利用分子标记鉴定白菜细胞质雄性不育类型的研究

Identifying Cytoplasmic Male Sterile Types of Chinese Cabbage(Brassica campestris L.)by Molecular Markers

通过对9份白菜细胞质雄性不育材料的不育胞质类型进行分子标记鉴定,以期为今后更好的利用分子标记来进行辅助育种奠定基础。通过CTAB法提取白菜的基因组DNA,根据Gen Bank中orf138基因保守序列设计2对特异引物,对9份白菜基因组DNA进行PCR扩增,将扩增出的条带进行测序,在NCBI中进行同源性比对,最终确定其不育胞质类型。结果表明:2对特异引物PI/PII和PIII/PIV只在4份白菜雄性不育基因中均扩增出条带,在另外5份材料中未扩增出条带,与田间育性鉴定相符,获得4份白菜Ogura胞质雄性不育的分子标记;在Gen Bank中用BLAST进行同源性比对分析,发现L1-CI、L3-CI、L3-F1 3个不育材料的特异片段与已报道的青花菜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:HQ149728)同源度高达100%,L1-F1的同源度达99%,出现了一个变异位点,另外5份材料的胞质不育类型有待进一步研究。通过该方法鉴定出9份材料中有4份材料是萝卜胞质雄性不育的材料,得到了4条分子标记。

Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identifiedby molecular marker in the study in order to better use molecular marker to conduct the assisted breeding inthe future.Genomic DNA was isolated from Chinese cabbage by CTAB method.The design of two pairs ofspecific primers was performed on conserved flanking region of orf138 gene in the Gen Bank.PCR wereperformed with genomic DNA of nine Chinese cabbage materials.The bands were sequenced.The homologouscomparison was conducted in NCBI,and finally,the type of sterile cytoplasm was determined.The resultsshowed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs ofspecific primers PI/PII and PIII/PIV,while the other five materials did not obtain the relative bands.The resultwas consistent with the field sterility identification.And then four molecular markers of Chinese cabbageOgura cytoplasmic male sterility（CMS） were obtained.After conducting a homologous comparative analysis with BLAST in Gen Bank,it was found that the homologous degree was 100% in specific segments of the threesterility materials（L1-CI,L3-CI and L3-F1） and Ogu orf138 gene（Gen Bank accession No.：HQ149728） ofthe reported broccoli Ogu CMS.The homologous degree of L1-F1 was 99% with a variation point.The type ofcytoplasmic male sterility of the other five materials needed further research.Four materials of the nine wereidentified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.