Biochemical And Molecular Markers Analyses On The Cytoplasmic Male Sterile Trait In Pepper (Capsicum Annuum L.)

Pepper (Capsicum annuum L.) is a worldwide vegetable with great importance to flavoring industry. It is the second largest vegetable crop in China. The cytoplasmic male sterility (CMS) of pepper is a very important pathway for heterosis breeing. Using CMS lines to produce hybrid F₁ is one of current issues for pepper improvement. Biochemical and molecular markers analyses on CMS pepper have an important value for enrichment of CMS mechanism.In the present study, expression of Peroxidase (POD) isozyme, esterase (EST), glutamate dehydrogenase (GDH), glutamate oxalocacetate transaminase (AAT) and malate dehydrogenase (MDH) isozyme and soluble protein in young leaves and flower buds with various sizes in cytoplasmic male sterile hot pepper line (21A) and its maintainer line (21B) was comparatively analyzed. Furthermore, the differences in genomic DNA of cytoplasmic male sterile pepper 21A and its maintainer line 21B were studied by means of ISSR and AFLP technique. The results are as following:1. Isozyme analysis at male gametes developmental stages between CMS 21A and its maintainer line 21BExpression of Peroxidase (POD) isozyme in young leaves and flower buds with various sizes in cytoplasmic male sterile hot pepper line (21A) and its maintainer line (21B) was comparatively analyzed. The results showed that generally more bands produced in flower buds than young leaves, indicating organ-specific expression. No difference was found in young leaves and small flower buds (0.8 - 1.5 mm long) between 21A and 21B, but some bands (POD_4b and POD_4c) in medium flower buds (2 - 3mm long) of 21B stain weekly. Two more bands (POD_2c and POD_4b) and one more band (POD_2c) were observed in the big flower buds (4-5 mm long) and super big flower buds (6.0 -7 mm long) of 21A, respectively, which suggested a close correlations between the expression of POD isozyme and microspores developmental stages, and the differences of POD zymogram in 21A and 21B occurred before the pollen abortion that observed in cytology. Analyses of esterase (EST), glutamate dehydrogenase (GDH), glutamate oxalocacetate transaminase (AAT) and malate dehydrogenase (MDH) isozyme showed that there were no differences of the four enzymes in young leaves between 21A and 21B. It was obviously to found that, however, there were more isozyme bands in flower buds than that in young leaves in the zymograms of EST, GDH and AAT isozymes, indicating organ-specific expression. With microspores development, significant difference was found between 21A and 21B among the isozyme zymograms. One more band (EST_3e) in 21B was observed than that of 21A since middle flower buds. And six bands in GDH isozyme during the big flower buds and super flower buds. Some difference expression of AAT were also seen between 21A and 21B. Futhre investigation indicated that the differences of GDH zymogram between 21A and 21B occurred at the same time as the pollen aborting stage observed by cytological analysies. While the differences of EST zymogram between 21A and 21B occurred before the pollen abortion.2. Protein Analysis between CMS Line and its Maintainer LineBy means of vertical slab SDS polyacrylamide gel electrophoresis, soluble protein in young leaves and various sizes flower buds of cytoplasmic male sterile hot pepper line (21A) and its maintainer line (21B) was comparatively analyzed. The results showed that more polypeptides produced in young leaves than flower buds, indicating organ-specific expression. Comparative analyses between the 21A and 21B found no difference in young leaves. But during male gametes development, the CMS line 21A presents a 28.6kD polypeptide that is absent in its maintainer line 21B. It suggested a close correlation between the distinct protein and microspores development of CMS line.3. ISSR and AFLP Analysis in CMS Line and Its Maintainer LineUsing 77 ISSR primers, the genomic DNA of 21A and its maintainer line 21B were studied by means of ISSR technique. There were 45 primers giving the amplified products on the genomic DNA between CMS line and its maintainer line. Among 45 primers, 7 primers presented the difference of ISSR pattern between CMS 21A and its maintainer line 21B. The number, location and depth of bands mainly expressed the difference. Furthermore, Special band ISSR-24₉₅₀ in CMS 21A was found.A special fragment CMS_21A169 was obtained from Cytoplasmic male sterility (CMS) line 21A and maintainer line 21B, by AFLP analysis. After recollection and sequencing, it was analysed with BLASTn, BLASTX and TBLASTX in GeneBank of NCBI. Partial DNA sequence of CMS_21A169 was the repeated sequence of Penaeus monodon sex and growth traits AFLP marker sequenc, which indicated that the special fragment CMS_21A169 is related to the sterility traits. The results of BLASTX and TBLASTX online showed that CMS_21A169 had high identities and positives with partial protein sequence of Solanum demissum putative gag-pol protein, Solarium demissum putative retrotransposon protein, Solanum demissum putative retrotransposon gag protein and Solanum demissum putative integrase, respectively. It suggested that CMS_21A169 is related to retrotransposon closely.