大麦分蘖角度动态型基因(BDTA1)的克隆与功能鉴定

Tiller number and tiller angle are two agronomically important traits for ideotype breeding in gramineous crops. A barley mutant BDTVla, which showed mutated characteristics of prostrated tillers in vegetative growth, erected tillers after booting stage, and multiple tillers, was controlled by single gene named as barley dynamic tiller angle controlling gene1 (BDTA1). In this study, strategies of positional cloning and Mutmap will be utilized to isolate the BDTA1 gene. The DH population of BDTVla/Buloke will be used to preliminarily map the BDTA1 gene. The F2 population from cross of BDTVla/Buloke will be applied to fine map the BDTA1 gene. A total of 50 individuals with mutated phenotype will be screened out from BDTVla/Buloke F2 population to construct a DNA pool. A 10X deep sequencing will be performed to the mixed DNA pool. The sequence information of the mixed DNA pool and the parent Buloke will be analized. SNP (InDel) index will be used to predict candidate genes of BDTA1. Both BDTVla/Buloke and BDTVla/Vlamingh F2 population will be used to further test the candidate genes. The overexpression and the RNAi vectors for BDTA1 gene will be constructed to transfer barley and rice, respectively. The function of BDTA1 gene will be verified. The haplotype and the expression pattern of BDTA1 gene will be investigated. Meanwhile, molecular diagnostic markers will be developed for barley molecular breeding. The cloning and the dissection of BDTA1 gene would facilitate the programs of ideotype breeding in barley and other gramineous crops.

分蘖数目和角度是禾本科作物株型育种的两个重要农艺性状。大麦突变体BDTVla具有早期匍匐生长、拔节后挺立的动态分蘖角度和多分蘖的突变表型，该突变性状由单基因BDTA1控制。本研究拟采用图位克隆和Mutmap相结合的方法克隆该基因并进行功能鉴定。利用BDTVla/Buloke的DH群体对BDTA1基因进行初步定位，加密分子标记，利用该组合F2群体进行精细定位，同时构建突变单株混合测序池，进行深度测序，与Buloke的基因组序列进行比对，预测候选基因。利用BDTVla与Buloke和Vlamingh的两个F2群体检验候选基因。构建BDTA1基因的超表达及RNAi载体，分别转化水稻和大麦，鉴定该基因的功能。分析BDTA1基因单体型及表达模式，开发该基因分子诊断标记，用于株型分子育种。克隆大麦分蘖角度动态型基因BDTA1，将为禾本科作物的理想株型育种提供理论支持和技术支撑。

大麦是世界第四大谷物，也是我国主要种植作物之一。分蘖数目和分蘖角度是禾本科作物的两个重要农艺性状，当分蘖与主茎间夹角增大到极致，分蘖的负向重力性减弱或者消失，作物就形成匍匐生长。作物匍匐生长能减少叶片之间相互覆盖，增大截光面积，从而增加光能利用；也能覆盖地表，保持水土和养分，减少杂草的生长。大麦中苗期匍匐生长相关遗传机制尚不明确。本研究采用的大麦突变体V-V-HD具有早期匍匐生长，拔节后挺立直立生长的动态表型，这种表型被认为是作物的理想株型。本研究采用大麦突变体亲本V-V-HD和野生型亲本Buloke杂交构建的双单倍体系群体共208个系作为作图群体，利用图位克隆和QTL定位方法来定位控制大麦匍匐生长的基因。通过分析处理V-V-HD/Buloke双单倍体系群体的表型数据，经卡方测验验证，证实大麦匍匐生长的表行是由单基因控制。通过选取47个已经确定表型数据的双单倍体单株和大麦已经发布的单核苷酸多态性（SNP）标记，经过试验证明目标基因位于大麦3号染色体上，在SNP标记2_0343附近。通过查找已经发布的SSR标记和设计新的插入/缺失标记，最终目标基因被定位在插入/缺失标记InDelz3028 (631.84Mb)和InDelz3032 (635.37Mb)之间，大小为3.53Mb，共有52个候选基因，并被分为9大类。其中控制赤霉素20-氧化酶3的矮化基因、控制锌离子结合位点基因，被确定为匍匐生长候选基因。候选基因功能验证正在进行中。本研究结果将有助于我们了解匍匐生长这个性状，本研究所设计的引物将有助于推动大麦匍匐生长性状的分子标记辅助选择育种进程。