatives of crops. Understanding the composition of collections is important but may never guide collection strategies, which are typically opportunistic due to regulatory and financial constraints and often proceed without the benefit of date-driven analysis. The growing awareness that genomic tools are useful to guide collection management has led to better interactions between research and curation within the National Plant Germplasm System (NPGS) and calls for more investment in bioinformatics approaches to characterize useful diversity. Strategies to back up valuable germplasm from clonal repositories or from important species producing difficult-to-store seeds remains one of our largest challenges. Basic cryopreservation principles are increasingly understood, and so the challenge is less about survival following exposure to liquid nitrogen and more about obtaining sufficient numbers of healthy propagules to process and store, as well as reliable recovery procedures that ameliorate damage in stressed cells, promote healthy organ development and are broadly applicable across diverse genotypes and species within a collection. Our work shows that it takes between 35 and 45 hours of skilled technician time to cryopreserve a single in vitro accession to ensure high probability of 60-70 viable propagules. Our goal is to reduce processing time to 9-12 hours per accession per technician before transferring technology for implementation. Examples of highly successful application of PGPRU approaches are observed in Prunus (plums, cherries and peaches), Citrus, Vitis (grapes), Carica (papaya), and Quercus (oak). We were less successful in work with Saccharum (sugar cane) and Persea (avocado). One strategy that reduces the number of clonal accessions needing back-up and reduces processing time per accession is to store seeds or pollen, which are usually more stress tolerant and do not require an in vitro step. This method, which requires controlled pollination among conspecific accessions, would only be applicable to accessions representing species related to the crop species. The approach of collecting seeds has proven feasible for some collections traditionally considered clonally propagated. However, collections often have only one or two specimens of a species – if the latter, they may be separated in the field – making controlled pollinations difficult and increasing risk of genetic erosion. Viability assessments are considered the ‘gold standard’ for monitoring germplasm health. However, this approach only tests the proportion of alive versus dead in a population that has been stressed by desiccation, temperature or time. It lacks a quantitative assessment of propagule response to stress and any predictive power of response if stress were increased. PGPRU continues efforts developing quantitative assays that reveal stress before mortality and that have the power to predict future response reliably. One of these tests, the RNA integrity assay, is moving towards technology transfer and routine use with the seed curation group.

1. Cryopreservation of Vitis (grape) cultivars. United States genetic resource collections for grape are primarily held as field plantings that are vulnerable to environmental and biological threats. ARS scientists in Fort Collins, Colorado have developed methods to backup these valuable collections as cryopreserved shoot tips from vine cuttings. The procedure is applicable to genetically diverse grape plants and is highly flexible and labor-saving. Next steps are to implement the procedure using the most valuable accessions of Vitis. These efforts will ensure vital germplasm that is required by grape-growing industries is secured and available in perpetuity.

2. The mechanism of seed aging. Germination assays only describe whether or not a seed is alive. Understanding the cause of mortality is necessary to predict when a seed becomes no longer viable. Yet reliable predictions are needed to improve processing and storage treatments as well as flag the need for regeneration of genebanked seeds in United States genetic resource collections. ARS scientists in Fort Collins, Colorado have shown that aging is the accumulation of minor damage within cells that eventually reaches a tipping point beyond which the cell cannot recover. By developing general assays that “count” the number of damaging events, we can now measure how quickly the biological clock ticks for stored seeds and therefore predict longevity. The work can now be applied as a reliable assessment of seed aging. Ultimately this discovery will increase genebanking efficiency by reducing both the cost of testing seeds and the risk of losing valuable collections.

3. Does genetic diversity correlate with geography or genomics? Curators and plant collectors frequently use geographic distance as a proxy to find genetic diversity that is not included in genetic resource collections. Geography or environmental characteristics have also been used to develop core subsets of the collection intended to maximize diversity with the fewest number of samples. ARS scientists in Fort Collins, Colorado discovered that these assumptions do not hold and that genetic diversity is best estimated and structured using bioinformatic tools that measure diversity through the relationships of genes (i.e., through specific gene ontology groups). This new approach will increase the over-all utility of plant genetic resources to future scientists through new methods to find novel diversity in nature and to locate valuable genes that are hidden in weedy accessions.

4. Development of a National Plant Germplasm System Genebank Training Program. There is a critical need of sustained training for future genebank managers. ARS scientists in Fort Collins, Colorado hosted a planning conference in April 2018 that brought numerous experts in plant genebank management and germplasm use together to identify training education formats. The initiative began the process of successional planning to ensure there is the expertise available to maintain plant germplasm collections that are vital to agriculture in the United States and the world.

Review Publications
Richards, C.M., Reeves, P.A. 2017. Capturing haplotypes in germplasm core collections. Genetic Resources and Crop Evolution. doi:10.1007/s10722-017-0549-6.
Smykal, P., Hradilová, I., Trnený, O., Brus, J., Rathore, A., Bariotakis, M., Das, R., Bhattacharyya, D., Richards, C.M., Coyne, C.J., Pirintsos, S. 2017. Genomic diversity and macroecology of the crop wild relatives of domesticated pea. Nature Scientific Reports. doi:10.1038/s41498-017-17623-4.
Stevenato, P., Brocenello, C., Pajola, F., Richards, C.M., Panella, L.W., Hassani, M., Formentin, E., Chiodi, C., Concheri, G., Heidari, B. 2017. Targeted next-generation sequencing identification of mutations in disease resistance gene anologs (RGAs) in wild and cultivated beets. Genes. 8(10):264. doi:10.3390/genes8100264.
Hardegree, S.P., Moffet, C., Walters, C.T., Sheley, R.L., Flerchinger, G.N. 2017. Hydrothermal germination models: Improving experimental efficiency by limiting data collection to the relevant hydrothermal range. Crop Science. 57(5):2753-2760. doi:10.2135/cropsci2017.02.0133.
Hardegree, S.P., Roundy, B., Walters, C.T., Reeves, P.A., Richards, C.M., Moffet, C., Sheley, R.L., Flerchinger, G.N. 2018. Hydrothermal germination models: assessment of the wet-thermal approximation of potential field response. Crop Science. 58(5):2042-2049.
Bi, W., Hao, X., Cui, Z., Volk, G.M., Wang, Q. 2018. Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.). In Vitro Cellular and Developmental Biology - Plants. 54:590-599.
Wang, M., Chen, L., Liu, J., Teixeira Da Silva, J.A., Volk, G.M., Wang, Q. 2018. Cryopreservation of apple (Malus spp.): development, progress and future prospects. Plant Cell Reports. 37(5):689-709.
Bettoni, J.C., Costa, M.D., Souza, J.A., Nickel, O., Volk, G.M., Da Silva, F.N., Kretzschmar, A.A. 2018. Cryotherapy by encapsulation-dehydration is effective for in vitro eradication of latent viruses from ‘Marubakaido’ apple rootstock. Journal of Biotechnology. 269:1-7.
Byrne, P.F., Volk, G.M., Gardner, C.A., Gore, M.A., Simon, P.W., Smith, S. 2018. Sustaining the future of plant breeding: The critical role of the USDA-ARS National Plant Germplasm System. Crop Science. 58(2):451-468.
Gross, B.L., Martinez, M., Wedger, M.J., Volk, G.M., Hale, C. 2018. Identification of unknown apple cultivars demonstrates the impact of local breeding program on cultivar diversity. Genetic Resources and Crop Evolution. 65:1317-1327.
Reeves, P.A., Bowker, C.L., Fettig, C.E., Tembrock, L.R., Richards, C.M. 2016. Effect of error and missing data on population structure inference using microsatellite data. bioRxiv.
Bassil, N.V., Bidani, A., Hummer, K.E., Rowland, L.J., Olmstead, J., Richards, C.M., Lyrene, P. 2017. Assessing genetic diversity of wild southeastern North American Vaccinium species using microsatellite markers. Genetic Resources and Crop Evolution. 65(3):939-950.
Gaggiotti, O., Chao, A., Peres-Neto, P., Chiu, C., Edwards, C., Fortin, M., Jost, L., Richards, C.M., Selkoe, K. 2018. Diversity from genes to ecosystems: A unifying framework to study variation across biological metrics and scales. Evolutionary Applications. 11:1176-1193.