Molecular Genetic Analysis of Cytoplasmic Male Sterility by Mitochondrial Genome Sequencing and Isolation of a Restorer-of-fertility Candidate Gene in Pepper (Capsicum annuum L.)

Abstract

Cytoplasmic-genic male sterility (CGMS), which involves the interactions and conflicts between mitochondrial and nuclear genomes, has been widely used for hybrid seed production in crops including chili peppers. Although the candidates for CMS-associated mitochondrial gene were isolated and molecular markers linked to Restorer-of-fertility (Rf) have been developed in pepper, the understanding of CGMS mechanism and the utilization of reliable molecular breeding system have been limited. Therefore, comparative analysis of CMS and normal mitochondrial genomes, isolation of an Rf candidate gene, and deduction of the origin of CMS cytoplasm were performed in this study. In the first chapter, comparative analysis between mitochondrial genomes from a CMS pepper line, FS4401 and a fertile pepper line, Jeju was conducted. The complete mitochondrial genomes which were 507,450 and 493,911bp in length were assembled in FS4401 and Jeju, respectively. Although most of gene contents were conserved between two genomes, extensive rearrangement of genome structure resulted in the generation of eighteen blocks of sequences which were syntenic between two mitochondrial genomes and unaligned sequence segments between them. The CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments which were specific to FS4401. Although severe rearrangements and lack of any similarity with reported sequences in this region hampered the elucidation of detailed mechanism, presence of repeated and overlap sequences on DNA segments implied that extensive rearrangements by nonhomologous end-joining followed by substoichiometric shift due to recombination through repeated sequence might be involved in generation and integration of this region on the master DNA molecule. Further analysis using mtDNA pairs of CMS-normal cytoplasms in other plant species showed common features of DNA regions around CMS-associated genes. In the second chapter, three kinds of mapping strategies were used to isolate pepper Rf gene. Firstly, pepper BAC clones which contain sequences homologous to petunia Rf gene were screened and mapped. Secondly, AFLP analysis was performed using more than one thousand primer combinations. Finally, comparative mapping was conducted using tomato genome sequence. As the result, a group of selected BAC clones and AFLP markers were mapped on pepper DNA region that was co-segregated with the Rf gene. By six times of chromosome walking started from this region, the sequence which spanned DNA region containing Rf was obtained. Prediction of expressed genes in this sequence using transcriptome analysis screened an Rf-candidate gene, PPR6. The PPR6 gene encoded a pentatricopeptide repeat protein in which degenerative 35 amino acid motif was repeated for fourteen times. Specific expression of this gene in restorer lines showed that PPR6 is a strong candidate for Rf in pepper. In the third chapter, the origin of CMS cytoplasm was deduced by using plastid DNA markers. The complete sequence of plastid genome in a pepper line FS4401 was assembled and used as the source for marker development. Two plastid sequences, trnH-psbA and rpl16-rpl18 intergenic sequences, were used to analyze cytoplasm types of pepper germplasms which include six Capsicum species. Plastid barcode analysis revealed that cytoplasm types can be divided into six types and four types for trnH-psbA and rpl16-rpl18, respectively. The sequences on these two regions in CMS pepper lines were identical to the sequences of a cytoplasm type of a particular clade in C. annuum. For further investigation, two molecular markers were designed from TrnL-TrnF and rpl16-rpl18 intergenic regions, respectively. Application of these markers to a larger number of germplasm confirmed that the cytoplasm type of CMS is identical to the cytoplasm type of the particular C. annuum clade. These results suggest that the CMS cytoplasm of pepper may originate from an interspecific cross in which the seed parent belonged to the C. annuum clade. The results of this study are expected to contribute to reliable and efficient molecular breeding for CGMS system as well as provide insights in evolution of CMS cytoplasm and Rf gene in pepper.