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Title: The impact of genotyping-by-sequencing pipelines on SNP discovery and identification of markers associated verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa l.)

item Yu, Long-Xi
item ZHENG, PING - Washington State University
item BHAMIDIMARRI, SURESH - S&w Seed Company
item LIU, XIANQPING - Washington State University
item MAIN, DORRIE - Washington State University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/2017
Publication Date: 2/7/2017
Citation: 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.

Interpretive Summary: Verticillium wilt can cause forage yield loss by 50% in US and Canada. In the present study, we identified marker loci associated with VW resistance in the alfalfa population using genotyping-by-sequencing and genome-wide association approaches. Although different markers were identified by different pipelines, most significant resistance loci located on chromosome 6 have been identified by all pipelines, confirming the consistency of SNP discovery and marker identification by GBS pipelines used in the present study. Sequence alignment to the M. truncatula genome revealed multiple resistance loci, several of which are linked to known resistance genes. These putative resistance loci identified had similar chromosomal locations as those previously reported in tetraploid alfalfa and in its diploid relative, M. truncatula. With further validation, those markers closely linked to the VW resistance can be used for MAS to accelerate the development of new alfalfa cultivars with improved resistance to VW.

Technical Abstract: Verticillium wilt (VW) of alfalfa is a soilborne disease that causes severe yield loss in alfalfa. To identify molecular markers associated with VW resistance, an integrated framework of genome-wide association study (GWAS) with high-throughput genotyping by sequencing (GBS) was used for mapping loci associated with VW resistance in a F1 segregating alfalfa population. Phenotyping was performed using manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Genotyping was done by GBS, followed by genotype calling using three GBS pipelines including the TASSEL-GBS pipeline (TASSEL), the Universal Network Enabled Analysis Kit (UNEAK), and a haplotype based FreeBayes pipeline (FREEBAYES). The resulting number of SNPs, marker density, minor allele frequency (MAF) and heterozygosity were compared among the pipelines. The TASSEL pipeline generated more markers with the highest density and MAF, whereas the highest heterozygosity was obtained by the UNEAK pipeline. SNP markers generated from each pipeline were used independently for marker-trait association using linkage disequilibrium. Markers significantly associated with VW resistance were identified by each pipeline and were compared. Similar marker loci were found on chromosomes 5, 6 and 7, whereas different loci on chromosome 1, 2, 3 and 4 were identified by different pipelines. Most significant markers were located on chromosome 6 and they were identified by all three pipelines. Of those identified, several loci linked to known genes had functions involved in the plants resistance to pathogen.