Long-Amplicon Single-Molecule Sequencing Reveals Novel, Trait-Associated Variants of Homoeologs in Hexaploid Wheat.

The gene () is a key controller of vernalization requirement in wheat. The genome of hexaploid wheat () harbors three homoeologous loci on chromosomes 5A, 5B, and 5D. Structural sequence variants including small and large deletions and insertions and single nucleotide polymorphisms (SNPs) in the three homoeologous genes not only play an important role in the control of vernalization requirement, but also have been reported to be associated with other yield related traits of wheat. Here we used single-molecule sequencing of barcoded long-amplicons to assay the full-length sequences (∼13 kbp plus 700 bp from the promoter sequence) of the three homoeologous genes in a panel of 192 predominantly European winter wheat cultivars. Long read sequences revealed previously undetected duplications, insertions and single-nucleotide polymorphisms in the three homoeologous genes. All the polymorphisms were confirmed by Sanger sequencing. Sequence analysis showed the predominance of the winter alleles , , and across the investigated cultivars. Associations of SNPs and structural variations within the three genes with 20 economically relevant traits including yield, nodal root-angle index and quality related traits were evaluated at the levels of alleles, haplotypes, and copy number variants. Cultivars carrying structural variants within genes showed lower grain yield, protein yield and biomass compared to those with intact genes. Cultivars carrying a single copy and a unique haplotype with a high number of SNPs were found to have elevated grain yield, kernels per spike and kernels per m along with lower grain sedimentation values. In addition, we detected a novel SNP polymorphism within the G-quadruplex region of the promoter of that was associated with deeper roots in winter wheat. Our findings show that multiplex, single-molecule long-amplicon sequencing is a useful tool for detecting variants in target genes within large plant populations, and can be used to simultaneously assay sequence variants among target multiple gene homoeologs in polyploid crops. Numerous novel haplotypes and alleles were identified that showed significantly associations to economically important traits. These polymorphisms were converted into PCR or KASP assays for use in marker-assisted breeding.