Positional-based cloning is a foundational method for understanding the genes and gene networks that control valuable agronomic traits such as grain yield components. In this study, we sought to positionally clone the causal genetic variant of a 1000-grain weight (TGW) quantitative trait loci (QTL) on wheat (Triticum aestivum L.) chromosome arm 5AL. We developed heterogenous inbred families (HIFs) (>5,000 plants) for enhanced genotypic resolution and fine-mapped the QTL to a 10-Mbp region. The transcriptome of developing grains from positive and negative control HIF haplotypes revealed presence–absence chromosome arm 5AS structural variation and unexpectedly no differential expression of genes within the chromosome arm 5AL candidate region. Evaluation of genomic, transcriptomic, and phenotypic data, and predicted function of genes, identified that the 5AL QTL was the result of strong linkage disequilibrium (LD) with chromosome arm 5AS presence or absence (HIF r² = 0.91). Structural variation is common in wheat, and our results highlight that the redundant polyploid genome's masking of such variation is a significant barrier to positional cloning. We propose recommendations for more efficient and robust detection of structural variation, including transitioning from a single nucleotide polymorphism (SNP) to a haplotype-based approach to identify positional cloning targets. We also present nine candidate genes for grain yield components based on chromosome arm 5AS presence or absence, which may unveil hidden variation of homoeolog dosage-dependent genes across the group five chromosome short arms. Taken together, our discovery demonstrates the phenotypic resiliency of polyploid genomic structural variation and highlights a considerable challenge to routine positional cloning in wheat.

中文翻译：

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基于位置的克隆“故障安全”方法被小麦染色体结构变异所压制

基于位置的克隆是了解控制有价值的农艺性状（如谷物产量成分）的基因和基因网络的基本方法。在这项研究中，我们试图在小麦（Triticum aestivumL.) 染色体臂 5AL。我们开发了异源近交系 (HIF)（> 5,000 株植物）以提高基因型分辨率，并将 QTL 精细定位到 10 Mbp 区域。来自阳性和阴性对照 HIF 单倍型的发育谷物的转录组显示存在 - 不存在染色体臂 5AS 结构变异，并且在染色体臂 5AL 候选区域内出乎意料地没有基因差异表达。对基因组、转录组和表型数据的评估以及基因的预测功能，确定 5AL QTL 是强连锁不平衡 (LD) 与染色体臂 5AS 存在或不存在 (HIF r ²= 0.91）。结构变异在小麦中很常见，我们的结果强调，冗余多倍体基因组掩盖了这种变异是定位克隆的重要障碍。我们提出了更有效和稳健检测结构变异的建议，包括从单核苷酸多态性 (SNP) 过渡到基于单倍型的方法来识别定位克隆目标。我们还提出了基于染色体臂 5AS 存在与否的谷物产量成分的九个候选基因，这可能揭示了组 5 染色体短臂中同源剂量依赖性基因的隐藏变异。总之，我们的发现证明了多倍体基因组结构变异的表型弹性，并突出了小麦常规定位克隆面临的巨大挑战。