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Genomic Science Program

2006 Awardee

Using Association Mapping to Identify Markersfor Cell Wall Constituents and Biomass Yield inAlfalfa

INVESTIGATOR(S): Brummer, E. C.; Doyle, J.J.; Moore, K. J.

INSTITUTION: University of Georgia

NON-TECHNICAL SUMMARY: Alfalfa ( Medicagosativa ) is a potential biofuel crop because it produceshigh yield, its leaves can be used as a high value, highprotein coproduct, it fixes atmospheric nitrogen, and ithas beneficial effects on the environment. Improvingalfalfa as a biofuel crop will entail breeding forincreased biomass yield and altered cell wall composition.While traditional phenotypic selection can be successful,the perennial nature of alfalfa requires that a selectioncycle lasts for several years. Decreasing the cycle timewould increase genetic gain for all traits. This could beachieved using marker assisted selection for the traits ofinterest, but marker identification research conductedpreviously has not focused on representative alfalfabreeding populations nor has it examined wild germplasm asa source of new alleles to improve agronomically importanttraits. Our experiment will address these issues bystudying both wild germplasm not typically used in alfalfabreeding programs and also a cultivated breeding populationcurrently under selection. We will evaluate biomass yieldand cell wall composition in the field. Concurrently, wewill evaluate the genotype of each plant using geneticmarkers selected throughout the genome. We will alsodevelop markers based on DNA sequence variation in genes ofpossible involvement in cell wall synthesis. Ultimately,this project will improve the efficiency of selection forenhanced bioenergy characteristics in alfalfa, producenumerous new markers at important candidate genes, andidentify potentially useful alleles in wild germplasm.

OBJECTIVES: Our objectives are to usegenomics approaches to identify chromosomal regions, andultimately genes, controlling the two most importantbioenergy traits, biomass yield and composition, and todevelop genetic markers that can be used directly inapplied plant breeding programs to improve the bioenergyqualities of alfalfa. We will pursue two complementaryobjectives to attain our goals: 1. Identify loci, andspecific alleles, that control the concentration of alfalfastem cell wall constituents and that are associated withbiomass production using whole genome and candidate geneassociation mapping across a diverse set of natural diploidalfalfa accessions, and 2. Extend the analysis and methodsused in the first objective to a tetraploid alfalfabreeding population currently under selection. As a resultof this project, we (a) will have identified novel allelesin wild alfalfa germplasm that may be useful to improvecultivated alfalfa; (b) will have developed and used SNPmarkers in genes known to be involved in the biosynthesisof cell wall composition; (c) will be able to selectindividuals within a breeding population on the basis ofthese markers, and (d) will identify new alleles from wildgermplasm useful for improving cultivated alfalfa. Thisexperiment will provide the first estimate of linkagedisequilibrium (LD) in alfalfa, both in a broadcross-section of wild diploid germplasm and in apractically important cultivated breeding population, bothon a genome-wide and on an individual gene basis.Additionally, we will have applied association mapping tothis important crop legume for the first time.

APPROACH: We will use association mappingto identify genome regions and candidate genes that areassociated with biomass production and cell wallcomposition in both diploid and tetraploid alfalfapopulations. We are proposing to begin by screening a broaddiversity of diploid germplasm (three individuals from eachof 96 plant introductions) in order to identify new geneticvariation for these traits that could be useful in alfalfaimprovement. We will begin by analyzing diploid genotypesbecause they likely harbor a reservoir of unexploitedgenetic diversity and are more tractable for associationmapping experiments than tetraploid genotypes. Subsequentlywe will extend the results to tetraploids. The tetraploidpopulation we will examine is a breeding populationcurrently under clonal selection at four locations, with200 individuals being evaluated. As a breeding population,markers associated with traits could be immediately used ina recurrent selection program leading to the development ofimproved cultivars. Phenotypic analysis will be conductedbased on field grown plant material clonally replicated toenable assessment of individual genotypes. In addition tobiomass production and plant height measurements, we willconduct a through analysis of the stem cell wallcomposition of all entries. All plants will be genotypedthroughout the genome with simple sequence repeat (SSR)markers, some of which will be selected based on theirassociation with quantitative trait loci (QTL) for biomassyield, stem cell wall cellulose, hemicellulose, and ligninconcentration, or agronomic traits that we have identifiedin other experiments. Concurrently, we will sequenceportions of up to 100 genes that are candidate lociinvolved with cell wall biosynthesis. The sequencing willlead to the identification of single nucleotidepolymorphisms (SNP), which we will develop into markers forthose specific genes. All plants (288 diploid and 200tetraploid) will be genotyped with the SNP markers.Association mapping will be conducted using the recentlydescribed mixed-model method that will account forunderlying population structure within our two groups ofgenotypes (diploid and teatraploid), which will be analyzedseparately. We will test for associations based on bothgenome-wide SSR molecular markers, as well as on SNPmarkers for 20 candidate genes, which will be developedfrom sequence data on 96 diploid and 20 tetraploidindividuals.


Name: Brummer, E. C.



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