Location: Plant Germplasm Introduction and Testing Research
2022 Annual Report
Objectives
Objective 1: Identify DNA markers associated with resistance to soil borne diseases in alfalfa to clearly define the genetic basis of resistance to disease and accelerate breeding programs. (NP215 2A)
Objective 2: Identify alfalfa DNA markers and germplasm associated with drought and salt tolerance to clearly define the genetic basis of resistance to these stressors and accelerate breeding programs. (NP215 2A).
Approach
Approach 1: Marker-assisted selection for disease resistance will increase selection accuracy and reduce selection cycles in alfalfa breeding programs. First, genome-wide association mapping will be used to identify loci associated with VW resistance. Then, genetic regions responsible for VW resistance will be sequenced and compared among different genotypes using haplotyping and comparative genomics approaches. Significant SNP markers linked to VW resistance loci will be validated in various breeding populations provided by collaborators. High throughput platforms such as Kompetitive Allele Specific PCR (KASP) (www.lgcgenomics.com) or Taqman (www.thermofisher.com) assays will be used to test the cosegregation of marker loci and disease resistance scores. Flanking sequences for the significant SNP markers will be used for designing specific primers for array-based genotyping platforms (KASP or Taqman). Multiplex primer combinations will be used for evaluating the resistance locus or candidate gene, and all markers will be scored in a given genotype. Single markers with two character states will be tested for significant phenotypic differences between genotype groups by the t test for each trait, and Mann–Whitney U test for chip quality. Marker combinations will be analyzed using analysis of variance (ANOVA) for each trait, and Kruskal–Wallis test for chip quality. Statistical analyses will use SAS software (SAS Institute Inc. 2011, SAS OnlineDoc 9.3, Cary, NC, USA).
Approach 2: Breeding for abiotic stress tolerance is challenged by genotype x environment interactions (G x E). Genomic selection provides greater gain and increased selection accuracy than conventional breeding. To develop a genome-wide marker platform and statistical models for genomic selection of drought tolerant alfalfa. BC1 populations have been developed and will be screened for drought tolerance. Selected plants will be randomly intermated in the greenhouse in order to generate an elite base population. The population will used for associated mapping and genomic selection for alleles that affect drought tolerance, salt tolerance, forage quality and other economical traits. We will test statistic models by using the majority of the training population to create a prediction model, which is then used to predict a Genomic Estimated Breeding Value (GEBV) for each of the remaining individuals in the training population based only on their genotype data. Once validated, the model can then be applied to a breeding population to calculate GEBVs of each individual based only on a plant’s genotype information. Such GEBVs represent the overall predicted value of an individual as a potential parent for crossing.
Progress Report
In support of Objective 1, research continued on determining the function of genes associated with resistance to Verticillium wilt (VW) of alfalfa, which is a devastating disease that reduces forage yields by up to 50% in the northern United States and Canada. The best method for controlling the disease is to develop and grow resistant varieties. To understand the genetic basis of resistance in alfalfa to Verticillium wilt an ARS scientist in Prosser, Washington, used DNA markers to identify genes associated in disease resistance. Two linked R genes associated with VW resistance have been identified and their functions were characterized using yeast-2-hybrid and mutants derived from diploid alfalfa M. truncatula. We found that one of the R genes identified function positively in defense against VW while another responded negatively. The results have been reported in the 2022 Plant and Animal Genome Conference and published in Plant Biotechnology Journal.
In support of Sub-objective 2A, progress was made on developing molecular markers associated with drought resistance. In the western United States, the great majority of alfalfa is produced with supplemental irrigation water, which represents a large part of the total costs for a producer. An ARS scientist in Prosser, Washington, in collaboration with scientists from alfalfa seed companies and universities, tested 600 alfalfa breeding lines in the field for drought resistance. Biomass yield, plant height, and growth vigor under drought were measured and analyzed with statistic models for phenotypic variation in the populations. Alfalfa lines with resistance to drought were selected and used as breeding materials for developing new alfalfa varieties with enhanced resistance to drought.
In support of Sub-objective 2B, progress was made on developing molecular markers associated with salt resistance. Many agricultural lands in the western United States are composed of soil with high concentrations of salt, which are detrimental to alfalfa survival and production, especially under limited water conditions. Developing salt tolerant varieties is imperative for sustainable alfalfa production with increasing soil salinity. An ARS scientist in Prosser, Washington, screened 200 alfalfa germplasm accessions and identified a group of salt tolerant lines for developing cultivars with enhanced resistance to high salinity soil. Additionally, in collaboration with another ARS scientist, the ARS scientist in Prosser, Washington, used alfalfa breeding populations and identified 49 loci associated with salt tolerance using a genome-wide association study (GWAS). Twenty-one candidate genes underlying the intervals of the resistance loci have been reported to play roles in response to salt stress.
Accomplishments
1. DNA markers developed for Verticillium wilt resistance in alfalfa. Verticillium wilt (VW) is an alfalfa disease that reduces forage yields by up to 50 percent. Current breeding strategies primarily rely on field or greenhouse screening to identify disease resistant plants, which is a time-consuming process requiring specific conditions to produce reliable results. An ARS scientist in Prosser, Washington, identified VW resistance genes and developed high-throughput DNA markers that accurately identify alfalfa plants that are resistant to VW. Three DNA markers have been transferred to a major alfalfa commercial producer to accelerate breeding efforts to develop new varieties with enhanced resistance to Verticillium wilt.
2. Selection of alfalfa lines with drought tolerance for pre-breeding. In the western United States, the great majority of alfalfa is produced with supplemental irrigation water, which represents a large part of the total costs for a producer. An ARS scientist in Prosser, Washington, in collaboration with scientists from alfalfa seed companies and universities, tested 600 breeding lines in the field for drought tolerance. Drought resistance lines were selected, and seeds were transferred to a major seed company and ARS Western Regional Plant Introduction Station for distribution.
3. Selection of alfalfa lines with salt tolerance for pre-breeding. In the western United States, the impacts of soil salinization on alfalfa production will become more pervasive and severe in the future. An ARS scientist in Prosser, Washington, in collaboration with another ARS scientist in Logan, Utah, tested 400 alfalfa breeding lines in the field for salt resistance. Biomass yield under salt stress was measured and analyzed with statistic models for phenotypic variation in the populations. Alfalfa lines with salt resistance have been selected and can be used as pre-breeding materials for developing new varieties with improved resistance to high salinity soil.
Review Publications
Lin, S., Niu, Y., Medina, C., Yu, L. 2022. Two linked resistance genes function divergently in defence against Verticillium wilt in alfalfa. Plant Biotechnology Journal. 20(4):619-621. https://doi.org/10.1111/pbi.13779.
Medina, C., Samac, D.A., Yu, L. 2021. Pan-transcriptome identifying master genes and regulation network in response to drought and salt stresses in alfalfa (Medicago sativa L.). Scientific Reports. 11. Article 17203. https://doi.org/10.1038/s41598-021-96712-x.
Medina, C., Kaur, H., Ray, I., Yu, L. 2021. Strategies to increase prediction accuracy in genomic selection of complex traits in alfalfa (Medicago sativa L.). Cells. 10(12). Article 3372. https://doi.org/10.3390/cells10123372.
