油菜野芥NSa细胞质雄性不育系的特异性分子鉴定

Molecular Identification and Distinctness of NSa Male Sterile Cytoplasm in Brassica napus

常规细胞质类型鉴定方法花费时间长、工作量大、鉴定效率低、应用性较差。基于基因组重复序列的PCR技术(rep-PCR)在重复序列丰富的线粒体和原核生物的基因组鉴定中效果较好,本研究对8份油菜资源的细胞质进行分子鉴定,结果表明rep-PCR可以明确区分甘蓝型油菜中常见的6种雄性不育细胞质。同时对甘蓝型油菜和野芥(Sinapis arvensis)体细胞杂交创建的、育性较为稳定的NSa野芥雄性不育胞质进行了分子特异性鉴定,获得了2条NSa不育胞质的特征DNA条带。但波里马和萝卜质不育细胞质的特异性引物在NSa中不能扩增出特异性片段,说明NSa不属于这2种细胞质类型。本研究结果为该不育胞质系统的利用和知识产权保护提供了理论依据和技术支撑。

Cytoplasmic male sterility （CMS） has been widely used as an efficient pollination control system in rapeseed （Brassica napus） hybrid production. With the exploitation of wild species of Crucifer and the application of biotechnology, several cytoplasrnic male sterile systems of oilseed rape have been reported in recent years. Traditional methods used for the identification of cytoplasm, however, are time and labor consuming with low efficiency. Thus, it is highly desirable to develop an efficient and accurate identification method for cytoplasm classification. Repetitive sequence based polymerase chain reaction （rep-PCR） has proved to be a good method for the identification of mitochondrial and procaryotic organisms which contain abundant repetitive sequences in genome. Fingerprints of six cytoplasmic male sterility materials including Nsa, Pol, nap, No, Ogu, and Ath were mapped using rep-PCR. Based on the PCR amplification band patterns by two primer pairs, six male sterile cytoplasms in rapeseed could be distinguished unambiguously. Two bands, ERIC750 and ERIC4000, were unique to NSa CMS which was derived from somatic hybrids between B. napus and S. arvensis. The PCR results using gene specific primers also showed that NSa is a novel cytoplasmic male sterility system in oilseed rape. Thus, a method for cytoplasm classification in oilseed rape was established. The two specific markers for NSa cytoplasm not only confirmed the distinctness of NSa cytoplasm from other male sterile cytoplasm at molecular level, but also provided useful tools for the protection of intellectual property right of this potential CMS material in rapeseed breeding and production.