Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing

Abstract

Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. Identification of genes involved in drought tolerance will facilitate breeding for improving drought resistance and water use efficiency in alfalfa. Our objective was to use a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces to identify genes involved in drought tolerance. The panel was selected from the USDA-ARS National Plant Germplasm System alfalfa collection and genotyped using genotyping by sequencing. A greenhouse procedure was used for phenotyping two important traits associated with drought tolerance: drought resistance index (DRI) and relative leaf water content (RWC). Marker-trait association identified nineteen and fifteen loci associated with DRI and 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 all chromosomes while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5, 6 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 QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. 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.

Fig 1. Phylogenic analysis of 198 alfalfa accessions using the marker data generated by GBS and neighbor-joint tree.

Their origins are indicated outside of the phylogenic tree.

Fig 2. Phenotypic evaluations of drought resistance index (A) and leaf relative water content (RWC).

Phenotypic data from three replications were analyzed using a mixed model and least square means were obtained and normal distributions were obtained for both traits (DRI in A and RWC in B). A drought tolerance cultivar, “Wilson” was used as the resistance control for both DRI and RWC experiments.

Fig 3. The distribution of minor allele frequency of valid variants calls by genotyping by sequencing.

Fig 4. The distribution of heterozygosity of valid variants after genotyping by sequencing pipeline.

Fig 5. Manhattan and quantile-quantile plots resulting from GWAS for drought resistance index (A, B) and leaf relative water content (C, D) in alfalfa.

A false discovery rate of 5% was used for significant cutoff (dot lines). Significant markers (above the cutoff lines) associated with DRI and RWC were listed in Tables 3 and 4, respectively. The chromosome position was based on the reference genome of M. truncatula sequence (Version 3.5). “U” represents unknown chromosome. The abbreviations at the top of plots are putative candidate genes and the arrows indicate their genetic positions in the reference genome. SDD, subtilisin-like protease; ALDH7A1, Aldehyde dehydrogenase family 7 member A1; EIG-J7, Elicitor inducible protein EIG-J7; AGP-L, Glucose-1-phosphate adenylyltransferase large subunit; NYC1, Oxidoreductase NYC1; SRF, strubbelig-receptor family 3-like protein; SIN3, SIN3 component histone deacetylase complex; KAPP, Kinase-associated protein phosphatase; PPR, pentatricopeptide repeat-containing protein.