Long-Amplicon Single-Molecule Sequencing Reveals Novel, Trait-Associated Variants of VERNALIZATION1 Homoeologs in Hexaploid Wheat.
Long-Amplicon Single-Molecule Sequencing Reveals Novel, Trait-Associated Variants of VERNALIZATION1 Homoeologs in Hexaploid Wheat.
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DOI：
10.3389/fpls.2022.942461
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发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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基因VERNALIZATION1(VRN1)是控制小麦春化的关键基因。六倍体小麦(Triticum Aestivum)基因组在5A、5B和5D染色体上含有三个同源的VRN1基因座。结构序列变异包括3个同源VRN1基因的大小缺失和插入以及单核苷酸多态(SNPs)，不仅在春化需求控制中起重要作用，而且已被报道与小麦的其他产量相关性状相关。在这里，我们使用条形码长扩增片段的单分子测序来分析192个主要来自欧洲的冬小麦品种的三个同源VRN1基因的全长序列(∼13kbp加上启动子序列的700bp)。长阅读序列揭示了三个同源VRN1基因以前未被检测到的复制、插入和单核苷酸多态。Sanger测序证实了这些基因的多态。序列分析表明，越冬等位基因VRN-A1、VRN-B1和VRN-D1在供试品种中占优势。在等位基因、单倍型和拷贝数变异水平上分析了3个VRN1基因的SNPs和结构变异与产量、节根角指数和品质相关性状等20个经济性状的关联。携带VRN1基因结构变异的品种与携带完整基因的品种相比，籽粒产量、蛋白质产量和生物量都较低。携带单一VRN-A1拷贝和带有大量SNP的独特单倍型的品种具有较高的产量、每穗粒数和每平方米粒数，并具有较低的籽粒沉降值。此外，我们在VRN-A1启动子的G-四联体区域检测到一个新的SNP多态，该多态与冬小麦较深的根相关。我们的发现表明，多重、单分子长扩增序列是检测大植物群体中目标基因变异的有用工具，并可用于同时分析多倍体作物中多个目标基因同源基因之间的序列变异。许多新的VRN1单倍型和等位基因被鉴定出来，这些单倍型和等位基因与重要的经济性状显著相关。这些多态被转化为PCR或KASP分析，用于分子标记辅助育种。
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.
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