Genetic Analysis And Gene Expression Profiling Research On Fertility-Related Genes Of Male Sterile Line With Aegilops Kotschyi Cytoplasm In Wheat

Male sterility of wheat with Aegilops kotschyi cytoplasm is the generic name of wheat K-CMS and Ven-CMS.They are not only heritable and restorable but also seed plump,theirs maintainer line and restorer line derive from popularized variety in wheat.More studies showed that male sterility of wheat with Aegilops kotschyi cytoplasm was ideal sterile lines.According to the research with plasmatic effect,fertile restoration and transfer mode of androgamete and megagamete in male sterility of wheat with Aegilops kotschyi cytoplasm, male sterility of wheat with Aegilops kotschyi cytoplasm had the character with single sterile gene,thorough sterility and high restoration,which provided advantageous material foundation for the study of male sterility in wheat and the utilization of heterozyous advantage.Using above-mentioned materials, Genetic Analysis of the Male Sterility of Wheat with Aegilops kotschyi Cytoplasm was researched and molecular markers linked to restoring gene was Screened, which may provide a basis for choosing and breeding assistance;this study constructed differential expression SSH cDNA library under sterile and fertile conditon through SSH. Through sequencing of ESTs and BLASTx analysis of ESTs sequences in the two libraries, this study discussed the type of expressed gene, gene function and different biochemical metabolic pathway in male sterility of wheat with Aegilops kotschyi cytoplasm between sterile library and fertile library so as to analyze molecular mechanism of male sterility of wheat with Aegilops kotschyi cytoplasm.The results were as follows:1. The experiment materials are CMS line ms(Kots)-90-110, restore line rk5451and F₂ ,BF₁ and BC₁F₁ population ms(Kots)-90-110/rk5451 combination. The fertile isolations data of continuous two years were analysed. The analysis result showed there was one pair of dominant major gene. The fertility restoring exits quantity character and quality character. The results indicated that the fertility restoring of K-type CMS was controlled by the one pair of major genes together with some minor genes.The imitate near-isogenic lines (INIL) and near-isogenic lines (NIL)of Rf gene were developed utilizing crossing, backcrossing and self-pollinating.2.The manner of gametes transmission of male sterile wheat with Aegilops kotschyi cytoplasm and seed set of F₁ is analyzed in this paper. Obviously, male sterile wheat with Aegilops kotschyi cytoplasm have a characteristic of gametophte sterility; Fertility Separation ratio of ms(Kots)-90-110/rk5451//90-110 depatured seriously 1:1. In F₂ population of ms(Kots)-90-110/ rk5451 completely appear fertile without sterile.Test on hybridization F1 and F₂ indicates that average seed set of F2 is higher than F1 These results prove again that the hybridization F₁ of male sterile line with Ae. Kotschyi cytoplasm.Bicornis cytoplasms inherits in mode of gametophyte sterility.3.In order to probe into the fertility characteristics of male sterility with Aegilops kotschyi cytoplasm, SSR markers which were located on the chromosome 1BS and 6B were employed to tag the major restorer gene in BC₁F₁ of ms(Kots)-90-110/ rk5451//90-110. Two primer pairs(Wms273 and Wmc406) displayed polymorphism of the restorer gene . By analyzing the polymorphic markers in these segregating populations, the microsatellite locus Wmc406 located on chromosome 1BS was found to be linked to the restorer gene with the estimated genetic distance of 7.6cM. Wmc406 can be used in MAS directly.4.To further reveal the genetic mechanism of male sterility with Aegilops kotschyi cytoplasm, a male sterile line ms(Kots)-90-110(A) and its near isogenic line BC4F1 (fertility restored by rk5451) were employed to construct sterility and fertility cDNA libraries by suppression subtractive hybridization (SSH). Wheat anthers with pollen mother cells at dikaryophase were sampled for RNA extraction via microscopic examination. The two cDNA libraries were evaluated to be of good quality with high efficiency of SSH. Total 120 positive clones were randomly selected and sequenced from the 2 libraries, and 100 high quality sequences were obtained. According to BLAST screening and functional annotation, energy-related genes were identified with high frequencies in fertility cDNA library, while MADS-box transcriptional factor TaAGL7, ubiquitin-conjugating enzyme, and adenosine diphosphate glucose pyrophosphatase were detected in sterility cDNA library. The MADS-box transcription factor TaAGL7 plays important roles in regulating flower development. Immature spike ubiquitin-conjugating enzyme is likely to relate to apoptosis. The expression of adenosine diphosphate glucose pyrophosphatase directely affectes starch synthase and normal supply of energy. The genes detected in our study are probably important in fertility of wheat with Aegilops kotschyi cytoplasm.5.The four differentially expressed genes, Which were supposed to involve in fertility of wheat, were selected and tested by means of PT-PCR. The amino acid sequence, structure domain, hydrophobicity, multiple alignment and homology phylogenetic tree of the genes were studied.