大麦灌浆期耐热基因HvHTT1的定位与克隆

During grain filling，high temperature stress impacts seed development to decrease yield and quality in barley, wheat and rice. Barley high temperature tolerant (BHTT) mutant was identified from Vlamingh irradiated 7Li-beam. The BHTT mutants showed plump grain and higher thousand kernel weight compared to Vlamingh after high temperature stress in grain filling period. Genetic analysis identified a single gene HvHTT1 controlling the high temperature tolerance. In this study, positional cloning and BSR-seq will be combined together to isolate the HvHTT1 gene. A DH population of BHTT/Buloke will be used to preliminarily map the HvHTT1 gene. An F2 population from cross of BHTT/Buloke will be used to fine map the HvHTT1 gene. 50 individuals with mutated phenotype and 50 individuals with wild type will be screened out from BHTT/Buloke DH population to construct two RNA pools, respectively. A 20X deep sequencing will be performed to the mixed RNA pool. The sequence information of the mixed RNA pool and the parents Buloke and Vlamingh will be analyzed. SNP (InDel) index and different expression will be used to predict candidate genes of HvHTT1. Both BHTT/Buloke and BHTT/Vlamingh F2 population will be used to further test the candidate genes. The haplotype and the expression pattern of HvHTT1 gene will be investigated. Diagnostic molecular markers will be developed for barley molecular breeding. Meanwhile, homology-based cloning will be used to isolate homologous genes from wheat, rice and maize and develop their molecular diagnostic markers. Cloning and characterization of HvHTT1 gene would facilitate progressing high temperature tolerance breeding in barley and other cereal crops.

热害严重威胁稻麦类作物灌浆期生长发育，导致产量和品质下降。通过锂离子辐射诱变大麦Vlamingh，获得耐热突变体BHTT。灌浆期进行高温胁迫，结果发现，该突变体在高温胁迫下籽粒饱满、千粒重显著高于亲本。遗传分析表明，该突变体耐热性状受单基因HvHTT1控制。本研究拟采用图位克隆和BSR-seq相结合的方法克隆该基因。利用BHTT/Buloke的DH群体对其进行初步定位，加密分子标记，利用该组合F2群体进行精细定位，同时构建该组合DH群体突变单株RNA混合文库，进行深度测序，比对Buloke 和Vlamingh重测序数据库，预测候选基因。利用BHTT/Buloke和BHTT/Vlamingh两个F2群体验证候选基因。分析HvHTT1基因单体型及表达模式，在小麦、水稻、玉米中克隆HvHTT1的同源基因，并开发其分子标记，为禾本科作物耐高温分子育种提供突破口。

热害严重威胁稻麦类作物灌浆期生长发育，导致产量和品质下降。通过锂离子辐射诱变大麦Vlamingh，获得耐热突变体BHTT，后命名为VVY。高温胁迫下，分蘖期VVY叶片中叶绿素a、b降解速度比Vlamingh快，提示目标基因vvy即HvHTT1是一个温度诱导的叶绿素含量调控基因。利用VVY/Buloke的DH群体对其进行初步定位，加密分子标记，精细定位该基因于0.428Mb的物理区间内，有11个候选基因。构建VVY/Buloke组合BC2F3群体进行图位克隆，成功克隆了vvy基因。对高温处理前后VVY 和Vlamingh进行转录组测序，进一步验证了候选基因，并比较vvy基因的表达调控网络。分析vvy基因单体型，在小麦、水稻、玉米中克隆vvy的同源基因，并分析了其进化。VVY × Buloke DH群体产量分析显示，与vvy基因的定位区间重合的QTL能够解释10.4%的产量性状变异。单体型分析表明，耐热突变体重vvy基因缺失6个碱基，此Indel有望开发为大麦耐热分子标记。后续将进行转基因，进一步研究温敏叶绿素调控基因vvy的功能，为禾本科作物耐高温分子育种提供突破口。