小麦芒长抑制基因B1的精细定位及其调控芒发育相关基因的分离

Wheat awnedness, the product of natural evolution and its adaptations to environment, is one of wheat cultivar characteristics. At the same time, it acts as a high effective organ for photosynthesis and plays a major role in the course of water and mineral transportation to kernels, grain filling and elaboration of yield in wheat. Therefore, it is very important to conduct the research on the inheritance of awnedness related genes and their effects on the development of awn, especially for wheat genetics and breeding. . In the study, the near-isogenic lines (NILs) with highly consistent genetic background and the only different awn length were produced. Genetic analysis demonstrated that the awn length of the NILs was controlled by the awn inhibitor gene B1, located on the long arm of 5A chromosome of common wheat. Molecular markers technology and bulked segregation analysis (BSA) were used to screen for specific markers linked with gene B1 in the awn length segregation populations derived from the NILs. To complete its fine mapping, high density linkage map of gene B1 on 5A chromosome will be constructed and novel molecular markers for gene B1 will be developed. The genes mediated by awn inhibitor gene B1 in awn differentiation stage, will be dissected by virtue of the technology of gene chip, cDNA-amplified fragment length polymorphism (cDNA-AFLP) and rapid-amplification of cDNA ends (RACE). The bioinformation analysis of these genes will be carried out by real time PCR characterization and gene ontology (GO) annotation tool. This will illustrate the molecular mechanism that gene B1 regulates awn development. The study benefits to establish assisted molecular marker selection system of gene B1 in practice and elucidate the molecular regulation mechanism underlying awn development mediated by gene B1 on theory.

麦芒是小麦进化与适应环境的产物，既可作为小麦品种特性之一，又是小麦穗部重要的光合器官，在水分与矿物质营养向籽粒的运转、籽粒灌浆以及产量形成中起关键作用。开展芒性相关性状的遗传、发育及其功能等研究，是小麦遗传育种重要研究内容。. 本研究在对获得的一对仅芒长长度存在差异的近等基因系进行遗传分析的基础上，证明该性状受小麦5AL染色体末端芒长抑制基因B1控制；研究以该系为材料，利用分子标记技术对构建的芒长分离群体进行BSA分析，绘制B1基因区段间高密度连锁图谱，筛选并开发出与之紧密连锁、特异的分子标记，对其进行精细定位；利用生物芯片、cDNA-AFLP、RACE等技术分离B1调控芒发育的相关基因，并对相关基因进行实时定量PCR验证与GO注释分析，初步阐明B1调控芒发育的作用机理。该研究对建立B1分子标记辅助选择体系具有重要的应用价值，对于了解B1调控芒发育的分子机制也具有重要理论意义。

小麦的芒是小花外稃的纤维状延伸，属于叶的变态；是小麦重要形态标记，又是穗部重要光合器官。本研究以芒抑制基因B1的近等基因系SN051-1（有芒）和SN051-2（无芒）为材料，对其芒发育动态、性状特点、千粒重等进行了调查，进行了遗传分析、Super BSA分析及转录组测序分析，结果如下：.（1）体视显微镜和扫描电镜观察表明，小麦芒原基出现在雌雄蕊分化期；在药隔形成期，芒长出现差异。SN051-1的千粒重高于SN051-2的千粒重，F2、BC1群体中有芒单株的平均千粒重高于无芒单株，差异达显著水平；表明芒在籽粒同化产物的积累中具有重要作用，从而造成二者千粒重的差异。.（2）遗传分析证明SN051-1和SN051-2的芒长性状由单基因控制，无芒对有芒为显性，其基因型分别为b1b1、B1B1。分子标记筛选获得一个与芒抑制基因B1紧密连锁、位于5AL染色体上的SSR标记Xgwm291，遗传距离为1.90cM。.（3）对亲本和F2样品进行Super BSA分析，共获得216,192个SLAF标签，其中多态性标签有3,679个，占1.7%。将SLAF标签与小麦ABD基因组进行比对，考虑标签中等位基因亲本来源，有34个差异标签定位在参考基因组上，有2个差异标签定位在小麦5AL染色体上；不考虑亲本来源，有140个差异标签定位在参考基因组上，有6个差异标签定位在小麦5AL染色体上。.（4）对近等基因系及其F1、F2中的有芒单株和无芒单株进行转录组测序得到Clean data总碱基数为48.06Gb。无参考基因组的转录组测序结果通过序列组装共得到96,360个Unigenes，亲本和F1之间差异表达基因有180个，其中无芒作为对照下调基因有134个。结合F2测序结果，得到22个共同差异基因，只有4个下调基因；qRT-PCR结果表明c18073.graph_c0，c55090.graph_c0和c73118.graph_c0在SN051-1中的相对表达量显著低于其在SN051-2中的相对表达量，推测这3个基因可能与芒发育有关。.（5）将转录组测序结果与中国春基因组进行序列比对，比对到参考基因组唯一位置的比对效率从60.80%到63.33%不等。筛选到亲本和F1之间差异表达的基因有431个，无芒作为对照的下调基因有304个；结合F2的测序结果，得到共同下调基因有2个，均为新发现基因。