Analysis Of MtDNA Variation Of A Special CMS And RAPD Of MtDNAs From A Hybrid Variety Breed By Chemical Hybridizing Agents In Wheat

Mitochondrion is one of significant organelles of eukaryotic biology and plays an important role in the conversion of energy, so it is one of main fields people are interested. Specially, there are a lot of proofs which show close relation between mitochondrion and CMS in plants, thus mitochondrion becomes the focus to study CMS mechanism of plants. In the study, the efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. Secondly, mtDNAs of the common fertile wheat with bicornis cytoplasms,1BL/1RS sterile lines in wheat with bicornis cytoplasms (bicornis sterility with anther uncovered)as well as the variation of the bicornis sterility with anther uncovered (bicornis sterility with anther covered) were analyzed with RAPD(Random Amplified Polymorphic DNA) technique to explore the differences of two sterile types in molecular mechanism. In addition, possible relation of the sterility and mtDNA was to be discovered, to provide a theoretical basis for studying CMS mechanism in wheat and using male sterile lines with bicornis cytoplasms.At present, it is not found the report of whether there is direct relation between mitochondrion and physiological sterility induced by external factors, eg CHA, high temperature, etc. Therefore, the variability of mtDNAs coming from hybrid wheat Xiza No.5 using CHA technique, female parent FP2 and male parent MP2 were studied by RAPD analysis. The main results are as follows:1.An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, DNaseⅠtreatment, lysis with SDS and proteinase K, removing proteins by TE-saturated phenol/chloroform and chloroform/Isoamyl alcohol extraction and a final RNase A treatment for obtaining mtDNA . It was proved that the mtDNA isolated by this method not only had the high yield at 21.84μg/g ,20kb in length of segment ,but also contained no impurities, such as nuclear DNA, RNA and protein. The result shows that this high quality mtDNA can be successfully used in molecular biological technique such as restriction digestion and PCR amplification. This way creates the rapid technique of mtDNA isolation to research into the relation between wheat CMS and mtDNA as well as the variation and polymorphism of mtDNA.2. Directing at bicornis sterility with anther covered, nuclear genome, chloroplastic genome and mitochondrial genome of cytoplasmic male sterility(CMS)lines in wheat were analyzed. The results show: differences were found between the mtDNA of CMS lines in wheat, while there were no difference in their chloroplastic genome and nuclear genome. Special bands were amplified by three primers in 50 primers, all of which were bicornis sterility with anther covered having one more special band than bicornis sterility with anther uncovered and common fertile wheat: 1565bp band amplified by S32, 2173bp band by OPDO5 and 1735bp by OPAA16.This indicates change of wheat sterile types possibly due to some variation of mtDNA ,which was possibly caused by re-arrangement of mtDNA in natural condition.3. In order to investigate the relation between superior combination of wheat hybridization and mtDNA differences of its parents, as well as the relation between male sterility induced by CHA in wheat and mtDNA, mtDNAs coming from superior hybrid Xiza No.5,parents MP2 and FP2 were compared.6 primers were amplified to different bands between female parent and Xiza No.5. This indicates that mtDNA of female parent produced certain variation when passed to offspring, which probably induced by the happening of nuclear DNA of male parent or by CHA. Further study is needed to realize whether it is one of chemical sterility mechanism.4. There were a lot of differences between the mtDNAs of female parent and male parent.20 primers were amplified to different bands between female parent and male parent in 50 primers, for instance, in bands amplified by S1126, male parent MP2 had one less 1091bp band than female parent FP2. This difference is high possible to be the forecasting target for choosing superior hybrid wheat to deeper study or utilize.