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United States Department of Agriculture

Agricultural Research Service

Research Project: RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS
2011 Annual Report


1a.Objectives (from AD-416)
Objective 1: Develop physiological and biophysical approaches and tools to assess changes in plant germplasm viability and the potential causes during genebank preservation. Objective 2: Develop statistical genetic strategies and tools to sample and preserve plant genetic diversity in genebank collections and in situ reserves.


1b.Approach (from AD-416)
The Preservation of Plant Genetic Diversity in Ex Situ Genebank program scientists conduct research to improve the biological and genetic integrity of genebanked germplasm and to standardize procedures for handling accessions and reporting associated data. Interrelated research goals will allow curators to preserve viability of conservation targets (Objective.
1)and rationalize and validate the genetic diversity and integrity of those targets (Objective 2). Using taxa that are empirically tractable systems, we will • define tolerances to preservation stresses of selected propagules, • develop methods to improve survival or reliably predict loss of viability over time, • model the effects of mortality and regeneration on genetic composition, and • develop sampling strategies for wild-collected germplasm that maximize genetic diversity while minimizing curator inputs for storage and regeneration.

A central theme is identifying appropriate conservation targets that capture desired genetic diversity, remain viable during storage and are available to the user when needed. A conservation target is a group of propagules (such as seeds or pollens) or an individual propagule (such as an explant) that comprises an accession valued for specific genes, genetic richness (number and frequency of alleles) or an allelic combination (genotype). PGPRU scientists and their collaborators will investigate major conceptual issues of repository biology and standardization using within-unit expertise in biophysics, plant physiology, cell and molecular biology and population genetics and National Plant Germplasm System (NPGS) curators’ expertise on reproductive biology, phenotypic diversity, history and cultivation of their assigned collections. Our central position within NPGS allows us to develop protocols and predictive tools that are applicable to a wide variety of species and propagules.


3.Progress Report
Interrelated research allows curators to preserve viability of conservation targets and rationalize and validate the genetic diversity and integrity of those targets. Survival of germplasm exposed to cryostorage conditions becomes increasingly possible through research on mechanisms of damage and protection of cells, tissues and organs. Genotypes vary significantly in response to stress, protection and recovery media and this variation requires considerable effort to optimize preservation procedures for individual accessions. Understanding the basis for different responses among plant cells will lead to greater efficiency in cryopreservation efforts. Major advances in developing recovery conditions for diverse citrus and avocado shoot tips has helped to streamline assessments of cryopreservation procedures. Efforts continue to describe the early stage of seed aging that is currently regarded as “asymptomatic.” Kinetics of aging and other reactions that occur under dry conditions, such as dormancy breaking and color changes, are correlated with biophysical measurements related to molecular mobility and phase behavior and biochemical changes such as emission of volatiles and degradation products of lipids, proteins and nucleic acids. Moisture and temperature effects of these reactions are used to predict the duration of the asymptomatic phase. Aging of preserved inventories is inevitable and older inventories must be regenerated. Both the aging and regeneration process can lead to loss of genetic identity of inventories. Population genetic tools are an essential component of NCGRP research to link efficiency and accountability to preservation methods. The extent and mechanisms of genetic change during genebanking are studied by changes in quantitative traits and genomic analysis of neutral markers and expressed sequences. Consequences of seed aging and regeneration have been modeled, and these models are now being tested with inventory grow outs. The expected product is an assessment of the degree to which random changes and directed selection affect genetic integrity of genebanked samples and the time period in which these changes are expected to occur. This assessment of the effectiveness of genebanking is critical to accommodate the increased attention of wild-collected accessions. As collections become larger and larger, it becomes increasingly important to develop tools to express, quantify, distill and communicate the genetic diversity of NPGS collections. These tools necessarily require integration of genetic and geospatial data. Advanced methods in statistical genetic structure estimation and GIS mapping play a critical role in visualizing patterns of diversity and help locate potential gaps in collections.


4.Accomplishments
1. Strategies to build core collections. A core collection distills a large collection into fewer accessions, providing a ‘snapshot’ of the total diversity and complexity of the larger collection. Usually only a single core is developed, even for large collections, and there is no way to know whether this single snapshot provides the perspective of the larger collection needed to efficiently access genes of interest. ARS researchers in Fort Collins, CO evaluated different strategies for making core collections using the Beta nana collection as an exemplar. The research shows that different subsetting rules highlight different aspects of genetic diversity and that a single physical core collection should be replaced with flexible organization of genetic diversity to meet user needs. These subsetting tools and methods to relate genetic diversity of cores to total diversity within the collections are now available. The work will enhance curators’ abilities to describe the complexity of their collections and to identify subsets of accessions likely to have desired properties. Ultimately, this will lead to targeted use of germplasm collections.

2. Recovery metabolism after cryopreservation. ARS researchers in Fort Collins, CO provided the first report of how plant shoot tips recover from cryopreservation treatments. Gene expression patterns reveal cells that were stressed by desiccation. Surviving cells have the capacity to express stress-related genes such as heat shock proteins, antioxidants, dehydrins and other housekeeping genes. This research contributes to the understanding of variation in response among gentopyes to cryopreservation treatments as well as therapeutic treatments for recovering germplasm.

3. Using structural mechanics to evaluate seed quality. Engineering tools that are commonly used to explain performance of thermal plastics and polymers were applied to seeds. ARS researchers in Fort Collins, CO used dynamic mechanical analysis (DMA) to quantify the effects of moisture and temperature on structural stability of seeds. The work contributes to a growing cadre of tools developed by ARS researchers in Fort Collins that enable seed biologists to directly probe life in a dry seed. The work demonstrates that characteristics of the seed change as they are dried and so processes that affect seed quality (such as deterioration or dormancy release) are best understood by probing the dry seed. DMA is expected to lead to assays that predict seed longevity during storage in genebanks and warehouses and that identify genetic and environmental factors that contribute to variation in seed quality among seed lots.


Review Publications
Reeves, P.A., Richards, C.M. 2011. Species delimitation under the general lineage concept: An empirical example using wild North American hops (cannabaceae: Humulus lupulus). Systematic Biology. 60:45-59.

Gross, B.L. 2011. MADS-box out of the black box. Molecular Ecology. 20:25-26.

Gross, B.L., Strasburg, J.L. 2010. Cotton domestication: Dramatic changes in a single cell. BioMed Central Biology. 8:137-139.

Volk, G.M. 2010. Advantages for the use of standardized phenotyping in databases. HortScience 45:1310-1313.

Bassil, N.V., Volk, G.M. 2010. Standardized Phenotyping: Advantages to Horticulture, Introduction to the Workshop. HortScience. 45(9):1306.

Richards, C.M., Lockwood, D.R., Volk, G.M., Walters, C.T. 2010. Modeling demographics and genetics in ex situ collections during seed storage and regeneration. Crop Science. 50:2440-2447.

Walters, C.T., Ballesteros, D., Vertucci, V. 2011. Structural mechanics of seed deterioration: Standing the test of time. Plant Science. 179:565-573.

Njuguna, W., Hummer, K.E., Richards, C.M., Davis, T.M., Bassil, N.V. 2011. Genetic diversity of diploid Japanese strawberry species based on microsatellite markers. Genetic Resources and Crop Evolution. http://www.springerlink.com/content/c404m461448gp6r3. 58:1187-1198.

Ballesteros, D., Estrelles, E., Walters, C.T., Ibars, A.M. 2011. Effect of temperature on green spore longevity for the ferns Equisetum ramosissimum and Osmunda regalis. CryoLetters. 32:89-98.

Thorp, K.R., Dierig, D.A., French, A.N., Hunsaker, D.J. 2010. Analysis of hyperspectral reflectance data for monitoring growth and development of lesquerella. Industrial Crops and Products. 33(2):524-531.

Postman, J.D., Volk, G.M., Aldwinckle, H. 2010. Standardized plant disease evaluations will enhance resistance gene discovery. HortScience. 45(9):1317-1320.

Mira, S., Gonzalez-Benito, E., Hill, L.M., Walters, C.T. 2010. Characterization of volatile production during storage of lettuce (Lactuca sativa) seed. Journal of Experimental Botany. 61:3915-3924.

Last Modified: 10/24/2014
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