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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

2014 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
Objective 1: Verticillium wilt (VW), caused by the soilborne fungus Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To address this, USDA-ARS Prosser, the Samuel Roberts Noble Foundation, Forage Genetics International (FGI), Pioneer Hi-bred, and Alforex Seeds agreed on a joint effort to identify publically available markers for selection for resistance to Verticillium wilt and stem nematode. Pioneer and Alforex generated and phenotyped biparental populations consisting of 188 and 164 individuals, respectively. An association study was conducted using autotetraploid alfalfa populations composed of 352 individuals. The samples were genotyped by high-resolution melting analysis of single nucleotide polymorphism (SNP) markers. Polymorphic SNPs were identified based on the melting profiles and up to five clusters were obtained, representing the various allelic combinations in tetraploid alfalfa. Phenotyping was done by inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale (e.g. 1-5). Marker-trait association was analyzed by the TASSEL software using the general and mixed linear models. Ten markers were significantly associated with VW resistance and they were located on three chromosomal regions. Five significant markers on chromosome 8 contributed a total of 36% of the phenotypic variation explained and represent novel loci associated with VW resistance. Additional significant markers were identified on chromosomes 2 and 7, and they co-locate with the regions of VW resistance loci reported in M. truncatula. This study highlights the value of SNP genotyping to identify the disease resistance loci in tetraploid alfalfa. The markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa. To increase the power and resolution of marker-trait association, we are using a genome-wide genotyping platform so-called “genotyping by sequencing (GBS)” which will increase thousands of SNP markers throughout the whole genome. We have genotyped the Pioneer VW population by GBS and obtained nearly 10,000 meaningful GBS markers. Improved methods increased the probability for identifying molecular markers associated with VW resistance. Preliminary results showed strong association of a group of GBS markers with the trait at p<0.001, with the highest phenotypic variation explained (PVE) of 19%. To confirm this finding, we are going to validate them for VW resistance in different populations. After validation, they can be used in marker-assisted selection for identification of resistance sources. It will facilitate breeding progress and provide alfalfa seed companies with breeding materials for use in the production of improved commercial varieties of alfalfa. It will accelerate the breeding program of resistant varieties, reduce economic losses to growers, increase sustainability, and reduce costs to consumers. Similar procedures will be used for identification of markers associated with stem nematode resistance. Objective 2: Enhancing drought resistance and water use efficiency of alfalfa varieties are important to meet the challenges of finite available water resource. However, no rapid and precise methods have been developed to identify the resistance resources for the alfalfa breeders. Development of molecular markers associated with drought resistance and improved water use efficiency would be helpful for improving the accuracy in detection, accumulating the major and minor genes, and accelerating the breeding process. Toward this end, in this study, a panel of accessions comprised of 202 alfalfa cultivars and landraces with potential drought tolerance were selected from the USDA-ARS National Temperate Forage Legume Germplasm Resources Center (http://www.ars-grin.gov). They were evaluated for drought resistance in field and greenhouse in dry season of 2013 and 2014. Agronomic and physiological traits including relative leaf water content, osmotic adjustment, canopy temperature, and biomass and forage quality under water deficit were measured. A greenhouse procedure for phenotyping drought tolerance index was established. The index of individuals were scored using a scale of 1 to 5, with 1=susceptible with plant death, 5=high resistant with minimal damage. Generally, the frequency stem of phenotypic data exhibited a trend of normal distribution with the mean of 3.10 and variance of 1.17. And the p-values of the Shapiro-Wilk (2.382e-09) and Kolmogorov-Smirnov (8.654e-05) tests were far less than W (0.915) and D (0.158) values, respectively, conforming a normal distribution of drought resistance scores of this panel. Using this protocol, we have identified a group of accessions with high resistance scores. With further characterization, they can be used as gene resources for breeding and developing populations for mapping quantitative trait loci (QTL) for drought tolerance and enhanced water use efficiency. Industrial seed companies such as Alforex Seeds, S & W Seeds and Pioneer Hi-Bred have expressed their interests in using these germplasm for developing alfalfa cultivars with improved drought/salt resistance and water use efficiency.


Accomplishments
1. Molecular markers for Verticillium wilt (VW) resistiance 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. 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. In collaboration with Alforex Seeds, Pioneer Hi-Bred, Forage genetics and the Noble Foundation, ARS scientists in 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.


Review Publications
Yu, L., Hugues, B., Rouse, M.N., Singh, S., Singh, R.P., Bhavani, S., Huerta-Espino, J., Sorrells, M.E. 2014. A Consensus Map for Ug99 Stem Rust Resistance Loci in Wheat. Theoretical and Applied Genetics. Available: http://link.springer.com/article/10.1007%2Fs00122-014-2326-7.
Yu, L., Parthasarathy, M. 2014. Molecular and cellular characterization of the tomato pollen profilin, LePro1. PLoS One. DOI:10.1371/journal.pone.0086505.
Mccord, P.H., Gordon, V.S., Saha, G., Hellinga, J., Vandemark, G.J., Larsen, R.C., Smith, M., Miller, D. 2014. Detection of QTL for forage yield, lodging resistance and spring vigor traits in alfalfa (Medicago sativa L.). Euphytica. 10:2134.