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ARS Home » Pacific West Area » Pullman, Washington » Plant Germplasm Introduction and Testing Research » Research » Research Project #425169

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

Location: Plant Germplasm Introduction and Testing Research

2017 Annual Report

The overall goal of this project is to develop improved alfalfa breeding strategies, germplasm, and molecular tools to enhance resistance to disease and abiotic stress. The desired outcomes are molecular markers and high throughput strategies that can be used in marker-assisted breeding to develop improved alfalfa varieties with resistance to disease and abiotic stress to increase alfalfa production and reduce costs. To achieve the long-term goal, the research for the next 5 years will focus on two objectives: Objective 1: Identify molecular markers in alfalfa associated with resistance to, Ditylenchus dipsaci (alfalfa stem nematode), and Verticillium albo-atrum (Verticillium wilt). Objective 2: Identify alfalfa molecular markers and germplasm associated with drought tolerance and increased water use efficiency, as evaluated by biomass yield under a deficit irrigation gradient.

Objective 1: Identify molecular markers in alfalfa associated with resistance to, Ditylenchus dipsaci (alfalfa stem nematode), and Verticillium albo-atrum (Verticillium wilt). Approach 1: Two segregating populations were used for mapping pest resistance loci. Penotyping will be conducted by industrial collaborators. Genotyping and will be conducted by ARS-Prosser using GBS. Raw sequence data will be filtered to remove sequencing errors. The filtered sequence reads will be aligned to M. truncatula genome. The sequence tags and SNPs will be identified using the UNEAK pipeline. GBS tags will be mapped and HapMap containing SNP sites will be used for genome-wide association analysis using TASSEL. Linkage disequilibrium (LD) between markers will be assessed by calculation of r2 between markers, Significant SNP markers linked to the resistance loci identified will be validated in various breeding populations provided by the collaborators. Contingencies: If the development of a mapping population for SN fails, we will use breeding lines segregating for resistance to SN for BSA. This will allow us to identify the resistance trait. If the marker cosegregates between the resistant and susceptible lines, validation will then be expanded to various breeding populations and varieties as described. Objective 2: Identify alfalfa molecular markers and germplasm associated with drought tolerance and increased water use efficiency, as evaluated by biomass yield under a deficit irrigation gradient. Approach 2: Two hundred alfalfa accessions with potential drought tolerance were selected will be used for screening drought tolerance. A split plot design will be used with three irrigation treatments as main plot treatments. Since field conditions are difficult to control, a highly controlled greenhouse assay would be used for selecting germplasm for drought tolerance and improved WUE. We will plant a second set of the same accessions in the USDA-ARS greenhouse in Prosser and it will be used for phenotyping traits associated with drought tolerance and improved WUE. In the first phase, we will develop a greenhouse protocol for measuring water usage and biomass. As transpiration efficiency (TE) refers to the amount of biomass produced per unit water transpired, it would be practicable to measure TE for plants grown in pots. An imaging system will be used for monitoring plant growth and biomass development, which can be performed non-destructively several times a week. Agronomic and physiological traits including biomass, root characteristics, flowering time, relative leaf water content and osmotic adjustment are highly correlated with drought tolerance and will also be measured in the mapping population. Similar genotyping and mapping strategies used in Objective 1 will be used for identifying QTL and linked markers associated with drought resistance and enhanced WUE. Contingencies: If the development of mapping population for drought tolerance fails, we will use breeding populations composited of 26 half-sib families developed at the USDA-ARS, Logan, UT for mapping of QTLs associated with drought tolerance and enhanced WUE using similar strategies as described

Progress Report
Progress was made on both objectives and their sub-objectives, under National Program 215, Pasture, Forage, and Rangeland System. This project focuses on Problem Statement C: 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. Since marker-assisted selection (MAS) can greatly expedite the process of crop genetic improvement, research is focused on identifying DNA markers associated with resistance to diseases and drought/salt stresses for using in alfalfa MAS. Verticillium wilt (VW) is one of the most serious diseases of alfalfa worldwide. Two alfalfa populations were used for mapping the disease resistance genes. Markers associated with VW were identified by Trait Analysis by aSSociation, Evolution and Linkage (TASSEL) software using the mixed linear models. Eleven markers were significantly associated with VW resistance and were located on three chromosomal regions in the alfalfa genome. Six significant markers on chromosome 8 could explain 40% of the total phenotypic variation and represent novel loci associated with VW resistance. Additional markers associated with VW resistance were identified on chromosomes 2 and 7, and co-located with regions of VW resistance loci reported in barrel medic, a distantly related wild relative of alfalfa with a small genome and used as a model plant in forage legume genomic research. This study highlights the value of single nucleotide polymorphism (SNP) genotyping to identify disease resistance loci in tetraploid alfalfa. Three research papers have been published in peer review journals. Enhancing drought resistance and water use efficiency of alfalfa is important to meet the challenges of finite available water resources. We developed eight advanced populations in collaboration with scientists from universities and selected from more than 3,000 individual populations evaluated for drought and salt resistance in the field in the dry season of 2016. We collected evaluation data on agronomic, physiological and quality traits as well as an integrated drought resistance index (DRI). These eight new lines had a higher level of drought resistance than the known resistant control and are being used in alfalfa breeding. Our study of marker-trait association also identified genetic loci associated with drought and salt tolerance. Most loci associated with drought and salt resistance in this work overlap with the previously reported quantitative trait loci (QTLs) associated with biomass under drought 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 resistance and improved water use efficiency. The results have been reported at professional conferences and published in peer-reviewed journals. Based on this work, we developed two proposals on developing molecular markers for enhancing resistance to drought and high salinity in alfalfa and they were funded by the National Institute of Food and Agriculture (NIFA) and Alforex Seeds Company. The additional funding will greatly strengthen our research towards developing tools and germplasm for enhancing the resistance to abiotic stresses in alfalfa crop.

1. Developed drought tolerant alfalfa lines and identified molecular markers associated drought tolerance. Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. An ARS scientist in Prosser, Washington, phenotyped and genotyped a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces. Marker-trait association identified 19 and 15 loci associated with drought resistance index (DRI) and relative leaf water content (RWC), respectively. Eight resistant alfalfa lines resulting from this project have been transferred to Alforex Seed Company under an ARS material transfer agreement (MTA) as breeding materials for developing alfalfa cultivars with improved drought/salt resistance and water use efficiency.

2. Identified DNA markers associated with Verticillium wilt (VW) resistance in alfalfa. 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, S & W Seed, Forage Genetics International and the Noble Foundation, an ARS scinetist in Prosser, Washington, identified 11 molecular markers associated with VW resistance in two alfalfa populations. The markers 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.

Review Publications
Yu, L. 2017. Identification of single-nucleotide polymorphic loci associated with biomass yield under water deficit in alfalfa (Medicago sativa L.) using genome-wide sequencing and association mapping. Frontiers in Plant Science. 8:1152.
Yu, L., Chao, S., Singh, R., Sorrells, M. 2017. Identification and validation of single nucleotide polymorphic markers linked to Ug99 stem rust resistance in spring wheat. PLoS One. doi: 10.1371/journal.pone.0171963.
Liu, X., Yu, L. 2017. Genome-wide association mapping of loci associated with plant growth and forage production under salt stress in alfalfa (Medicago sativa L.). Frontiers in Plant Science. 8:853.
Yu, L., Zheng, P., Bhamidimarri, S., Liu, X., Main, D. 2017. The impact of genotyping-by-sequencing pipelines on SNP discovery and identification of markers associated verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa l.). Frontiers in Plant Science. 8:89.