Study On Differences Of Mitochondrial Genome Between Cytoplasmic Male Sterile Line And Maintainer Line In Cotton (Gossypium Hirsutum L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait. It has been widely used in breeding programs to produce F1 hybrid seed in crops. The"three-line system"(involving a CMS line, maintainer line, and restorer line) can allow for large-scale cultivation of cotton hybrids. In cotton, there are several different types of CMS lines: CMS-D2 (G. Harknessii), CMS-D8 (G. trilobum), 104-7A, JinA, XiangyuanA and so on. CMS line P30A (cytoplasm originated from 104-7A) has been used in practical production.CMS-determining factors are believed to be located at the mitochondrial genomes. But CMS-associated genes in cotton are almost unknown. In this study, CMS line P30A, maintainer line P30B, restorer line Y18R, were used as the main materials. Mitochondrial gene probes were used to identify the differences between the P30B normal fertile (N-) and the P30A male sterile (S-) cytoplasm with Southern blot method. The differential sequences between N- and S-cytoplasm were cloned, and cytoplasm-specific RFLP, SCAR and SSR markers were developed. Further expression analysis was performed on the differential sequences. These work made foundation for cloning CMS-determining genes in cotton.Conserved sequences of 31 mitochondrial genes were obtained from mitochondrial genome of cotton by homolog cloning strategy. 20 mitochondrial genes were selected to perform Southern blot analysis on mtDNA of CMS line P30A, maintainer line P30B, and restorer line Y18R. Among the 20 genes, atpA, atp9, ccmB, nad1bc, nad6, nad7c and rrn18 revealed restriction fragment length polymorphisms (RFLPs) between N- and S-cytoplasm. The differences on atpA locus were most obvious.All of the EcoRI restriction fragments of atpA were cloned by inverse PCR (IPCR) technique for the first time. And further, all of the HindIII fragments were cloned with TAIL-PCR method. In N-cytoplasm, the EcoRI restriction fragments were 2225 bp and 5083 bp in length, the HindIII restriction fragments were 11689 bp and 8501 bp in length. In S-cytoplasm, the EcoRI restriction fragments were 2194 bp and 3297 bp in length, the HindIII restriction fragments were 11658 bp and 9139 bp in length. Both N- and S-cytoplasm had an intact atpA copy and a 3'truncated copy. The full length of atpA was 1524 bp.The truncated copies in N- and S-cytoplasm were truncated at 1352 bp and 1336 bp of atpA coding region, respectively.At the 3'flanking, and 161-212 bp downstream of, the intact atpA gene, a simple sequence repeat (SSR) locus was found between N- and S-cytoplasm. In N-cytoplasm, the locus was (TAA)7(TA)6; in P30A, JinA, XiangyuanA and CMS-D2, the locus was (TAA)3(TA)2; in CMS-D8, the locus was (TAA)4(TA)3. And the SSR locus was developed to be a molecular marker: SSR160.As far as the 3'truncated atpA copy was concerned, the truncating site (breakpoint) was different in N- and S-cytoplasm. Following the breakpoints, chimerical fragments of 515 bp ("N515") and 555 bp ("S555") were found in N- and S-cytoplasm, respectively. According to the chimerical sequences, two cytoplasm-specific sequence characterized amplified region (SCAR) markers were successfully developed. Three of the PCR markers can be used to distinguish N- and S-cytoplsam in cotton.Additionally, based on SSR polymorphisms and SNPs in truncated region, five CMS lines can be classified into two major groups: one corresponds to CMS-D8 and the other consists of other four CMS lines. Furthermore, a new SCAR marker was identified to distinguish CMS-D8 from other CMS lines.RT-PCR analysis showed that these atpA copies in N- and S-cytoplasm were all transcribed. There were six RNA edit sites in full length atpA coding sequences.The RNA editing rates of the full length atpA gene were the same between the two cytoplasms, but when the truncated copies were conserned, the RNA editing rates in S-cytoplasm were obviously higher than the N-cytoplasm. In S-cytoplasm, the RNA editing rates of truncated copy were close to 100%, while in N-cytoplasm the values were less than 60%. It indicated that the selective pressure upon the truncated copy of S-cytoplasm is heavier than that of N-cytoplasm, and further implied that the truncated copy of N-cytoplasm had lost function, while the truncated copy of S-cytoplasm was likely to possess new function. The full-length cDNAs of the intact and truncated atpA copies in S-cytoplasm were obtained by the cRT-PCR method. The results showed that the transcript of intact atpA gene covered the SSR160 sequence, and the transcript of truncated atpA gene covered the S555 sequence.We found that at the 3'downstream of atpA gene, there were several short repeat sequences which belonged to the 3'coding sequences of nad6 gene. In S-cytoplasm, these short repeat sequences were located at the"breakpoint"loci, this implied that the short repeat sequences played an important role in rearrangement of mitochondrial genome in S-cytoplasm. According to the short repeat sequences, a SCAR marker was developed to distinguish N- and S-cytoplasm of cotton. Northern blot analysis showed that there was an identical transcript of nad6 in"three line"and F1 cultivars. In addition, full length transcript of nad6 gene was obtained with cRT-PCR method, the mRNA of the nad6 gene was found to be processed -14 bp and -15 bp upstream of the inframe stop codons.rrn18-rrn5 gene cluster was found located at the 5'upstream of atpA gene, and an rrn18 copy was found to be deleted in S-cytoplasm when compared with N-cytoplasm. This indicated that rrn18 gene may be associated with CMS in cotton.In this study, all of the EcoRI and HindIII restriction fragments of atpA in N- and S- cytoplasms of cotton were cloned for the first time. SCAR and SSR molecular markers were developed to distinguish N- and S-cytoplasm. And the differential sequences between CMS-D8 from the other four CMS lines were determind. And parts of nad6 coding sequences were found to be participated in the rearrangement of cotton mitochondrial genome, especially in the S-cytoplasm. And one rrn18 copy was found to be deleted in S-cytoplasm when compared with N-cytoplasm. Transcription of atpA and nad6 was analysed. And RNA editing rates of truncated atpA gene were different between N- and S-cytoplasm. The results made foundation for further cloning CMS-determining genes in cotton.