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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

2018 Annual Report


Objectives
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.


Approach
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
This is the final report for this project which reached its five-year maximum term in March of 2018. It has been replaced by a bridging project, 2090-21000-035-00D, "Enhancing Resistance to Diseases and Abiotic Stresses in Alfalfa", while the new five year project plan undergoes Office of Scientific Quality Review. Please see the report for the bridging project for additional information. Progress was made on both objectives and their sub-objectives, under National Program 215, Pasture, Forage, and Rangeland Systems. 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. 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 marker-trait association using the mixed linear models. Eleven markers were significantly associated with VW resistance in the Forage Genetics International (FGI) population and they were located on three chromosomal regions in the alfalfa genome. Six significant markers on chromosome 8 explain 40 percent of the total phenotypic variation and represent novel loci associated with VW resistance. Additional markers associated with VW resistance were also identified on chromosomes 2 and 7, which were co-located with regions of VW resistance loci reported in barrel medic, a distantly related wild relative of alfalfa. Most significant markers associated with VW in the pioneer population were located on chromosome 6. We identified a group of putative candidate genes associated with VW resistance in alfalfa populations. One of these genes encodes an adenosine triphosphate (ATP), ATP binding cassette (ABC) transporter. The function of ABC transporters in pathogen resistance has been reported in plants. Validation of the marker linked to this gene has shown co-segregation with the VW resistance allele in alfalfa populations. Validations of other markers associated with other resistance genes such as nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes are in progress. After validation, these markers can be used for marker-assisted selection for breeding alfalfa cultivars with improved resistance to the disease. We reported this finding in the North American Alfalfa Improvement Conference, Logan, Utah, June 2018. Drought and high salinity are two important factors affecting alfalfa production worldwide. Enhancing alfalfa resistance to drought and high salinity is important to meet the challenges of finite water availability and increasingly saline soil. An ARS scientist in Prosser, Washington, developed advanced alfalfa populations in collaboration with scientists from ARS, industry and universities. They have been evaluating for drought and salt resistance in both greenhouse and field plots. Agronomic, physiological and quality traits data have been collected. Of these evaluated, eight new lines had a higher level of drought resistance than the known resistant control and are being used in alfalfa breeding in the Alforex Seeds. The same populations have been genotyped using genotyping by sequencing (GBS) and more than 10 thousand markers have been obtained and used for genome-wide association study (GWAS). 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 quantitative trait loci (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/salt resistance and improved water use efficiency.


Accomplishments
1. 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 only allows for slow and inefficient genetic improvement. In collaboration with S & W Seed Company, an ARS researcher in Prosser, Washington, identified a group of markers and putative candidate functional genes associated with Verticillium wilt resistance. One of these genes encodes an adenosine triphosphate (ATP), ATP binding cassette (ABC) transporter. The ARS researcher used the gene to develop a marker linked to the specific gene. A high co-segregation with the VW resistance allele in the S & W populations was obtained. The marker can be used for marker-assisted selection for breeding alfalfa cultivars with improved resistance to the disease.

2. Developed drought/salt tolerant alfalfa lines and identified molecular markers associated with drought tolerance. Drought and salt resistance are important breeding targets for enhancing alfalfa productivity in arid and semi-arid regions. An ARS scientist in Prosser, Washington, identified genetic loci associated with drought and salt tolerance in alfalfa using genome-wide association studies. Most markers associated with drought and salt resistance in this work overlap with the previously reported quantitative trait loci (QTL) associated with biomass under drought in alfalfa. The geneticist used a Basic Local Alignment Search Tool (BLAST) and revealed homology to several annotated genes with functions in stress tolerance. These markers may be used for breeding new alfalfa varieties with drought and salt resistance.


Review Publications
Zhang, T., Kesoju, S., Greene, S.L., Frasen, S., Hu, J., Yu, L. 2017. Genetic diversity and phenotypic variation for drought resistance in alfalfa (Medicago sativa L.) germplasm collected for drought tolerance. Genetic Resources and Crop Evolution. 65(2):471-484. https://doi.org/10.1007/s10722-017-0546-9.