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

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2017 Annual Report


Objectives
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.


Approach
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
(Objectives 1 and 4) Plant Germplasm Preservation Research Unit scientists are actively engaged with different groups to develop methods to use genomic and environmental data to guide collections of crop wild relatives (CWR) and find the useful genes in plants with “weedy” phenotypes. Geographic information is frequently used as a proxy for genetic diversity of wild populations; however, we have shown that genetic diversity does not correlate well with geographic distance. Wild populations are usually collected as they are discovered. For this reason, PGPRU has shifted focus from predicting where to find novel diversity in the wild to where to find useful genes within the collection. Developing algorithms and approaches that help to prioritize accessions for curation and cryopreservation continues to be a major focus. (Objective 2) Major advances in our abilities to cryopreserve germplasm are exemplified for diverse species and tissues. Concerted efforts were applied in an important collaboration among three Units to back-up citrus shoot tips of disease-indexed plants in the Riverside, California repository. About 85% of the processed materials met viability standards of 40% or more, based on normal growth after a micrografting technique introduced by PGPRU. Capacity to back-up citrus in field collection using seeds and pollen is progressing by characterizing tolerance to desiccation and cryo-exposure of diverse species. PGPRU continues work to identify germplasm that stores poorly under freezer conditions and to develop methods that increase survival in cryogenic storage through optimizing moisture, cryoprotectant concentration, and cooling and warming rates to and from, respectively, liquid nitrogen temperatures. (Objective 3) This objective is designed to evaluate changes to germplasm as a result of the genebanking process. The ability to quantitatively detect the effects of dry aging using an RNA integrity assay will make viability monitoring activities more efficient. These assays also distinguish between seeds that are dormant (high RNA integrity) and have died during storage (low RNA integrity). Controlled pollination among parent apple trees selected for maximum diversity captures diversity of wild populations in seed germplasm that maintains viability with minimum manipulation. Genetic erosion in the wild as a consequence of climate change was addressed in wild barley in a large international project. Rather than an attrition of diversity, these populations appeared to “share” genes that had been parsed into separate populations 30 years ago.


Accomplishments
1. Cryopreserved back-up of citrus genetic resources. The USDA citrus collection in Riverside, California is vulnerable to pathogens, pests and inclement weather. To protect this priceless collection, a collaborative effort led by ARS scientists in Fort Collins, Colorado focused on cryopreserving shoot tips from over 350 accessions using new technologies. Based on this effort, about 80% of greenhouse-grown, disease-free citrus genetic resources are safely backed up at the National Laboratory for Genetic Resources Preservation in Fort Collins. This project serves as a model for how to safely duplicate collections of other clonally-propagated crops in the U.S. and worldwide. Now, genes needed for crop improvement are safe and available in perpetuity, ensuring the sustainability of the citrus industry in the U.S.

2. Efficient marker for seed aging. Monitoring seed viability during storage is an essential curation activity for plant genetic resource collections, but it is labor-intensive, consumes valuable germplasm, and does not predict when seeds begin to die. ARS scientists in Fort Collins, Colorado have introduced a new method to detect seed aging that measures the integrity of RNA within seed cells. The assay has the potential for automation, is highly quantitative, closely corresponds to germination potential and consumes about 1/10 of the seeds used during germination tests. Implementing this technology will ensure that early effects of deterioration will be detected in seed collections at the National Laboratory for Genetic Resources Preservation with a reduced labor force and with minimal consumption of seeds.

3. Archived collections reveal wild plant responses to climate. How do wild plant populations respond to changing climates? Comparisons between genetic diversity of contemporary populations with genebanked samples can reveal plant adaptations. ARS scientists in Fort Collins, Colorado showed that wild barley from Jordan adjusted to intensifying heat and drought over a 30 year period by greater mixing of genes among populations, resulting in an overall increase in within-population diversity. This means that genetic diversity of collections of crop wild relatives may be represented by fewer populations, reducing the overall number of hard-to-curate accessions in the genebank. In addition, the discovery that wild plants tend to mix genes among populations in response to stress contrasts with breeders’ methods to consolidate genes for tolerance and water use efficiency and supports land manager efforts to restore U.S. lands using seed mixes containing different ecotypes.

4. Capturing genetic diversity in wild apple collections through planned pollinations. A current paradigm is that genetic diversity of clonal crops must be preserved by vegetative propagation, particularly for cultivars. However, labor required to process germplasm from wild accessions of clonally-propagated crops can be reduced to approximately 1/15th by using seeds as the germplasm form. ARS scientists in Fort Collins, Colorado demonstrated that genetic diversity of wild-collected accessions can be fully captured by making planned crosses and collecting seeds. This strategy can be applied to nearly 1/3 of the accessions in clonal collections and will significantly decrease the backlog for safety duplication of these invaluable genetic resources. This directly benefits U.S. and International fruit and nut producers by providing security of genetic resources for their crops, achieved at lower cost and greater speed.


Review Publications
Gross, B.L., Henk, A.D., Bonnart, R.M., Volk, G.M. 2016. Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips. Plant Cell Reports. 36:459-470. doi:10.1007/s00299-016-2095-7.
Volk, G.M., Henk, A.D., Forsline, P.L., Szewc-McFadden, A.K., Fazio, G., Aldwinckle, H., Richards, C.M. 2016. Seeds capture the diversity of genetic resource collections of Malus sieversii maintained in an orchard. Genetic Resources and Crop Evolution. doi:10.1007/s10722-016-0450-8.
Volk, G.M., Henk, A.D., Jenderek, M.M., Richards, C.M. 2016. Probabilistic viability calculations for cryopreserving vegetatively propagated collections in genebanks. Genetic Resources and Crop Evolution. doi:10.1007/S10722-016-0460-6.
Volk, G.M., Bonnart, R.M., Shepherd, A.N., Yin, Z., Lee, R.F., Polek, M., Krueger, R. 2016. Citrus cyopreservation: viability of diverse taxa and histological observations. Plant Cell Tissue and Organ Culture. doi:10.1007/211240-016-1112-4.
Zhang, J., Han, L., Lu, X., Volk, G.M., Xin, X., Yin, G., He, J., Wang, L., Chen, X. 2016. Improved droplet-vitrification and histological studies of cryopreserved shoot tips of cultivated Jerusalem artichoke genotypes. Plant Cell Tissue and Organ Culture. 128:327-334.
Fleming, M.B., Richards, C.M., Walters, C.T. 2017. Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential. Journal of Experimental Botany. 68: 2219-2230. doi: 10.1093/jxb/erx100.
Walters, C.T., Crane, J., Hill, L.M., Michalak, M., Carstens, J.D., Conrad, K.P. 2016. Preserving oak (Quercus sp.) germplasm to promote ex situ conservation. International Oaks. 27:255-266..
Sebastian, D.J., Fleming, M.B., Patterson, E.L., Sebastian, J.R., Nissen, S.J. 2017. Indaziflam: A new cellulose biosynthesis inhibiting herbicide provides long-term control of invasive winter annual grasses. Pest Management Science. doi:10.1002/ps.4594.
Ballesteros, D., Hill, L.M., Walters, C.T. 2017. Variation of desiccation tolerance and longevity in fern spores. Journal of Plant Physiology. 211:53-62 doi.org/10.1016/j.jplph.2017.01.003.
Tembrock, L., Simmons, M., Richards, C.M., Reeves, P.A., Reilley, A.A., Curto, M.A., Meimberg, H., Ngugi, G., Demissew, S., Wali Al Khulaidi, A., Mansoor, A., Simpson, S.A., Varisco, D. 2017. Phylogeography of the wild and cultivated stimulant plant qat (Catha edulis, Celastraceae) in areas of historic cultivation1. American Journal of Botany. 104: 538-549.
Volk, G.M. 2017. Ensuring the genetic diversity of apples. In: Evans, K.Achieving sustainable cultivation of apples. Burleigh Dodds Science Publishing,Cambridge, UK. p.3-21.
Volk, G.M., Samarina, L.K., Kulyan, R., Vyacheslav, G., Malyarovskaya, V., Ryndin, A., Polek, M., Krueger, R., Stover, E.W. 2017. Citrus genebank collections: International collaboration opportunities between the U.S. and Russia. Genetic Resources and Crop Evolution. 65:433-447. https://doi.org/10.1007/s10722-017-0543-z.