| Male sterility of wheat with Aegilops cytoplasm sourced from Ae. Kotschyi, Ae.variabilis, Ae. Ventricosa and Ae. bicornis is dominated by single-gene, completely sterile, easy to restore, and therefore provides the favorable material basis of male sterility research and utilization of heterosis in wheat. In this research, we took male-sterile line ms (kots)-90-110(A) and its near isogenic line BC5F2 as experimental materials. The primers were designed according to the chimeric gene orf 256 sequence of T-wheat, and detected in the male sterile wheat with Aegilops kotschyi cytoplasm and its near-isogenic lines. Meanwhile, to further explore the relationship between expression of chimeric genes and pollen abortion of the male sterile wheat with Aegilops kotschyi cytoplasm, the expression patterns of transcripts of chimeric gene analyzed in different developmental stages in leaves and anthers of sterile wheat and its near isogenic male line via semi-quantitative RT-PCR and real-time PCR. Besides, to find the source of fertility different gene, using the two materials ms (kots) -90-110 (A) and its near isogenic line BC4F1 as test materials, mitochondrial DNA library of male sterile wheat with Aegilops kotschyi cytoplasm was constructed by suppression subtractive hybridization (SSH) technology, The results were obtained as follows:1. The primers were designed according to sequence of chimeric gene orf256tim CMS in T-wheat , and detected in the leaves of mtDNA and its anther during binucleate stage in cDNA of male sterile wheat with Aegilops kotschyi cytoplasm and its near-isogenic lines, the genes orf256kot and orf260kot, were obtained but the length of transcripts was changed, and the other genetic background was exactly the same except the difference of fertility, so the orf256kot was mediated and changed into orf260kot . The further analysis show that 256 amino acids were coded by orf256kot, but then changed into orf260kot that encoded 260 amino acids in the same cytoplasmic fertile lines. And the changing and missing of amino acid were mostly concentrated in the transmembrane region, explaining that these inserted amino acids may be related to differences of fertility phenotype in wheat. 2. Using cDNA template of the early-uninucleate stage , later-uninucleate stage, binucleate stage and dinucleate stage of male sterile wheat with Aegilops kotschyi cytoplasm and its near isogenic lines, the expression pattern of orf 256kot and orf 260kot with neighbor coxI bicistronic transcripts and coxI single polycistronic transcript were detected by semi-quantitative RT-PCR in CMS and fertile lines. And the analysis of the single-cistron transcripts expression of orf 256kot and orf 260kot by quantitative PCR was performed at the same time. The results showed that: the expression of orf 256kot and orf 256kot-coxI in the sterile lines during the binucleate stage was highest. However, the expression of coxI was lower than fertile lines, showing that orf 256kot involved in regulation of pollen development. In the fertile lines, expression of orf 260kot-coxI bicistronic transcripts in binucleate stage of male sterile line was significantly lower which further explained orf 256kot mediating anther abortion, and the restoration of fertility was executed by restorer gene through inhibiting expression of orf 260kot-coxI.3. In this study, we constructed subtractive mtDNA library of male sterile lines by SSH with mitochondrial DNA of sterile wheat ms (kots) -90-110 (A) as a tester and its near-isogenic lines BC4F1 as a driver. Then we cloned and transformed all amplified products of given library and 6 single-band amplified products, the analysis of their sequences showed approximately 90% of single PCR products in SSH Library were from non-coding region of mitochondrial genes nad1 and nad5 .
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