Long-Amplicon Single-Molecule Sequencing Reveals Novel, Trait-Associated Variants of VERNALIZATION1 Homoeologs in Hexaploid Wheat

Abstract

The gene VERNALIZATION1 (VRN1) is a key controller of vernalization requirement in wheat. The genome of hexaploid wheat (Triticum aestivum) harbors three homoeologous VRN1 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 VRN1 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 VRN1 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 VRN1 genes. All the polymorphisms were confirmed by Sanger sequencing. Sequence analysis showed the predominance of the winter alleles vrn-A1, vrn-B1, and vrn-D1 across the investigated cultivars. Associations of SNPs and structural variations within the three VRN1 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 VRN1 genes showed lower grain yield, protein yield and biomass compared to those with intact genes. Cultivars carrying a single vrn-A1 copy and a unique haplotype with a high number of SNPs were found to have elevated grain yield, kernels per spike and kernels per m2 along with lower grain sedimentation values. In addition, we detected a novel SNP polymorphism within the G-quadruplex region of the promoter of vrn-A1 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 VRN1 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.

Transgressive and parental dominant gene expression and cytosine methylation during seed development in Brassica napus hybrids

“…The observed genotype-specific bias is potentially a result of different genomic, transcriptomic and epigenomic factors. Firstly, genomic rearrangements gene copy number variations and other structural variants are known to affect various traits in B. napus and other polyploid plants (Schiessl et al 2017 ; Vollrath et al 2021 ; Makhoul et al 2022 ) and could have led to potential biases in expression patterns. Transcriptomic aspects such as gene isoforms, gene network interactions and allele expression bias might also be involved in favoring the up- or downregulation from a certain genotype or haplotype (Fan et al 2020 ; Schiessl et al 2020 ; Golicz et al 2021 ).…”

Transgressive and parental dominant gene expression and cytosine methylation during seed development inBrassica napushybrids

“…The observed genotype-specific bias can be a result of the following genomic, transcriptomic and epigenomic factors. Firstly, genomic rearrangements such as structural variations and gene copy number variations are known to affect various traits in polyploid plants (Schiessl et al, 2017;Vollrath et al, 2021;Makhoul et al, 2022) and could have led to potential biases in expression patterns.…”

“…The observed genotype-specific bias is potentially a result of different genomic, transcriptomic and epigenomic factors. Firstly, genomic rearrangements gene copy number variations and other structural variants are known to affect various traits in B. napus and other polyploid plants (Schiessl et al, 2017; Vollrath et al, 2021; Makhoul et al, 2022) and could have led to potential biases in expression patterns. Transcriptomic aspects such as gene isoforms, gene network interactions and allele expression bias might also be involved in favoring the up- or down-regulation from a certain genotype or haplotype (Fan et al, 2020; Schiessl et al, 2020; Golicz et al, 2021).…”

Wild emmer wheat, the progenitor of modern bread wheat, exhibits great diversity in the VERNALIZATION1 gene

“…Of the four known vernalization response genes, the VRN1 gene is considered the most important one (Yan et al, 2003). Sequence analysis of the VRN1 gene in bread wheat revealed only minor sequence variation (Strejcǩováet al, 2021;Makhoul et al, 2022). This may reflect the limited gene pool of progenitors, which contributed to the original population(s) of bread wheat.…”