Identification Of Self-Incompatibility And Its Related Genes In Raphanus Sativus

The ascendent of crossbreed is very obvious in Raphanus sativus. self-incompatibility of Raphanus sativus is one of the important routes in ascendent of crossbreed using. The self-incompatibility (SI) of advanced inbred lines were tested with the traditional index method and the fluoroscope observation method. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene,SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG-and SRK-specific primers. The DNA fragments of R.sativus SLG alleles and R.sativus SRK alleles were cloned and determinded. These establish base for self-incompat--ibility ascendent of crossbreed using in Raphanus sativus, understanding the evolution of SLG alleles and SRK alleles in Cruciferae, and researching of self-incompatibility mechanism in Raphanus sativus.1. Using the advanced inbred lines as the materials, the interaction of pollen-stigmas at different periods was investigated in radish of self-pollination done both in the bud and at anthesis with the fluoroscope. The results indicated that there were obvious differences in the germination of pollen grains in stigmas and growth of pollen tubes in styles between the self-incompatible and self-compatible lines. Few pollen grains of open flower self-pollina--tion attached and germinated in the stigma of self-incompatible individuals, and intensive callose reaction occurred on the surface of stigma. The germinated pollen grains could not grown naturally and fail to penetrate into the stigma with the abnormality of distorting of pollen tubes, swelling of the tops and failure to further growth. Most pollen grains of self-pollination both in the bud and at anthesis germinated naturally and penetrated into the stigmas and finally succeeded in reaching to the ovary in self-compatible individuals, and theses also occurred in the bud self-pollination in self-incompatible dividuals. The self-incompatibility (SI) indexes of all the lines were tested with the traditional index method, and it is high accordant with the results from the fluoroscope observation method, indicating that self-incompatibility of radish lines can be identified with the fluoroscope observation method. Therefore fluoroscope observation method is a good efficiently tool for development of elite self-incompatibility in radish breeding program.2. Polymorphism of the S-locus glycoprotein gene, SLG and the S locus receptor kinase gene, SRK which participate in the pollen-stigma interaction of self-incompatibility in Raphanus sativus were analyzed by PCR-RFLP using SLG- and SRK- specific primers. 19 inbred lines of R.sativus could be grouped into 12 S haplotypes by PCR with class I SLG specific primers showed different profiles up polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. 20 inbred lines of R.sativus could be grouped into 13 S haplotypes by PCR with SRK specific primers. 17 inbred lines of R.sativus could be grouped into 6 S haplotypes by PCR with class II SLG specific primers. Therefore the PCR-RFLP technology is very important in identification and classification of S haplotypes in Raphanus sativus. The nucleotide sequences of the DNA fragments of 5 R.sativus SLG alleles and 6 R.sativus SRK alleles were determinded. Degrees of similarity of the R.sativus SLG deduced amino-acid sequences to a Brassica SLG6 were 80.72%~84.26%. Phylogenetic analysis of the SLG sequence from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggested that diversification of the SLG alleles of Raphanus and Brassica before differentiation of the genera.