| As the agent of one of the most disastrous rice disease, Magnaporthe oryzae causes tremendous damage to rice yielding every year all over the world. Besides its commercial importance, well studied genetic engineering platform, solid inoculation and assessment system and published genomic sequence information make M. oryzae one of the important model microorganisms not only for the study of molecular mechanism of genes function of pathogen but also an important system for the interaction research between pathogen and host.Based on the published genome sequence of M. oryzae, Magnaporthe oryzae Release 5 and Magnaporthe oryzae Assembly 6, we developed many SSR markers which were used in the construction of the physical map of 4 avirulence genes in the strain CH63. We also constructed vectors for gene disruption of 3 cell wall related genes and 3 Eghl6 gene family members of M. oryzae for their further studies of their gene function. The following is a brief introduction of the relevant research, the progress and the significance of these studies:With combined use of SSRIT tool, SSRHunter software and online primer designing tool-Primer3, we developed 106 SSR markers in total based on Magnaporthe oryzae Release 5 geneome sequence. Through polymorphic screening,18 markers exhibited polymorphism between CH63 and TH16. The 18 polymorphic SSR markers were then used to detect the genetic structure of F1 progenies and the physical position of these SSR markers based on Release 5 were also investigated.Finally, the physical maps of 3 of the total 4 avirulence genes were constructed. Avr-Zh201 was located on the interval between the SSR marker SSR-5.195-79/80 and the telomere region, which was 628 kb in length. The physical map of Avr gene Avr-C105TTP1 indicated the gene was mapped to the region delimited by SSR-5.17717/18 and pyms409-410 with the physical distance of 892 kb and the recombination events of these two markers was 17 and 24, respectively. Avr-Zh156 was located on the chromosome 7, and the map interval was defined by pyms271-272 and SSR-5.125-17/18 which was spanned 175 kb.Because the research work based on the Magnaporthe oryzae Release 5 was failed to identify the physical position of the Avr gene Avr2-K59, we used the sequence data of Magnaporthe oryzae Assembly 6 to reconstruct the physical map of these 4 genes. In current study, we developed 379 SSR markers based on Magnaporthe oryzae Assembly 6, 37 of which showed polymorphism between CH63 and TH16. And 1 polymorphic PATE marker was also obtained by screening of 17 PATE markers used in the gene isolation of AvrPiz-t. After inspection of the genetic structure of F1 mapping population including 84 progenies, we constructed physical maps for four AVR genes. Avr2-K59 was located on an interval defined by two SSR makers SSR-6.9-69\70 and SSR-6.27-87\88 with the physical distance of 165 kb. Avr-Zh201 and Avr-Zh156 were both mapped to the telomere region, and the nearest linked markers was SSR-6.29-41\42 and SSR-6.23-43\44, and the physical interval was 109 and 808 kb in length, respectively. Avr-C105TTP1 linked with SSR-6.12-103\104, was delimitated toward to the telomere region, and spanned a physical distance of 616 kb.The physical maps of the same Avr gene exhibited obvious dissimilar because of the diversity between different strains, and the difference between various versions of the Magnaporthe oryzae genome sequence. And in further study, a BAC library was needed for the accuracy analysis of the physical map and accelerates the isolation of Avr genes. |
PDF Full Text Request