Bhoite Roopali, Smith Rosemary, Bansal Urmil, Dowla Mirza, Bariana Harbans, Sharma Darshan
Grains Genetic Improvement Department of Primary Industries and Regional Development South Perth Western Australia Australia.
The UWA Institute of Agriculture The University of Western Australia Perth Western Australia Australia.
Plant Direct. 2023 Aug 18;7(8):e520. doi: 10.1002/pld3.520. eCollection 2023 Aug.
Targeted exome-based genotype by sequencing (t-GBS), a sequencing technology that tags SNPs and haplotypes in gene-rich regions was used in previous genome-wide association studies (GWAS) for sodicity tolerance in bread wheat. Thirty-nine novel SNPs including 18 haplotypes for yield and yield-components were identified. The present study aimed at developing SNP-derived markers by precisely locating new SNPs on ~180 bp allelic sequence of t-GBS, marker validation, and SNP functional characterization based on its exonic location. We identified unknown locations of significant SNPs/haplotypes by aligning allelic sequences on to IWGSC RefSeqv1.0 on respective chromosomes. Eighteen out of the target 39 SNP locations fulfilled the criteria for producing PCR markers, among which only eight produced polymorphic signals. These eight markers associated with yield, plants m, heads m, and harvest index, including a pleiotropic marker for yield, harvest index, and grains/head were validated for its amplification efficiency and phenotypic effects in focused identification germplasm strategy (FIGS) wheat set and a doubled haploid (DH) population (Scepter/IG107116). The phenotypic variation explained by these markers are in the range of 4.1-37.6 in the FIGS population. High throughput PCR-based genotyping using new markers and association with phenotypes in FIGS wheat set and DH population validated the effect of functional SNP on closely associated genes-calcineurin B-like- and dirigent protein, basic helix-loop-helix (BHLH-), plant homeodomain (PHD-) and helix-turn-helix myeloblastosis (HTH myb) type -transcription factor. Further, genome-wide SNP annotation using SnpEff tool confirmed that these SNPs are in gene regulatory regions (upstream, 3'-UTR, and intron) modifying gene expression and protein-coding. This integrated approach of marker design for t-GBS alleles, SNP functional annotation, and high-throughput genotyping of functional SNP offers translation solutions across crops and complex traits in crop improvement programs.
基于靶向外显子组测序的基因型分析（t-GBS）是一种在富含基因区域标记单核苷酸多态性（SNP）和单倍型的测序技术，此前在面包小麦耐盐碱全基因组关联研究（GWAS）中得到应用。共鉴定出39个新的SNP，包括18个与产量及产量构成因素相关的单倍型。本研究旨在通过在t-GBS约180bp等位基因序列上精确定位新的SNP、进行标记验证以及基于其外显子位置进行SNP功能表征，来开发基于SNP的标记。我们通过将等位基因序列与各染色体上的国际小麦基因组测序联盟（IWGSC）RefSeqv1.0进行比对，确定了显著SNP/单倍型的未知位置。目标39个SNP位置中有18个符合产生PCR标记的标准，其中只有8个产生了多态性信号。这8个与产量、每平方米株数、每平方米穗数和收获指数相关的标记，包括一个对产量、收获指数和每穗粒数具有多效性的标记，在聚焦鉴定种质策略（FIGS）小麦群体和一个双单倍体（DH）群体（Scepter/IG107116）中对其扩增效率和表型效应进行了验证。这些标记在FIGS群体中解释的表型变异范围为4.1%-37.6%。使用新标记进行基于PCR的高通量基因分型以及与FIGS小麦群体和DH群体中的表型关联，验证了功能性SNP对紧密相关基因——类钙调神经磷酸酶B、 dirigent蛋白、碱性螺旋-环-螺旋（BHLH）、植物同源结构域（PHD）和螺旋-转角-螺旋成髓细胞白血病（HTH myb）型转录因子的影响。此外，使用SnpEff工具进行全基因组SNP注释证实，这些SNP位于基因调控区域（上游、3'-非翻译区和内含子），可修饰基因表达和蛋白质编码。这种针对t-GBS等位基因的标记设计、SNP功能注释以及功能性SNP高通量基因分型的综合方法，为作物改良计划中的各种作物和复杂性状提供了转化解决方案。
