Cloning And Epigenetic Variation Analysis Of Gene Homologous To Ms2Bnap In Raphanus Sativus×Brassica Alboglabr Allopolyploids

Male sterility is an inheritable trait characterized by the inability of a plant to produce functional pollen. Pollen development is a complicated process and needs a lot of genes to express at certain time and space. In the process of pollen development in angiosperms, from stamens primordia differentiation to mature pollen, a series of morphological, physiological and biochemical changes have taken placed and if any abnormal change occurs, it will lead to male sterility. Data of different amount of gene expression can provide valuable information for anther development.F1 plants from intergeneric hybrids between Raphanus sativus L. (2n=18,RR) and Brassica alboglabra Bailey(2n=18,CC) were obtained by hand cross and through continuous selfing we get the stable genetic F10 generation. This new material has good prospects for the breeding of fodder crops or being used as a genetic bridge for intergenetic gene transfer. However, the lower seed fertility limited its practical utilization. Compared to F1 generation with complete sterility, F10 plants exhibited good fertility but there still had some sterile plants.The mechanisms of infertility are largely unknown.In present paper, paraffin and semi-thin sectioning were used to investigate the time of microspore abortion and characteristics of fertility alteration. Results showed that the male sterile line was abortive completely. The key stage of male sterility was from microsporocyte to microspores.The main characteristic was large vacuoles in tapetum. The tapetum of male sterile anther enlarged radial with a number of larger vacuoles; the surface of male sterile microspore was smooth without exine formation. Till the stage of later microspore, tapetum and microspores were disaggregated to be an empty pollen sac.RT-PCR was used to indentify the expressive stage of the gene homologous to ms2Bnap and the gene expression differences in F10 generation of Raphanus sativus×Brassica alboglabra allopolyploids male sterility and male fertility. The results showed that fragment homologous to ms2Bnap was steadily amplified from meiosis stage, while there was no PCR products using cDNA of single nucleus stage as template. These indicated that the gene homologous to ms2Bnap was not expressed in single nucleus stage. The gene expression amount in male fertility was higher than male sterility. Cloning and analyzing of sequence of gene homologous to ms2Bnap showed that there were four different nucleotides dispersed in the code region of the male sterility and male fertility, with two sites of synonymous mutations and two sites of missense mutation. This changes maybe related to sterility of F10 generation of Raphanus sativus×Brassica alboglabra allopolyploids.Bisulfite sequencing PCR was used to reveal DNA methylation variation of the gene homologous to ms2Bnap between male sterility and male fertility. The methylation ratio of fl-f5 fragments in sterile plants and fertile plants was 10.4% and 2.5%,respectively. This result indicate that the different methylation ratio between fertility and sterility may related to sterility of F10 generation of Raphanus sativus X Brassica alboglabra allopolyploids.