Molecular Marker, Cloning And Expression Analysis Of Genes Associated With Cytoplasmic Male Sterility In Pepper(Capsicum Annuum L.)

Pepper(Capsicum annuum L.) is the second largest vegetable crop in China, which plays an important role in the world. Pepper have obvious superiority in hybrid breeding. The cytoplasmic male sterility (CMS) of pepper is a very important pathway for the usage of heterosis. The application of CMS lines to produce seeds is one of the current issues, and marker assisted selection can enhance the application of CMS lines to produce hybrid Fi for pepper cultivar improvement. In this study, AFLP technique was employed to find the polymorphisms between the genomic DNAs of cytoplasm male sterile line21A and its maintainer line21B in hot pepper. We selected reliable reference genes by qRT-PCT in pepper, and cloned gene related to cytoplasmic male sterility using RGA technique. The expression of this gene was also analyszed in cytoplasm male sterile line21A and its maintainer line21B. The results were summarized as follows:1. Establishment and optimization of AFLP technique system for pepperAn optimized AFLP analysis system was established with pepper cytoplasmic male sterile Line21A and maintainer21B by optimizing several main factors of AFLP fingerprints, which included the method of genomic DNA extraction, enzyme digestion and ligation, pre-amplification and selective-amplification, electrophore-sis, dyeing conditions etc. By mean of this optimized system, a clear, credible AFLP fingerprints could be obtained for gene location of pepper cytoplasmic male sterility.2. AFLP analysis associated with cytoplasmic male sterility in hot pepperAFLP technique with64pairs primer combinations of Pst I-NNN+Mse I-NNN was employed to find the polymorphisms between the genomic DNAs of cytoplasm male sterile line21A and its maintainer line21B in hot pepper. It showed that5447bands were amplified in two lines with60pairs of primer combinations. There were4polymorphic loci in two lines and only2polymorphic fragments AGC/CGC378and ATC/GAG446were found in cytoplasm male sterile line. These specific AFLP fragments were cloned and sequenced. Sequencing analysis indicated that the length of the sequence were340bp and408bp, with94%and96%identities of its nucleotide sequence to a part of the nicotiana tabacum mitochondrial DNA genome.3. Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative Real-Time PCRQuantitative real-time polymerase chain reaction (qRT-PCR) has already been extensively used in several plant species as an accurate technique for gene expression analysis. And, accuracy in qRT-PCR requires the use of stable endogenous controls as the reference genes. Normalization with multiple reference genes is very important, thus we evaluated reference genes in a number of different experimental conditions. Using two distinct statistical algorithms softwares by geNorm and NormFinder, we evaluated a total of ten candidate reference genes (Actin,Actin1,Actin2,18S rRNA, PPR1, GAPDH, TEF1A, EF1α, UEP and CYC) in different experimental sets:four abiotic stress treatments (leaves of cold-and heat-stress treatments, leaves of salt-and drought-stress treatments), two hormone treatments(leaves of salicylic acid and gibberellic acid treatments), and four different pepper tissues (root, stem, leaf and flower). The results indicated that UEP, EF1α and Actin1are the most suitable combination of reference genes as internal control for reliable qRT-PCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with pepper plant samples under conditions and gene expression analysis other than those tested here.4. Cloning and expression analysis of gene related to cytoplasmic male sterility in hot pepperThe putative fragment was amplified by using specific primers designed according to the pepper gene orf456associated with cytoplasmic male sterility between the genomic DNAs of cytoplasmic male sterile line21A and its maintainer line21B in hot pepper. And the target fragment was found only in cytoplasmic male sterile line. Cloning and sequencing analysis indicated that the length of the sequence was292bp, named CMS292, with99%identities to the reported orf456nucleotide sequence, presumably encoding for97amino acids. The expressions of CMS292in roots, stems, leaves and flower buds at bud and flowering stages were analyzed by qRT-PCR. The results indicated that CMS292expressed in all tissues of cytoplasmic male sterile line. The expressions of CMS292in roots and flower buds were higher than in stems and leaves at bud stage, and its expression in stems and flower buds were lower than in roots and leaves at flowering stage. From bud stage to flowering stage, the expressions of CMS292in roots, stems and flower buds were downtrend, while its expressions in leaves were raised. It was thus speculated that CMS292regulated the expression of protein in anther, which might cause microspore abortion and lead to cytoplasmic male sterility.