Tagging Genes Relative To Cytoplas Micmale Sterility With Molecular Marker And Application In Sweet Pepper

Pepper (Capsicum annuum L.) is the most important vegetable crop in acreage after Chinese cabbage in China now. Pepper belongs to often cross-pollinated plant and has obvious superiority in hybrid breeding. The production of using excellent hybrid seeds is larger than the conventional ones by30%-50%. Pepper hybrid seeds production using male sterile could escape labor intensive hand emasculation procedures, lower cost and increase the genetic of the F1seeds. Domestic and overseas breeders are more and more interested in study of cytoplasmic male sterile (CMS) in pepper. Using CMS pepper to select hybrids is one of the most intense popular in the present world pepper breeding. In this study, SRAP and SCAR were analyzed to find the polymorphisms between restorer line5-2R1and its maintainer line5-2of Cytoplasmic Male Sterility (CMS) in sweet pepper. A pair of specific primers of SCAR were using to checking restoring gene in peppers. Then survey the fertility restoration of materials which contain restoring genes in the field. AFLP technique was employed to find the polymorphisms between the genomic DNAs of cytoplasm male sterile line T6A and its maintainer line T6B in sweet pepper. The results were summarized as follows:1. SRAP and SCAR marker linked to restoring gene for cytoplasmic male sterility in sweet pepperSRAP (sequence-related amplified polymorphism) was analyzed between restorer line5-2R1and its maintainer line5-2of Cytoplasmic Male Sterility (CMS) in sweet pepper. Using128pairs of SRAP primers,3796bands between100bp and800bp were amplified in these two lines, and averagely30bands per primer combination were amplified. There are4polymorphic loci in these two lines, but only one polymorphic fragment was found in CMS restoring line. This specific SRAP fragment was retrieved, cloned and sequenced, and the results indicate the length of the sequence is259bp. The BLAST shows that this fragment has81%identity with NADH dehydrogenase subunit5(ND5) gene and part of this fragment is highly homologous with part of BAC clone PEPBAC158K24in Capsicum frutescens. After cloning and sequencing, specific SCAR220primers were designed. By amplification testing the specific fragment was detected in restorer line but not in the maintainer line. This showed that the SRAP marker was successfully transformed to simple and stable SCAR marker. So it could be inferred that the SCAR220marker is likely related to CMS restoring gene.2. Application of marker of restoring gene SCAR220in sweet pepperSCAR220was used to analyze the96kinds of hot peppers and34kinds of sweet peppers introduced from both at home and abroad, specific band which was220bp amplified in32kinds of sweet (hot) peppers. Above130kinds of sweet (hot) peppers were crossed with cytoplasmic male sterile line21A and Liao A, and then identified the sterility of their hybrids in the field. The results showed that21kinds were detected specific bands in96kinds of peppers, but only6kinds had fertility restoration capacity and4kinds of the materials has SCAR220.8kinds were detected specific bands in14kinds of restorer line of transformation. Three were amplified specific band in another20kinds of sweet peppers, but only one mamaterial had fertility restoration capacity. This illustrated that the certain degree of stability restorer gene SCAR220could be used as a sweet pepper recovery system for early screening.3. SRAP marker linked to cytoplasmic male sterility in sweet pepperThe research amied to get specific fragment in cytoplasmic male sterile line by SRAP and lay the foundation for illuminating the molecular mechanism of the cytoplasmic male sterility in pepper further. SRAP (sequence-related amplified polymorphism) was analyzed between cytoplasmic male sterile (CMS) line T6A and its maintainer line T6B in sweet pepper. The specific SRAP fragment was reclamied, cloned and sequenced. DNA sequence of the specific fragment was analyzed with blastn and blastx in GenBank of NCBI. Total128pairs of SRAP primers were used.3844bands were amplified in two lines and31bands were amplified for an average primer combination. There were3polymorphic loci in two lines, but only one stable polymorphic fragment was found in CMS line. The specific SRAP fragment was retrieved, cloned and sequenced. Sequencing analysis indicated that the length of the sequence was201bp, named CMS T6A201. The results of blastn and blastx online showed that the fragment had higher homology with part of Ty3-gypsy-like retrotransposon sequence and had higher identities with partial protein sequence of Oryza sativa Japonica Group putative Ty3-gypsy retrotransposon protein, Medicago truncatula Reverse transcriptase, Oryza sativa Indica Group gag-pol polyprotein and Solanum demissum Putative gag-pol polyprotein, respectively. We inferred that the function of this specific fragement CMS T6A201is likely to be closely related to retrotransposon.