Cloning And Expression Analysis Of Genes Relative To Cytoplasmic Male Sterility In Pepper(Capsicum Annuum L.)

Pepper(Capsicum annuum L.) originated from middle-south America, is the second largest vegetable crop in China, which plays an important role in the world. According to the China ministry of Agriculture’s statistics, the planting area of pepper in China has been kept aroud1.4million hm2per year since2000. Because of it’s obvious heterosis, pepper hybrid F1seeds have been widely used to increase yields and improve the quality of peppers. Using cytoplasmic male-sterile (CMS) line of pepper to produce hybrids is the important pathway of hereosis utilization, and is one of the major objectives in the present world pepper breeding. Biochemical and molecular markers analyses on CMS pepper have an important value for enrichment of cytoplsmic male-sterile mechanism, however, basic research on the CMS mechanism in pepper is very limited.In this study, genomic DNA extracted with CTAB method while mtDNA extracted by sucrose sedimentating combined with differential centrifugation method, SRAP technique was employed to find the polymorphisms between the DNAs of cytoplasmic male sterile line21A and its maintainer line21B in hot pepper, qRT-PCT method was used to analyze the expression of those cytoplasmic male-sterile related genes. The results were summarized as follows:1. SRAP analysis between the genomic DNAs of cytoplasmic male sterile line and its maintainer line in hot pepper64pairs of SRAP (sequence-related amplified polymorphism) primer combinations was employed to detect the polymorphisms between the genomic DNA of cytoplasmic male sterile line21A and its maintainer line21B, it showed that1792bands between100-1000bp were amplified in two lines, everagely28bands per primer combination, there were4polymorphic loci between this two lines, two of those, polymorphic fragments, Me7Em4-280and Me2Em7-230were found in CMS line21A. These specific SRAP fragments were cloned and sequenced, the length of this two sequences were283bp and236bp, blast in NCBI indicated that Me7Em4-280has high identity with many plants’ NADH dehydrogenase subunit D (ndhD) gene, Me2Em7-230has74%identity with Oryza sativa Indica Group strain WA-CMS mitochondrion gene. Specific SCAR primers were designed and both were only detected in CMS line but not in the maintainer line, so it could be inferred that this two SCAR markers were likely related to CMS.2. SRAP analysis between the mitochondrial DNAs of cytoplasmic male sterile line and its maintainer line in hot pepperMitochondrial DNA was extracted from etiolated seedling using sucrose sedimentating combined with differential centrifugation method, SRAP(sequence-related amplified polymorphism) technique was detected between mtDNA of cytoplasmic male sterility line21A and its maintainer line21B of pepper, analysis showed that:(1) A total of3072amplified fragments between100-1000bp and nine polymorphic fragments were detected by applying128SRAP primer combinations, the polymorphic fragment percentage was0.29%, seven of the polymorphic fragments were detected in male sterility line21A.(2) The polymorphic fragments were isolated, cloned and sequenced,blast of the9clones in Genbank showed that it all have some homologous with energy metabolism genes.(3) According to the sequences,specific primers were designed to transform the SRAP marker to more stable SCAR marker, and four of them only detected in the21A line, this suggested that the newly detected fragments were likely related to cytoplasmic male sterility.3. Expression analysis of genes related to cytoplasmic male sterility in hot pepperQuantitative real-time polymerase chain reaction (qRT-PCR) has already been extensively used in several plant species as an accurate technique for gene expression analysis. The expression of CMS721, the specific fragment which can only found in CMS line21A, in different tissues were analyzed by qRT-PCR. The results indicated that CMS721only expressed in cytoplasmic male sterile line21A, and different tissue has different expression. The expressions of CMS721decreased quickly since mid-bud stage, it was thus speculated that CMS721regulated the expression of energy metabolism related protein in anther, which might cause microspore abortion and lead to cytoplasmic male sterility.