The Impact of Genotyping-by-Sequencing Pipelines on SNP Discovery and Identification of Markers Associated with Verticillium Wilt Resistance in Autotetraploid Alfalfa (Medicago sativa L.).

Long-Xi Yu,Suresh Bhamidimarri,Dorie Main,Ping Zheng,Xiang-Ping Liu

doi:10.3389/fpls.2017.00089

Abstract

Verticillium wilt (VW) of alfalfa is a soilborne disease causing severe yield loss in alfalfa. To identify molecular markers associated with VW resistance, we used an integrated framework of genome-wide association study (GWAS) with high-throughput genotyping by sequencing (GBS) to identify loci associated with VW resistance in an F1 full-sib alfalfa population. Phenotyping was performed using manual inoculation of the pathogen to 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 bioinformatics pipelines including the TASSEL-GBS pipeline (TASSEL), the Universal Network Enabled Analysis Kit (UNEAK), and the haplotype-based FreeBayes pipeline (FreeBayes). The resulting numbers 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. The FreeBayes pipeline generated tetraploid genotypes, with the least number of markers. SNP markers generated from each pipeline were used independently for marker-trait association. Markers significantly associated with VW resistance identified by each pipeline 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 were linked to known genes whose functions are involved in the plants’ resistance to pathogens. Further investigation on these loci and their linked genes would provide insight into understanding molecular mechanisms of VW resistance in alfalfa. Functional markers closely linked to the resistance loci would be useful for MAS to improve alfalfa cultivars with enhanced resistance to the disease.

Highlights

Verticillium wilt (VW) is a soil-borne disease caused by the fungal pathogen Verticillium alfalfae (Renormalized by Inderbitzin et al, 2011) in alfalfa (Medicago sativa L.)
Using the criteria mentioned in Section “Materials and Methods,” we have identified 13 genotyping by sequencing (GBS) markers from the FreeBayes pipeline significantly associated with VW resistance (Table 3, Figure 5A)
In the present study, compared to the TASSEL and Universal Network Enabled Analysis Kit (UNEAK) pipelines, a lower missing value was found in FreeBayes, fewer variants were obtained (Table 2) and only13 markers significantly associated with VW resistance were identified by the FreeBayes pipeline (Table 3)

Summary

Introduction

Verticillium wilt (VW) is a soil-borne disease caused by the fungal pathogen Verticillium alfalfae (Renormalized by Inderbitzin et al, 2011) in alfalfa (Medicago sativa L.) It causes severe forage yield loss in US and Canada (Graham et al, 1977; Atkinson, 1981; Grau et al, 1981; Gray and Roth, 1982; Gordon et al, 1989). The disease symptoms start with leaf tip chlorosis, leaf desiccation and abscission, and as disease progresses, infected plants eventually wilt and die (Leath and Pennypacker, 1990). Due to other dispersal factors including insects, manure, wind, and water, these control strategies are often ineffective For this reason, the preferred method of control of the disease is the use of resistant varieties (Peaden et al, 1985; Leath and Pennypacker, 1990). It has been suggested that cultivars require at least 60% resistant plants for reasonable protection against the disease (Grau, 1991)

Methods

Results

Discussion

Conclusion