Location: Plant Germplasm Introduction and Testing Research
2024 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
This is the final report that documents the progress for the life of project 2090-21000-036-000D, "Enhancing Resistance to Biotic and Abiotic Stresses in Alfalfa", which was replaced by new project 2020-21500-001-000D, "Genetic Improvement of Alfalfa for Enhanced Productivity Under Biotic Stress". For additional information, please see the new project report.
In support of Objective 1, research continued to determine the genes and their function associated with resistance to Verticillium wilt (VW) of alfalfa. To understand the genetic basis of resistance in alfalfa to Verticillium wilt, ARS scientists in Prosser, Washington, used DNA markers to identify genes associated with disease resistance. Two genes possibly associated with VW resistance were identified and confirmed using mutants of a relative of alfalfa, M. truncatula, which is widely used as a model plant for genetic studies. The two linked genes associated with VW resistance appear to be resistance (R) genes, and their functions were characterized using yeast-2-hybrid and mutant lines derived from diploid alfalfa M. truncatula. ARS scientists found that one of the R genes functions positively in defense against VW while the other responded negatively. Three DNA markers derived for the promising R gene have been developed and transferred to S&W Seed Inc. for rapid and reliable detection of resistance to VW in alfalfa via marker assisted selection. The markers can be used to identify resistance alleles in parents and progeny in alfalfa. The results were reported at the 2022 Plant and Animal Genome Conference and published in Plant Biotechnology Journal. VW is a devastating soilborne fungal disease that reduces forage yields by up to 50% in the northern U.S. and Canada. The best method for managing the disease is the development and use of disease resistant varieties, which this program is focusing on.
Under Sub-objective 2A, progress was made on developing molecular markers associated with drought tolerance. ARS scientists in Prosser, Washington, in collaboration with scientists from alfalfa breeding companies and universities, tested 200 alfalfa breeding lines in the field for drought resistance.
ARS scientists used genome-wide association studies (GWAS) and genomic selection to improve the breeding processes. The genotypes of the alfalfa breeding lines were determined using more than 10,000 DNA markers. Datasets were analyzed using new methods so each line could be selected based on genotypes associated with drought resistance, an approach generally referred to as “genomic selection”. This genomic selection approach allowed the identification of an ideal group of alfalfa lines (accessions) with high yield under drought conditions that can be used for developing improved varieties. This is the first report in which this new method of data analysis was shown to improve the accuracy of genomic selection, and this approach can be broadly applied to a wide range of other crops and traits. In addition, ARS scientists in Prosser, Washington, in collaboration with ARS scientists in Logan, Utah, and universities, developed a multiparent population using 32 drought resistant parents crossed with elite cultivar, Guardsman II. The F1 progeny were backcrossed with Guardsman II to create F1B1 populations. These populations were tested in the field for drought tolerance with yield (biomass), plant height, and vigor traits measured under drought conditions. Phenotypic traits were analyzed with the same genomic selection marker-based strategy described above. Drought tolerant alfalfa lines with good agronomic potential were selected using a combination of phenotypic traits and genomic selection (GS) models for further population development. In the western United States, the great majority of alfalfa is produced with irrigation, which represents a large part of the total costs for a producer. Identifying alfalfa germplasm that is tolerant to drought and developing markers that makes breeding for drought in alfalfa more effective are both goals of this program.
For Sub-objective 2B, progress was made on developing alfalfa populations with improved salt tolerance. ARS scientists in Prosser, Washington, in collaboration with ARS scientists in Logan, Utah, evaluated traits related to salt tolerance in alfalfa. They used alfalfa breeding populations and identified 49 loci associated with salt tolerance using GWAS. Twenty-one candidate genes underlying the intervals of the resistance loci have been reported to play roles in response to salt stress. The closely linked markers can be used for marker-assisted selection in developing alfalfa with enhanced resistance to high salinity soil. These markers and the prediction model created with this dataset (using methodology similar to the previous section on drought) have been published in peer-reviewed journals and reported at the World Alfalfa Congress and the Crop Science Society of America meetings. The markers and the identification of the best model to use for predicting salt tolerance are useful for more efficient development of new alfalfa varieties with improved salt tolerance. Especially in the western United States, where much of the nation’s alfalfa is grown, high salt concentrations are found in soils and irrigation water, negatively affecting alfalfa growth and production. Identification and incorporation of traits for salt tolerance from germplasm into modern alfalfa cultivars and developing expedited methods for breeding this trait would mitigate the losses experienced in these regions.
Accomplishments
Review Publications
He, X., Zhang, X., Deng, Y., Yang, R., Yu, L., Jia, S., Zhang, T. 2023. Structural reorganization in two alfalfa mitochondrial genome assemblies and mitochondrial evolution in Medicago species. International Journal of Molecular Sciences. 24(24). Article 17334. https://doi.org/10.3390/ijms242417334.
Moya, Y.S., Medina, C., Herrera, B., Chamba, F., Yu, L., Xu, Z., Samac, D.A. 2023. Genetic mapping of tolerance to bacterial stem blight caused by Pseudomonas syringae pv. syringae in alfalfa (Medicago sativa L.). Plants. 13(1). https://doi.org/10.3390/plants13010110.
