Variation On Mitochondrial DNA Of Male Sterile Lines With Aegilops Cytoplasms And Its Molecular Markers In Wheat

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the absence of functional pollen. Crop heterosis is often attained through the use of CMS. Wheat is one of the most important field crops in the world today. In spite of the progress made in well-studied species, the mechanism of CMS in wheat remains puzzling. So we should pay more attention to the mechanism of CMS in wheat. In several well-studied species, molecular studies of CMS have correlated the trait with mitochondrial DNA (mtDNA) mutations that disturbing mitochondrial function at a critical stage of anther development.In this thesis, mitochondrial DNAs (mtDNAs) of four Aegilops species (Ae.kotschyi, Ae.variabilis, Ae.ventricosa and Ae.bicornis), their corresponding cytoplasmic male sterile (CMS) wheat with the Aegilops cytoplasms and the fertility-restored F1 hybrids were studied by RAPD (Random amplified polymorphic DNA) analysis. RAPD fingerprint patterns were compared under a single cytoplasm background to analyse mitochondrial DNA (mtDNA) variation. The main results are as follows.(1) Reproducible differences in the mtDNAs were obtained from the Aegilops species and the corresponding CMS lines. With primer OPY-01 different fingerprint patterns were obtained from the mtDNAs of Ae.kotschyi and the male sterile line ms(Ae.kotschyi)-90-110. With primer S32 different fingerprint patterns were obtained from the mtDNAs of Ae.variabilis and the male sterile line ms(Ae.variabilis)-90-110. With primers S22 different fingerprint patterns were obtained from the mtDNAs of Ae.ventricosa and ms(Ae.ventricosa)-90-110. With primers S202, OPB-05, OPA-04, S153 and S21, different fingerprint patterns were obtained from the mtDNAs of Ae.bicornis and male sterile line ms(Ae.bicornis)-90-110. These results indicate that the genetic interactions between the common wheat 90-110 nucleus and alien cytoplasm of Aegilops species have affect the structure of the mitochondrial genome. The differences of fingerprint patterns between Aegilops species and the corresponding CMS wheat revealed that some structural alterations of the mitochondrial genomes occurred during the recurrent backcrossing program. These differences of mtDNAs can be used for molecular markers to locate CMS-associated gene regions.(2) Reproducible differences in the mtDNAs were obtained from male sterile lines and the corresponding fertility-restored F1 hybrids. With primer OPY-01 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.kotschyi)-90-110 and F1 hybrid ms(Ae.kotschyi)-90-110×5253. With primer OPD-05 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.variabilis)-90-110 and F1 hybrid ms(Ae.variabilis)-90-110×5253. With primer S21 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.ventricosa)-90-110 and F1 hybrid ms(Ae.ventricosa)-90-110×5253. With primer OPP-02 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.bicornis)-90-110 and F1 hybrid ms(Ae.bicornis)-90-110×5253. These results indicate that the mitochondrial genomes were changed in fertility restoration by the introduction of fertility restorer nucleus and fertility restoration involves a strong influence of nuclear restorer genes on mtDNA organization. And also, the differences of mtDNAs in Aegilops species, their corresponding CMS lines and the fertility-restored F1 hybrids indicate that mitochondrial genomes variation is controlled by different nuclear genomes. The differences of mtDNAs between male sterile lines ant the the corresponding fertility-restored F1 hybrids can be used for molecular markers to locate CMS-associated gene regions.(3) An efficient method for isolation of mtDNA from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, DNase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.