Genome-wide association study of agronomic traits in winter wheat (Triticum aestivum L.) using a custom SNP marker set.

Targeted genotyping by sequencing (GBS) of single-nucleotide polymorphisms (SNPs) is a relatively new high-throughput genotyping technique that combines target enrichment using polymerase reaction, followed by high-throughput sequencing of the resulting DNA fragments. Unlike traditional GBS, this approach requires prior knowledge of polymorphisms in the genome. Based on annotated polymorphisms in a wheat reference genome, we developed a panel of SNPs suitable for targeted GBS. Initially, 60,000 SNPs were proposed, including those tested by breeding markers, SNPs mentioned in previous genome-wide association studies (GWAS) publications, as well as mutations in protein-coding regions of genes. Due to the polyploidy of the wheat genome, the suboptimal GC content in surrounding regions and the requirements for high-quality allele calls, only 3500 SNP were retained for further analysis. These SNPs were used to genotype 200 winter wheat accessions maintained at the National Grain Center in Krasnodar, Russia. A population structure analysis based on genome-wide SNP data revealed two subpopulations in the collection corresponding to domestic and foreign cultivars. Genotyping results for specific SNPs revealed genes whose diversity can be enriched in the domestic gene pool to improve grain size (TaGW2-A1) and protein content (Gpc-B1). Additionally, no-call variants of SNPs obtained from SNP-seq procedures were used to detect chromosome rearrangements such as 1RS/1BL wheat-rye translocations. GWAS using mixed linear models identified new marker-trait associations for days to heading, plant height, visual grain score and disease resistance. KASP markers were developed for significantly associated SNPs and candidate genes based on their proximity and function were suggested. Additionally, a potential link was proposed between the presence of awns and increased calcium in grain. The GWAS also confirmed the known effects of reduced height (Rht) and photoperiod sensitivity (Ppd) genes in wheat, and the Ppd-D1 gene showed an almost significant association with grain yield. SNPs originating from breeding markers were the most effective in finding marker-trait associations, while those from other sources found associations as frequently as random SNPs. Of the 13 markers significantly associated with traits, seven did not overlap with markers from existing genotyping arrays, and could be considered novel.