Studies On Molecular And Physiological Mechanisms Of Pepper Cytoplasmic Male Sterility And Genetic Relationship In Capsicum

Producing Hybrid seeds using cytoplasmic male sterility (CMS) as female parent is a very important way to utilize heterosis in pepper. However, basic researches of cytoplasmic male sterility mechanism in pepper are still poor. In order to enrich the understanding of cytoplasmic male sterility, quicken the usefulness of CMS-9704A, the maintainer (M-9704B), restorer (R-9701) and F1 as experiment materials, the relationship of CMS-9704A with the metabolism of ROS was investigated, some molecular marks were established and a novel gene ORF168 associated with CMS was cloned and analyzed.Capsicum is a medium-sized genus with 5 cultivated spices and 20-30 wild spices. Different opinions on the circumscription of Capsicum and relationships among different species also occur. Studies on molecular phylogeny on Capsicum have not been reported until now. In this study?focus on Capsicum's molecular phylogeny based on palynology and DNA barcoding. It is really helpful for us to solve taxonomic problems of higher taxa in Capsicum, improve study level and technique in Capsicum. The followings were our experiment results.1. At the abortion stage (pollen mother cell meiosis stage), anthers of CMS-9704A had higher contents of O2-, H2O2 and MDA than those of the maintainer. Simultaneously, there were higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in scavenging ROS in the anthers of the CMS line than in the maintainer. Abundance of Pod, Mn-Sod, Cat1, Cat2 and Cat3 mRNA in anthers of CMS-9704A corresponded with the activities of these three genes. Therefore, it is possible that the sterility in CMS-9704A is related to the abortion of microspores, induced by chronic oxidative stress caused by an abnormal increase in ROS, and also by membrane lipid peroxidation in the mitochondria in the early stage of pollen development.2. At the abortion peak (uninucleate microspore stage), anthers of CMS-9704A had lower contents of ASA, DHA, Total ASA, GSH and Total GSH than those of the maintainer 9704B, except for that of GSSH. The ratio of ASA/(ASA+DHA) between the anthers of CMS-9704A and maintainer 9704B was litter, while the ratio of GSH/(GSH+GSSG) in anthers of CMS-9704A was lower than that of the maintainer. There were lower activities and e xpression level of APX, GPX, and GR in the anthers of the CMS line than in the maintainer. Therefore, the excessive ROS accumulation in the anthers of CMS-9704A is related with the lower contents of non enzymatic antioxidant system.3. The transcription of Mdh, NADP-Me, NADP-Icdh and G6pdh were remarkably down-regulated during the period of microspores abortion, except for that of Ndpk. Therefore, it was deduced that the reaction of tricarboxylic acid cycle and pentose phosphate pathway weakened due to the decreased transcription of Mdh, NADP-Me, NADP-Icdh and G6pdh genes, the energy was seriously in short supply during the course of pollen development, Hence insufficient energy production might cause male sterility of pollen in some extent.4. The mitochondrial DNA of cytoplasmic male sterility line, its maintainer line and restorer line of Pepper were studied by RAPD analysis. All of lines had obtained amplified products through 67 out of total 500 random primers. Hey emerged polymorphism in three lines through 19 primers. Some obvious differences in mitochondrial DNA were detected between CMS and its maintainer line, and 9 special bands in CMS line were found. The relationship of those different bands in CMS to cytoplasmic male sterility needs farther study.5. The Cox?and Atp6 gene fragments, Cox?-708 and Atp6-706 (GenBank accession numbers:FJ986190 and FJ986191), associated with cytoplasmic male sterility (CMS) were cloned from the mitochondrial DNA of a pepper (Capsicum annuum L.) cytoplasmic male sterile line using PCR. These fragments did not have complete encoding region but showed the homology of 95% with Cox?and Atp6 genes from the referred Solanaceae in GenBank. No band was cloned from the mitochondrial DNA of maintainer and restorer lines. The result suggested a close relationship of pepper CMS with the variation in DNA sequence of Cox?and Atp6 gene in CMS line.6. A DNA, homologous to ORF456 verified associated with CMS in chili pepper, named ORF168, was cloned from leaves of pepper 9704A. Results revealed that the ORF168 had one nucleotide deleted in codon 150 (nt450delC) and consists of a 507-bp single-coding exon which encodes 168 amino acid compared with the original sequence data of ORF456. The RT-PCR results showed that the ORF168 gene was transcribed in buds and leaves at all developmental stage of 9704A (CMS) and hybrid F1, while no band was detected in the maintainer line. The nuclear restorer gene may restore the CMS fertility by post-transcriptional processing or translational/post-translational modification, but did not interfere with transcription of ORF168 genes directly.7. Morphological studies of pollen of 6 varieties of Capsicum annuum L. in China were reported in this paper. The result showed that the pollen shapes were rectangular, or elliptic, or suborbicular from the equatorial view and three-lobed in polar view; the aperture was three-colpate, which extended to polar area and no converaged; the ornamentation of the exine was spinule-granular under SEM. There were some differences in pollen shape and the ornamentation of the exine among different varieties. The pollen shape and the ornamentation of the exine used in this experiment could be classified. This classification was comparable to the traditional subspecies classification.8. The results from most parsimonious trees showed that the relationships of the 5 cultivated species are C. annuum, C. frutescens, C. chinese, C. baccatum, C.pubesens. Our results also showed that the C. frutescens from China and NGRL have great differences, which provided us with new evidence to the arguments that the south of Yunnan has an important role in the origin and development of pepper.Capsaicin and dihydrocapsaicin content in pericarp placenta and total fruit of 5 different varieties of China were detected. The results showed that the germplasm belonging to C. frutescens have the higher capsaicin and dihydrocapsaicin content than those in the germplasm belonging to C. annuum. There are higher genetic diversity of capsaicin and dihydrocapsaicin content and the ratio of capsaicin/dihydrocapsaicin among the different pepper varieties.