Skip to main content
ARS Home » Pacific West Area » Pullman, Washington » Plant Germplasm Introduction and Testing Research » Research » Research Project #436020

Research Project: Enhancing Resistance to Biotic and Abiotic Stresses in Alfalfa

Location: Plant Germplasm Introduction and Testing Research

2020 Annual Report

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 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) ( or Taqman ( 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
Considerable progress was made on the two research objectives, both of which address Problem Statement 2A (Plant resilience and resistance to stressors) of Component 2 (Improve the physiology and genetics of plant materials to enhance health, vitality, and utility of pasture, biomass for feed and fuel, rangeland, and turf systems) of the National Program 215, Grass, Forage, and Rangeland Agroecosystems, Action Plan (2019-2023). Under Objective 1, progress was made on determining the function of genes associated with resistance to Verticillium wilt 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. Databases of DNA sequences were then examined to try to predict the function of the genes associated with disease resistance in alfalfa. We found that some of the genes identified may function to either detect the fungal pathogen or start other defense responses. The results have been reported at the American Societies of Agronomy, Crop Science and Soil Science Annual Meeting in San Antonio, Texas, November 7-11, 2019 and published in the peer-reviewed journal Plant Disease. Under Sub-objective 2A, progress was made on developing alfalfa varieties with improved 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 a diverse collection of alfalfa varieties in the field for drought resistance. Several varieties were discovered to be relatively resistant to drought and plants from these varieties are being used as parents to develop new varieties with enhanced drought resistance. These new varieties will contribute to more sustainable and profitable alfalfa production. Under Sub-objective 2B, progress was made on developing alfalfa populations with improved salt tolerance. Many agricultural lands in the western United States have high soil salt concentrations that are detrimental to alfalfa growth and production. Developing alfalfa varieties with salt tolerance is critical for sustainable production under increasing soil salinity. An ARS scientist in Prosser, Washington, in collaboration with another scientist in Logan Utah, evaluated agronomic and physiological traits related to salt tolerance in alfalfa. Genetic studies identified 49 DNA markers associated with salt tolerance. The markers will be useful for marker-assisted selection in breeding alfalfa cultivars with improved salt tolerance.

1. Identified candidate genes associated with Verticillium wilt resistance in alfalfa. Verticillium wilt 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, developed a set of DNA markers and identified two different markers that were reliably associated with resistance to Verticillium wilt. The markers were used to make a rapid polymerase chain reaction (PCR) test that can identify alfalfa seedlings that have disease resistance genes. This new test accelerates the development of improved varieties by making it easier for breeders to identify resistant plants because they can be detected as seedlings without having to conduct disease screening tests in the field or greenhouse.

Review Publications
Yu, L., Zhang, F., Culma, C., Lin, S., Niu, Y., Zhang, T., Yang, Q., Smith, M., Hu, J. 2020. Construction of high-density linkage maps and identification of quantitative trait loci associated with verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa L.). Plant Disease. 104(5):1439-1444.
He, F., Kang, J., Zhang, F., Long, R., Yu, L., Wang, Z., Zhao, Z., Zhang, T., Yang, Q. 2019. Genetic mapping of leaf-related traits in autotetraploid alfalfa (Medicago sativa L.). Molecular Breeding. 39.
Gul, A., Diepenbock, C., Munkvold, J., Paterson, A., Kucek, L.K., Souze, E., La Rota, M., Yu, L. 2019. Mark E. Sorrells: Plant breeder, geneticist, innovator, mentor. In: Goldman, I., editor. Plant Breeding Reviews, Volume 42. Hoboken, NJ: John Wiley & Sons, Inc. p. 1-38.
Lin, S., Medina, C., Boge, B., Hu, J., Fransen, S., Norberg, S., Yu, L. 2020. Identification of genetic loci associated with forage quality in response to water deficit in autotetraploid alfalfa (Medicago sativa L.). Biomed Central (BMC) Plant Biology. 20.