Mitochondrial Genome Of Annual Wild Rice And Rearrangement In The Progenies Of Wide-hybrids

Rice (Oryza sativa L.) is one of the most important cereal crops in china, and hybrid rice is the core element for rice yield increase in China. Of which, three-line hybrid rice still plays central roles for hybrid rice commercial production. Cytoplasmic male sterility (CMS) is the basis for the success of three line hybrid rice, and the fertility of CMS is controlled by fertility restorer genes. Thus, CMS works as a window to understand plant mitochondrial-nuclear communication. Research of rice mitochondrial genome will greatly help us to understand mitochondrial-nuclear interaction at molecular level. In this study, we sequcenced a annual wild rice (W20) mitochondrial genome which contained gametophtic CMS cytoplasm, and investigated mitochondrial recombination in the process of backcrosses between W20and YueTai B (YB), and further analyzed the characteristics, evolution of rice mitochondrial specific-sequences (MSS). The main results are summarized as follows:1. A typical annual wild rice (W20) mitochondrial genome was sequenced by Solexa sequencing platform. The size of W20mitochondrial genome is542,103bp, very close to that of O. rufipogon (CW). Nuclear-like sequence covered57.6%(312,096bp) in W20mtDNA,44.6%of W20mtDNA sequences are homologous to the sequences in a approximately250kb region (positions20597115-20845508) in chromosome12. Comparative analysis of6sequenced rice mitochondrial conservative sequences, found that the arrangement of mitochondrial conservative sequences are more disorder, we deduced that CMS have relevance to the acceleration of mitochondrial genome evolution. In W20mtDNA, it is shown that orf310as a chimeric gene composed of portions of orf288and reduced transcript when introducing restorer genes; There is a mitochondrial recombination via a invert178bp repeats caused W20mtDNA in a multiple molecules state. 2. Comparative analysis of W20and other five sequenced rice mitochondrial genomes, found that rice mitochondrial genome exist a large number of mitochondrial specific-sequences (MSS), which account for7.0%in W20. Phylogenetic analysis by25pairs of MSS primers which display different amplicons among38AA-genome rice species, different rice varieties can basically clustered into separated groups by MSS, and non-AA genome rice apparently distinguished from others. As a molecular marker, MSS is effective for mitochondrial evolution analysis. Additinaly, we detected some candidate ORFs in the MSS regions, and most of them could be transcribed.Meanwhile, we detected17CMS specific MSS by comparative analysis sporophytic CMS (WA) and gametophytic CMS (CW and LD) mitochondrial genome, of which,9were specific for sporophytic CMS,4for gametophytic CMS, and the other4were shared by all of the CMS types. This kinds of MSS can used as marker to differentiate different CMS types. Southern blot showed that the location of CMS specific MSS are conserved in mitochondrial genome. Additionally,11predicted ORFs in CMS specific MSS are found transcriptable, this kinds of ORFs may help to explain the mechanism of rice CMS.3. In order to analyze the mitochondrial recombination during the process wide-hybridization, we constructed sequential backcross populations between annual wild rice (W20) and cultivated rice YB. We detected change of recombination via four short repeats among progeny of hybridization between W20and YB, two repeats is direct repeats (Repeat A and Repeat B), the other two are inverted repeats (Repeat C and Repeats D), with sizes ranging between82and144bp; in the comparative analysis between W20and W20/YB mutant, we found that the copy number of orf79and sublimon which produced from recombination via short repeats between orf79and other regions dramatically decreased, and became SSS. We compared OsRECAl, OsRECA4, OsRECA7, OsMutsl and OsOSBl genes possibly related to rice mitochondrial recombination, cDNA sequencing results showed only OsRECA4and OsRECA7exist differences between W20and YB. After BC2, genotypes of the progenies derived from W20/YB are the same as YB at these two allelic sites.4. We compared the genomic structure of orf288and homologous gene orf290、 orf310and orf352, the difference of amino acid mainly exist in N terminal among these four chimeric genes, and the C terminal is almost same. Besides, the location in mitochondrial genome of these chimeric genes is variated. PCR analysis showed that orf288exists only in Hong-Lian CMS maintainer line YB, orf290exists gametophytic CMS lines YA、W15A and W46A, orf310exists in W20A, W34A, BaoYuanA, ChuJin23A and HeXi41A, all the7sporophytic CMS lines contained orf352. With the result of the transgenic test, we found that both orf310and orf290may be related to the fertility of rice.