Molecular Biology Studies On Taigu Genic Male-Sterile Gene Ms2

The Taigu Genie Male-Sterile Wheat is one of the most characteristic male sterile plants discovered up to the present, which is great valuable in genetic improvement by its stable inheritance, complete male abortion, and high cross-fertilize rate. Dwarfing-Sterile Wheat is a dwarf marked male sterile material, carrying the dominant gene ms2 for male sterility and dominant dwarf gene Rht10. The two genes are closely linked together on the short arm of chromosome 4D, with a crossing-over value as small as 0.18%. The material used in this study was the Dwarfing-Sterile Chinese Spring separated individual. In our research, several techniques such as RT-PCR, SSR, RFLP, cDNA-AFLP and in silic cloning were used to explore the co-separate marker of male sterile gene, clon the male sterile homology gene and to investigate the difference in gene expression between sterile and fertile anthers development of TGMSW. One 134bp fragment was amplified in male sterile and fertile wheat anthers using one pair of degenerated primer MSP designed based on conserved domain of MS2 gene in Arabidopsis thaliana and putative male sterile protein of Oryza sativa, and one l,604bp Contig was extended by in silico cloning based on the 134bp fragment. The comparison of sequence indicated that the putative amino acid encoded by the Contig include a 200bp conserved domain of male sterility, and this Contig expressed in male fertile wheat anthers, no expression in male sterile anthers, leaves and roots. This research demonstrated that the male steile homology sequence is specific to wheat anther development and the combination of candidate gene clon strategy based on the homologue sequence and in silico cloning technique can accelerated the gene clon.To find molecular markers tightly linked to the male sterility gene ms2 of wheat, 11 SSR markers on 4D chromosome, 8 RFLP probes, 4 PCR primers designed from EST on the short arm of chromosome 4D and 22 PCR primers designed from the BAG clon AC087797, which were located on the chromosome 3 of rice, were screened between the genomic DNA of male sterile and fertile plants of TGMSW. In the result,none polymorphism were found between the genomic DNA of male sterile and fertile plants of TGMSW. Reverse Northern blotting result indicated that the gene OsGAI of rice express in wheat anthers.The growing availability of EST sequences in wheat provides a potentially valuable source of new SSR markers. In this study, 444 SSR-ESTs were identified from 10,380 ESTs in the International Triticeae EST Cooperative database(ITEC), representing 4.1% of the total number of ESTs. 34 dinucleotide repeats and 347 trinucleotide repeats were found in these SSR-ESTs, representing 7.7% and 78% of the total. SSR-ESTs, respectively. 135 cSSR primers were designed to sequences flanking SSRs from 175 selected SSR-ESTs, of which 82 primers were efficient in wheat and 32 cSSR primers were located on 18 wheat chromosomes except 2D, 4B and 4D. In order to investigate the difference in gene expression between sterile and fertile anther development of Taigu Genie Male-Sterile(TGMSW), cDNA-AFLP was employed to compare the gene expression in pre-meiotic anthers between the male sterile and fertile plants of TGMSW NILs. In this study. 469 Pstl/Msel primer combinations were analysed in cDNA of male sterile and fertile anthers, as a result, a mean of 18.8 amplification products were detected from the above cDNA templates and 232 differentially expressed transcript derived fragments (TDFs) have been identified. Fifty differentially amplified PCR prodcuts specific to either male fertile or sterile anthers were excised from polyacrylamide gels, reamplified and directly sequenced following purification. Of the fifty sequenced fragments, thirty were confirmed by reverse northern hybridazation. Sequence analysis of the cloned PCR products revealed that twenty-two clones showed significant similarity to known genes in GenBank, of which seventeen sequences were specific to or shows high expression in male fertile anthers, Five sequences were spe...