大白菜CMS96细胞质雄性不育分子特性研究

大白菜CMS96细胞质不育系是由大白菜株系与甘蓝型油菜细胞质不育源杂交、多代回交后获得的。该不育系生长势旺、不育性稳定,不育度和不育株率均为100%,蜜腺正常,在大白菜杂种一代生产中具有广阔的应用前景。为了定位大白菜CMS96细胞质不育所属的类型和获得其不育有关的特异序列,依据atp9、coxI、orf138和orf224保守序列设计4对引物,对3组11份大白菜材料线粒体DNA进行PCR扩增,每组材料内包含同核异质PolCMS、OguCMS、CMS96三种大白菜不育系和一种共用保持系。结果表明,atp9和coxI引物在所有材料中均有扩增产物,供试材料间没有差异;而orf138和orf224引物扩增产物存在差异。orf138引物仅在全部OguCMS和CMS96不育系中扩增出309bp特异带,而保持系和PolCMS不育系没有扩增产物;orf224引物仅在所有PolCMS中扩增出689bp特异带,而保持系、OguCMS和CMS96不育系没有扩增产物。同时,对保持系和不育系花组织mRNA进行RT-PCR分析,进一步验证了orf138和orf224引物扩增产物的特异性和一致性。同源性分析结果表明,利用orf138引物所获得的309bp大白菜mtDNA特异片段均与萝卜OguCMS、甘蓝型油菜OguCMS萝卜体细胞杂种所具有的Oguorf138高度同源,二者有172个核苷酸完全相同,有58个氨基酸完全相同;orf224引物所获得的689bp大白菜mtDNA特异片段与甘蓝型油菜的Polorf224高度同源,二者有677个核苷酸完全相同,有225个氨基酸完全相同,同源性均达到100%。初步认为大白菜OguCMS和CMS96不育系具有相似性,OguCMS萝卜所具有的orf138是导致二者不育的原因;Pol甘蓝型油菜所具有的Polorf224是导致大白菜PolCMS不育的原因。

Molecular Identification of Cytoplasmic Male Sterile Line CMS96 in Chinese Cabbage

The cytoplasmic male sterile line CMS96 was transferred into Chinese cabbage by hybridization and subsequent backcrosses using CMS donor of B.napus. CMS96 are strong in vigor, stable in sterility and normal in nectar. The degree and percentage of male sterility are 100 percent. It showed significant value in Chinese cabbage hybrids production. To identify the male sterile type and study specific fragments related to CMS96, 4 primers according to atp9?coxI?orf138 and orf224 were designed for PCR analysis in three sets of eleven Chinese cabbage materials(including maintainer?Pol CMS?Ogu CMS and CMS96), which had same nucleus and different cytoplasm in each set. Results showed fragments could be amplified by atp9 and coxI primers in all materials, and there were no differences between them. While specific fragments could be amplified by orf138 and orf224 primers. 309bp fragments could only be amplified in all Ogu CMS and CMS96, and there were no products in maintainer and Pol CMS by orf138 primers. 689bp fragments could only be amplified in all Pol CMS, and there were no products in maintainer?Ogu CMS and CMS96 by orf224 primers. RT-PCR analysis showed 309bp fragments were expressed in flowers of Ogu CMS and CMS96, and 689bp fragments were expressed in flowers of Pol CMS. Sequences were aligned by BLAST method. Results showed that 309bp fragments shared high similarity with orf138 in Ogu CMS radish and Brassica cybrids of Ogu CMS radish, which 172 nucleotide sequences and 58 amino acids were same among them. And 689bp fragments shared high similarity (100%) with Pol orf224 in B.napus, which 677 nucleotide sequences and 225 amino acids were same among them. Therefore the initial male sterility transferred to Ogu CMS and CMS96 in Chinese cabbage came from Ogu CMS in radish. The possible reason related to them was similar to Ogu orf138 in radish, and for Pol Chinese cabbage it was similar to Pol orf224 in B.napus.