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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

2019 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
This project, which began in March of 2019, continues research from 2090-21000-035-00D, “Enhancing Resistance to Diseases and Abiotic Stresses in Alfalfa”. Please see the report of the previous project for additional information. Progress is being made on Objectives 1 and 2, both which fall under National Program 215, Grass, Forage, and Rangeland Agroecosystems. This project focuses on Problem Statement C: the need for greater fundamental understanding of ecological processes and interactions so science-based management practices, technologies, and germplasm can be improved to meet production, conservation, and restoration objectives under changing climatic conditions. Under Objective 1, we made significant progress in identifying molecular markers in alfalfa associated with resistance to Verticillium albo-atrum, more commonly known as Verticillium wilt (VW) of alfalfa, which is a devastating disease which causes forage yield reductions of up to 50 percent in the Northern U.S. and Canada. The best method for controlling the disease is through the development and use of resistant varieties. To understand the genetic base of VW resistance in alfalfa, in the present study, we used a full-sib population segregating for VW resistance for mapping quantitative trait loci (QTL) for the disease resistance. High density linkage maps for both resistant and susceptible parents were constructed using single-dose alleles (SDAs) derived from genotyping by sequencing (GBS) markers. Five QTL associated with VW resistance were mapped in four linkage groups. The QTL (qVW-8C) located on LG 8C contributed a major effect to VW resistance while the rest of the QTL had minor effects. The identification of multiple loci for VW resistance suggests its polygenetic inheritance in alfalfa. Two putative candidates, nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes were identified in the QTL intervals of qVW-6D2 and qVW-8C, respectively. The result agreed with our previous studies where similar resistance loci were identified. The results provide insight into the mechanistic basis of VW resistance in alfalfa. The closely linked markers or candidate genes identified in the present and previous studies can be used for marker-assisted selection to develop alfalfa varieties with enhanced resistance to the disease when they are validated. Validation of the marker linked to these genes has shown co-segregation with the VW resistance allele in alfalfa populations. We collaborated with a seed company for testing the marker’s robusticity in a broad-range of alfalfa populations. We reported this finding at the International Forage and Turf Breeding Conference in Lake Buena Vista, Florida, March 24-27, 2019. Under Objective 2, significant progress for identifying deoxyribonucleic acid (DNA) markers associated with resistance to drought and high salinity was made as these are two important factors affecting alfalfa production worldwide. Enhancing alfalfa resistance to drought and high salinity is important to meet the challenges of finite available water resources and increased saline soil. In collaboration with scientists from ARS, industry, and universities, we developed advanced alfalfa populations and evaluated for drought and salt resistance in greenhouse and field trials. Molecular markers and agronomic, physiological and quality traits were analyzed using statistic models. Marker-trait association identified a group of genetic loci associated with drought and salt tolerance. Most loci associated with drought and salt resistance in this work overlap with the previously reported QTL associated with biomass under drought and high salinity in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. A Basic Local Alignment Search Tool (BLAST) search (using their flanking sequences) revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be useful for marker-assisted breeding new alfalfa varieties with drought and salt resistance and improved water use efficiency. The results have been reported at professional conferences and published in peer-reviewed journals.

1. Identified deoxyribonucleic acid (DNA) markers and genes associated with Verticillium wilt (VW) resistance. VW is an alfalfa disease that reduces forage yields up to 50 percent. Current breeding strategies rely greatly on phenotypic recurrent selection that allows slow and inefficient genetic improvement progress. In collaboration with Alforex Seeds, an ARS scientist in Prosser, Washington, identified five quantitative trait loci associated with VW resistance in a biparental alfalfa populations. The markers and genes identified in this study could be used for improving resistance to VW in alfalfa breeding programs. Alfalfa seed companies are interested in using these markers in their breeding programs.

2. Developed backcross populations for drought tolerance in alfalfa to identify molecular markers associated with drought tolerance. Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. In collaboration with ARS scientists and researchers at Washington State University, an ARS scientist in Prosser, Washington, developed backcross populations for drought tolerance. The population contains more than 1000 lines and can be used for mapping genes associated with drought tolerance. These lines can also serve as breeding materials for developing alfalfa cultivars with improved drought resistance and water use efficiency.

Review Publications
Zhang, F., Kang, J., Long, R., Yu, L., Wang, Z., Zhao, Z., Zhang, T., Yang, Q. 2019. High-density linkage map construction and mapping QTLs for yield and yield components in autotetraploid alfalfa using RAD-seq. Biomed Central (BMC) Plant Biology. 19(165):1-12.
Liu, X., Hawkins, C., Peel, M., Yu, L. 2019. Genetic loci associated with salt tolerance in advanced breeding populations of tetraploid alfalfa using genome-wide association studies. The Plant Genome. 12:180026.