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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

2015 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 progress report addresses the work conducted in 2015 by an ARS research scientist of the Plant Germplasm Introduction and Testing Research Unit at the Prosser, Washington work site. Satisfactory progress was made on both objectives and their sub-objectives, all of which fall under National Program 215, Pasture, Forage, and Rangeland System. The two milestones were fully met. Progress on 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. Marker-assisted selection can greatly expedite the process of crop genetic improvement. This project focused on identifying DNA markers associated with resistance to diseases and drought stresses in alfalfa. Verticillium wilt is one of the most serious diseases of alfalfa worldwide. An ARS Research Geneticist at Prosser, Washington, led the project for identification of molecular markers associated with Verticillium wilt resistance. Manual inoculation of the pathogen to replicated cloned plants of each individual was performed and disease severity was scored using a standard scale. Marker-trait association was identified by Trait Analysis by aSSociation, Evolution and Linkage software (TASSEL) using the general and mixed linear models. Eleven markers were significantly associated with Verticillium wilt resistance and they 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 Verticillium wilt resistance. Additional markers associated with Verticillium wilt resistance were identified on chromosomes 2 and 7, and they co-located with regions of Verticillium wilt resistance loci reported in M. truncatula. This study highlights the value of Single Nucleotide Polymorphism (SNP) genotyping to identify disease resistance loci in tetraploid alfalfa. A research paper titled “Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting” has been published in PLoS ONE. This is the first report on the identification of molecular markers associated with Verticillium wilt resistance in alfalfa. The markers identified in this study are useful for improving resistance to Verticillium wilt in alfalfa breeding programs. Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. A panel of 200 alfalfa accessions with potential drought tolerance was evaluated for drought resistance in field and greenhouse in dry season of 2013 and 2014. Twenty agronomic, physiological and quality traits were evaluated. An integrated procedure for phenotyping drought resistance index (DRI) was used for evaluating 1,800 Individuals. We have identified 27 accessions with a higher level of resistance than the known drought resistant control that are useful in alfalfa breeding. Our study of marker-trait association identified twenty and fifteen loci associated with DRI and relative water content (RWC), respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on chromosomes 1, 2, 3, 4, 5, 6 and 7, while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the 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 Agignment 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 used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency. The results have been reported at professional conferences and submitted to peer-reviewed journals. Based on our research results, we developed a research proposal of a collaborative project between ARS and Cornell University. The proposal entitled “Developing molecular markers for enhancing resistance to drought and high salinity in alfalfa” was submitted recently to the National Institute of Food and Agriculture (NIFA) Alfalfa and Forage Research Program and has been selected for funding at $215,000 for three years. This additional funding will greatly strengthen our research towards developing tools and germplasm for enhancing the resistance to abiotic stresses in Alfalfa crop.


Accomplishments
1. Molecular markers for Verticillium wilt (VW) resistance in alfalfa. Verticillium wilt is an alfalfa disease that reduces forage yields up to 50%. Current breeding strategies rely greatly on phenotypic recurrent selection that allows slow and inefficient genetic improvement progress. In collaboration with Alforex Seeds, Pioneer Hi-Bred, Forage Genetics and the Noble Foundation, An ARS Research Geneticist at Prosser, Washington, identified 11 molecular markers associated with Verticillium wilt resistance in two alfalfa populations. After validation, the markers identified in this study can be used for improving resistance to Verticillium wilt in alfalfa. The identification of molecular markers and associated germplasm for disease and pest resistance will make the breeding process more efficient and facilitate rapid improvement in alfalfa cultivars with increased resistance.

2. Germplasm and molecular markers for improving alfalfa drought tolerance. Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. An ARS researcher at Prosser, Washington, conducted a replicated trial in both greenhouse and field and identified 27 accessions with higher level of resistance than the known drought resistant control. A laboratory study on marker-trait association identified 20 and 15 loci associated with drought resistance index and relative water content, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. These markers are potentially useful in marker-assisted breeding of new alfalfa varieties with drought resistance and enhanced water use efficiency.


Review Publications
Zhang, T., Yu, L., Mccord, P.H., Miller, D., Bhamidimarri, S., Johnson, D., Monteros, M., Ho, J., Reisen, P., Samac, D.A. 2014. Identification of molecular markers associated with verticillium wilt resistance in alfalfa (Medicago sativa l.) using high-resolution melting. PLoS One. 1-18. https://doi.org/10.1371/journal.pone.0115953.
Yu, L., Shen, X., Setter, T. 2014. Molecular and functional characterization of two drought-induced zinc finger proteins, ZmZnF1 and ZmZnF2 from maize kernels. Environmental and Experimental Botany. 111:13-20.
Greene, S.L., Kisha, T.J., Yu, L., Parra-Quijano, M. 2014. Conserving plants in gene banks and nature: Investigating complementarity with Trifolium thompsonii Morton. PLoS One. 9(8):e105145.