基于转录组测序的野生二粒小麦盐胁迫相关基因的鉴定与优异等位变异发掘

Wild emmer wheat (Triticum dicoccoide) is the direct progenitor of modern cultivated wheat, which could be utilized as elite genetic resources for wheat improvement. It’s very important to identify and discover the salt tolerance-associated genes from wild emmer wheat for developing the excellent salt tolerant wheat varieties. In previous study, we have systematically investigated the salt tolerance of the wild emmer lines collected from different origin regions, and the elite lines were obtained. Here, we performed the high throughput transcriptome sequencing to analyze the gene expression profiles of salt tolerant and sensitive lines under normal conditions and salt stress at whole genome-wide level. The differential expressed genes or gene clusters were obtained upon comparison of these transcriptome expression profiles, and then the prominent salt induced genes were identified and cloned. Furthermore, the genetic variation and nucleotide diversity of these selected genes were investigated in some natural populations of wild emmer wheat and cultivars to reveal the adaptive evolutionary mechanism of these salt induced genes. Association analysis was further performed to identify the key candidate genes by correlating the SNPs with the salt tolerance of the lines, and also to discover the elite alleles and haplotypes. Our study will provide helpful information for revealing the molecular mechanisms of salt resistance in wild emmer wheat, and could be useful for enriching the genetic resources for wheat improvement and overcoming the problem of gene homogenization in wheat and beyond.

野生二粒小麦是普通小麦的野生近缘祖先种，是小麦遗传改良的重要资源和基因库。挖掘、利用其优异耐盐基因对提高小麦耐盐性，培育优良耐盐小麦新品种具有重要意义。本研究在前期对不同来源地野生二粒小麦的耐盐性进行系统评价并筛选获得了优异耐盐品系的基础上，采用转录组测序结合生物信息学分析在全基因组水平研究耐盐和盐敏感品系在正常和盐胁迫条件下的mRNA表达谱，高效、快速获得差异表达基因，鉴定、克隆盐胁迫响应相关基因，进一步采用扩增子测序对这些基因的遗传变异和多态性进行分析，结合与耐盐性的关联分析，鉴定获得与盐胁迫响应相关的关键基因，并挖掘其中的优异等位变异及单倍型，以期为揭示野生小麦耐盐的分子机理奠定理论基础，为丰富小麦抗盐育种的遗传基础和克服基因资源同质化问题服务。

野生二粒小麦是普通小麦的野生近缘祖先种，是小麦遗传改良的重要资源和基因库。挖掘、利用其优异耐盐基因对提高小麦耐盐性，培育优良耐盐小麦新品种具有重要意义。本研究首先对收集保存的不同来源地野生二粒小麦群体的的耐盐性进行了系统的鉴定与评价，筛选获得了10份优异的耐盐野生二粒小麦品系；在此基础上，采用RNA-Seq技术对亲缘关系比较近的耐盐品系B-48和盐敏感品系A-4在苗期盐胁迫与正常条件下的mRNA表达谱进行了比较分析，共鉴定到了2816个差异表达基因，其中耐盐品系中特异差异表达的有95个，盐敏感品系中特异差异表达的基因有2,595个，共同存在的差异基因为126个，盐敏感品系中差异表达的基因数目显著高于抗盐品系，表明盐敏感品系在盐胁迫下应激了大量的基因来应对盐胁迫；进一步，选取TtHKT1,5-4B和 TtNCED1这两个差异最明显的基因，利用分子克隆技术从抗盐品系B-48克隆获得了其全长序列，并对其在盐胁迫不同时间节点的表达特性进行了初步分析；同时，构建了TtHKT1,5-4B过表达载体并转化拟南芥，获得了阳性转基因植株，T3代纯合株系的芽期和苗期耐盐性鉴定发现转基因株系的耐盐性较野生型显著提高，初步证实了TtHKT1,5-4B的生物学功能。最后，还选取了抗盐性不同的24个野生二粒小麦和12个硬粒小麦的HKT1,5-4B基因的序列多态性进行了分析，共检测到16种单倍型，其中在野生二粒小麦中发现了11种单倍型，在硬粒小麦中发现了9种单倍型，4种单倍型在两种材料间同时存在，表明野生二粒小麦具有更丰富的单倍型组成，遗传多态性较高；但没有找到与抗盐性显著关联的SNP位点，进一步的研究还在进行中。此外，还对野生二粒小麦中盐胁迫相关miRNA及其表达特性进行分析，为野生二粒小麦盐胁迫相关miRNA的发掘与功能研究奠定了基础。本研究在发掘耐盐种质基础上，对其盐胁迫相关基因进行了初步的鉴定和挖掘，这为深入揭示野生小麦耐盐的分子机理奠定理论基础，也为丰富小麦抗盐育种的遗传基础和克服基因资源同质化提高了重要资源。