近着丝粒区油菜隐性三系核不育上位基因Rf克隆和功能分析

The Recessive Genic Male Sterility is one of the most valuable resources for heterosis utilization in Brassica napus. The three-way recessive epistatic genic sterile system has been widely used in hybrid seed production for so many advantages as follows: the stable of sterility, easy transformation of sterile gene, broad spectrum of restoration and diverse cytoplasmic sources and high yield production of hybrid seeds. But the molecular basis of the genic sterile is not well known. That sterility is controlled by two pairs of recessive duplicated sterile genes (ms1 and ms 2) and one pair of recessive epistatic gene (rf).Our previous studies show that the epistatic gene Rf located in the centromeric region of A7 linkage group in Brassica napus. Based on the features that the recombination frequency is always low around the centromeric region, this research use three different BC1 segregative populations and some special germplsams for fine mapping and cloning the Rf gene through marker development, DNA sequencing of Rf gene's region, association analysis between fertility and sequence variations, and candidate genes transoformation. The results of this study, certainly, will facilitate understanding the genetic basis of genic male sterility and enhancing their applications in hybrid breeding program in Brassica napus. The results generated from this project will provide reference for Map-based cloning those genes nearby the centromeric regions.

隐性细胞核雄性不育是油菜杂种优势利用的重要途径。甘蓝型油菜隐性核不育三系因不育性稳定、不育基因易转育、恢复谱广、无胞质负效应、制种产量高等优点在生产上得到广泛应用，但其不育分子机理研究相对滞后。该系统不育性状受两对隐性重叠不育基因（ms1和ms2）与一对隐性上位基因（rf）互作控制。本项目在前期已将上位基因Rf定位于A基因组A7连锁群和甘蓝型油菜TN群体A7染色体近着丝粒区。本研究拟针对近着丝粒区重组交换率低的特点，利用含Rf基因的3个不同遗传背景BC1大分离群体和一批特异种质资源，通过近着丝粒区特异分子标记开发、上位基因所在区域DNA序列测定、序列变异与育性表现的关联分析等手段对Rf基因进一步精细定位，图位克隆并验证其功能。预计研究结果有助于进一步阐明核不育分子机制，提高核不育三系在生产中的利用效率，同时也为近着丝粒区或交换非活跃区的基因克隆提供借鉴和参考。

隐性细胞核雄性不育是油菜杂种优势利用的重要途径。甘蓝型油菜隐性核不育三系9012AB因不育性稳定、不育基因易转育、恢复谱广、无胞质负效应、制种产量高等优点在生产上得到广泛应用，但其不育分子机理研究相对滞后。该系统不育性状受1对隐性不育基因（ms3ms3）与一对复等位基因（Rfa、Rfb、Rfc）互作控制，其中显隐性关系为Rfa>Rfb>Rfc, Rfa能够恢复由Rfb造成的不育。本项目根据前期初步定位的结果，利用含Rf基因的不同遗传背景BC1大分离群体和一批特异种质资源，开发标记，对Rf基因进一步精细定位，图位克隆该基因并验证了其基因功能。研究结果表明， Rfa、Rfb和Rfc之间存在丰富的结构变异，相对于Rfa和Rfb，Rfc存在大片段缺失。转Rfb (G14)基因的拟南芥表现出和9012A一致的不育表型，并且由G14导致的不育性状能够被恢复基因Ms3恢复；G14编码一个核定位蛋白，G14在拟南芥中的异位表达负调控绒粘层发育相关的重要基因，延迟绒粘层细胞的程序化死亡，四分体释放受阻，从而引起不育。该项目的顺利实施，初步阐明了该基因的功能和表达模式，探明了甘蓝型油菜隐性三系核不育的分子机制，丰富了核不育的遗传理论， 同时分离该基因过程中产生的分子标记可应用于该系统的分子标记辅助选择，将大大促进该系统在生产中的应用。