Molecular Characterization Of Cms Related Genes In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.), belonging to the Brassicaceae family, is an important worldwide root vegetable crop, especially in East Asia. Cytoplasmic male sterility (CMS) is a widespread, maternally inherited trait of plants, which prevents the production of functional pollen. In many cases, specific dominant nuclear genes termed restorers of fertility (Rf) have been identified that can alters the expression of CMS-associated genes and restores fertility in plants carrying the CMS mitochondrial. The application of male sterility becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. The cultivation of multiple excellent CMS lines is very important for elite F1hybrid cultivars development. In Brassica plants, the Ogura CMS has been introduced into a variety of Brassica species through either conventional breeding or protoplast fusion. The technical system of cytoplasm identification of radish germplasm and the system of molecular marker-assisted developing elite maintainer has not been established, the occurrence and the mechanism of plant male sterility still remain mysteries that have not been unveiled thoroughly. We characterized the CMS cytoplasm and the restorer gene from morphology, cytology as well as molecular biology in radish, and differential gene expression analysis were used to characterize the pollen development and molecular regulation mechanism, moreover, we isolated some important genes from CMS line and its maintainer by means of homology-based cloning method. The results obtained are as follows.(1) The production of hybrid cultivars with identical sterile cytoplasm in the large area could cause the sterile cytoplasm simplification, which could brought the risk of irreparable damage to the plant production.80crosses of inbred lines and CMS lines and20cultivars were amplified by specific primers MSF1/R1, LwnomalF/R, LwNWBF/R, KOS1F/1R and LwDCGMSF/R. The radish cytoplasms were divided into seven types:Ogura, DBRMF1, DBRMF2, NWB, DCGMS, Chiasma-type and novel.65inbred lines/cultivars were Ogura and10were NWB, five were DCGMS type, three were Chiasma type; eight inbred lines were DBRMF1and two were novel type, seven were DBRMF2. Transverse anther sections from early to late stages of pollen development were examined by optical microscopy. No cytological differences were observed between the CMS and its maintainer in the meiosis until tetrad stage. At the tetrad stage, the tapetum cells of CMS was expanded and bulged by severe cytoplasmic vacuolation. After the microspores release of individual microspores, microspores of CMS began to degenerate and finally resulting in empty anthers with no pollen grains. Radish cytoplasm was identified and classified by specific molecular marker, which provide an important technical foundation for the rational utilization and evaluation of germplasm resources.(2) Cytoplasmic male sterility (CMS) is a maternally inherited trait, which was widely utilized in hybrid seed production. In this study, the Rs-Rf1gene and its allele, Rs-rf1gene, were isolated from radish CMS restorer line and maintainer, respectively. It was found that Rs-Rf1gene was the member of the pentatricopeptide repeat (PPR) family. With the RT-PCR and qRT-PCR, the floral buds at different development stages were used to characterize the expression feature of Rs-Rf1and orfl38, a unique transcribed gene in mitochondrion for male sterility in cytoplasm. The orfl38was up-regulated in CMS line and F1hybrid at three bud developmental stages including the meiosis, tetrad and microspore, but not expressed in maintainer and restorer line, suggesting that the Rs-Rf1gene could not block the orfl38transcription. As in the F1plant, the transcription of Rs-Rf1was gradually up-regulated in restorer line with bud development, but it was not transcribed in CMS line and maintainer. Moreover, different restriction sites for Sspl between the allele of Rs-Rf1and Rs-rf1was identified and the functional marker for distinguishing the genotypes of the individuals with the Rs-Rf1was developed. For its being co-dominant, this marker could be used to discriminate the heterozygous and homozygous fertility restoring allele. With this functional marker, the system of molecular marker-aided developing elite CMS lines was well established in radish, which would make great contribution for candidate maintainer identification and accelerate the process of elite CMS line development in late-bolting radish breeding programs.(3) We established male sterile line NAU-RsWA1and its maintainer NAU-RsWB1, differential display reverse transcription PCR (DDRT-PCR) was used to analyze the gene differential expression of different development stags of flower buds in male sterile line and its maintainer of radish.90different fragments were recovered from the gels, re-amplified with the same primers and sequenced. Genes were identified based on BLAST comparisons with NCBI. Compared to the maintainer line,30genes were up-regulated and60genes were down-regulated in male sterile line. We classified the differentially expressed gene detected in the flower buds transcriptome of the male sterile line and its maintainer into several categories, based on the putative function as well as gene ontology annotations derived from homologies. They were involved in cell wall biosynthesis and regulation, transporter and channel, flower development, signal transduction, protein metabolism, electron transport or energy pathways, defense mechanisms and stress response, etc. About39%of these genes have an unknown or hypothetical function, plenty of candidate genes involved in pollen development were identified and the number of genes specifically expressed in pollen and anther will be increased to some extent for the identification of these genes. We investigated the expression pattern of22fragments derived from the transcriptome in male sterile and its maintainer. The expression feature of these genes in four organs (stamens, petals, leaves, stalks) and different size (1-1.5,2-2.5and4-5mm) of floral buds corresponding to three stages of anther development (meiosis, tetrad and microspore) of male sterile line and its maintainer were different from each other, and each transcript had a unique expression level at different developmental stages. These results from the expression pattern analysis validate the complexity of gene expression, and the expression feature of these genes activation or silencing and orderly changes of expression level are consistent with the pollen development.(4) By means of DDRT-PCR, selected some important genes which were closely related with the male sterile pollen development. These genes involved in flower development (PCP, Bnm, TCTP, Rac). They were isolated from male sterile or its maintainer by means of TA clone, named as RsPCP1, RsPCP2, RsBnm, RsTCTP, and RsRac. RT-PCR was used to characterize the expression feature of these genes in different organ and flower buds developmental stages of male sterile line and its maintainer. RsPCP1, RsPCP2, RsTCTP and RsBnm were expressed in stamens, stalks and buds with different development in maintainer line, but they were not expressed in stamens, petals, leaves, stalks and buds with different development in male sterile line. RsRac was expressed in stamens, petals, leaves, stalks and buds with different development in male sterile line, but it was not expressed in maintainer line.(5) We obtained a differentially expressed cDNA fragment with TA clone and named as RsBHLH. RsBHLH encoded336amino acids, contained a bHLH domain, and shared high homology with AtBHLH060. RT-PCR and qRT-PCR was used to characterize the expression feature of RsBHLH in different organ and flower buds developmental stages of male sterile line and its maintainer. RsBHLH was expressed in stamens, petals and buds with different development in male sterile line, and the expression level in stamens was highest. The expression level of RsBHLH in buds with different development in maintainer line was lower than in male sterile line, but it was not expressed in stamens, petals, leaves and stalks in maintainer line.(6) In this study, DDRT-PCR was used to analyze the gene differential expression of different development stags of flower buds in male sterile line and its maintainer of radish. We obtained a differentially expressed cDNA fragment with TA clone and named as RsCHS. RsCHS encoded392amino acids, contained a CHS domain, and shared high homology with Brassica rape CHS. RT-PCR and qRT-PCR was used to characterize the expression feature of RsCHS in different organ and flower buds developmental stages of male sterile line and its maintainer. RsCHS was expressed in stamens, petals and buds with different development in maintainer.(7) We obtained a differentially expressed cDNA fragment with TA clone and named as RsPMEI and RsPG. RT-PCR and qRT-PCR was used to characterize the expression feature of RsPMEI and RsPG in different organ and flower buds developmental stages of male sterile line and its maintainer. RsPMEI was expressed in stamens, petals and buds with different development in maintainer, and the expression level in stamens was highest. The expression level of RsPMEI in buds with different development in male sterile line was lower than in maintainer line, but it was not expressed in stamens, petals, leaves and stalks in male sterile line. RsPG was only expressed in buds with different development in maintainer line.