Map-Based Cloning Of Fertility Restoring Gene Of CMS And Analysis Of PPR Gene Family In Cotton

Plant cytoplasmic male sterility (CMS) is a maternally inherited trait that is unable to produce viable pollens, which plays an important role in utilization of hybrid vigor. Comparing with hand-emasculation and pollination, and genetic male sterile lines, utilization of CMS lines is much more effective and economical in producing commercially hybrid seeds. Cotton is not only the world's most important natural textile fiber and a significant oilseed crop, but also a crop that is of significance for foil energy and bioengergy production. The heterosis in cotton hybrid is much significant, especially in improving yields, fiber qualities, disease-resistances and stress tolerance. Some hybrid strains of cotton with CMS have been applied in production, but the utilization of CMS is rather limited because of the low yield of restoring lines and the noxiousness of the exotic cytoplasm in the male sterile lines. Therefore, the cloning of restoring gene plays an important value on studying the mechanism of CMS and heterosis utilization.With the exception of Rf2 from maize, all cloned Rf genes are members of the pentatricopeptide repeat gene family (PPR).The PPR protein family is one of the largest families which was found recently. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. Up to now, PPR proteins have been identified in many eukaryotes, but there is still no report on PPR gene in cotton.A total of 2803 pairs of EST-SSR primers designed from cotton ESTs in Genbank were screened the restoring line 0-613-2R and the CMS line 104-7A with 22 small populations by genotypic difference analysis. The selected primers were amplified by using 613 individuals BC1 mapping populations, and 6 new EST-SSR primers were found to closely link to the Rf1 of cotton. Integrating other markers linked with the Rf1 gene identified by Yin et al (2006), fine map was made with Joinmap3.0 software. The genetic mapping contained 19 makers that were 13 for SSR,2 for RAPD, and 3 for STS and the total genetic distance was 0.65cM. The 6 EST-SSR primers (NAU2650,NAU292,NAU3205,NAU3652,NAU3938 and NAU4040)added to the genetic map of Rf1 in this study showed co-segregation, and all the 6 primers had a genetic distance of 0.327cM from Rf1.Among them, only NAU3205 was a dominant marker and other five markers showed co-dominance.In order to construct a fine physical map of Rf1, the 6 EST-SSR primers were used to screen the restoring line 0-613-2R BAC library. Finally 7 single positive clones were attained by PCR method. The 7 clones were digested with Not I enzyme indicating insert size of them with a range of 105 to 130 kb. After integrating with previous results of our laboratory, there are totally 56 single positive clones availably.31 clones selected from 13 markers tightly most linked to the Rf1 were used to digested with Hindâ…¢.The fingerprintings of these clones were used by Image 3.10b and FPC V7.2 software to construct physical map.The Rf genes of rice, radish and petunia were used as query probes for tBLASTn searches of the cotton EST database. All the hit sequences were assembled by CAP3 assembly tool and 63 contigs were attained. Blastx analysis was used for function prediction of these contigs, and 3 contigs were predicted to have a higher similarity with Rf genes of radish, petunia and rice. Specific primers of the 3 contigs were designed to screen the BAC clones in the physical map of the Rf1. We identified a clone Y43 screened by the primers of contig2, and presumed this BAC probably containing the Rf1 gene.24 ORF were acquired by sequencing and gene prediction of this BAC. ORF2,ORF3,ORF7and ORF18 were predicted to encode PPR protein and have a mitochondrially targeted signals in their N-terminal amino acids. Sequences alignment of the four ORF and the restoring line 0-613-2R and the CMS line 104-7A, the ORF3 was identified as a candidate ORF for the Rf1 gene which is almost entirely composed of 16 repeats of the 35-aa PPR motif.In order to study the number and distribution of PPR genes in upland cotton, we identified 6 ORF encoding PPR protein in 147 BAC sequences, and 309 PPR unigenes were found through extensive survey of the EST database of upland cotton. Furthermore, we isolated five full length cDNA of PPR genes from 0-613-2R which were named GhPPRl-GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPRl-5 contain from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPRl-2 belonged to PLS subfamily and GhPPR3-5 belonged to P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All the five GhPPR genes were constitutively expressed in roots, stems, leaves, pollens, and 15dpa fibers based on real-time quantitative RT-PCR, but the relative expression levels were a little different.The isolation and clone of restoring genes is concerned because it pays an important value on studying the mechanism of sterility and heterosis utilization. In our study, the strategy combining map-based cloning and homology cloning was used to isolate the Rf1 of cotton. On the basis of physical map construction of Rf1, specific primers were designed from homology EST of Rf genes to screen those BAC clones on the physical map of Rf1. The BAC clone containing Rf1 gene was identified to be sequenced, and finally a candidate ORF encoding PPR protein for Rf1 was identified through bioinformatics analysis and sequence alignment. This is the basic work for isolation and clone of restoring genes. We can breed new cotton restoring strains with excellent traits, break through the utilization of heterosis and greatly push cotton breeding and development of production through transgenic methods. The fertile restoring gene with property right also can protect our gene resources. The results of the research can be contributive to reveal the molecular machine about cotton CMS and establish a foundation for the study and use with cotton heterosis.