Effects Of OsMsh6 Gene Mutation On SSR Stability And Homologous Recombination In Rice

The mismatch repair (MMR) is a major pathway in DNA repair system, which is critical for maintaining genome stability and DNA fidelity in the replication, as it is responsible for recognizing and repairing erroneous insertions, deletions and mismatch bases newly arised during DNA replication and genetic recombination, as well as during repair of some forms of DNA damage. Some MMR genes have been annotated in Rice Annotation Project Database, one of which is the OsMsh6 (LOC_Os09g24220), a homologue to AtMsh6 (At4g02070) in the MMR system of A. thaliana. However no information is available for its function and response to y ray radiation. Therefore the effects of OsMsh6 gene mutation on SSR stability and homologous recombination were studied and the response of OsMsh6 insertion mutant to y-ray radiation was analyzed in this dissertation. The main results are as follows:(1) Effect of OsMsh6 gene mutation on SSR stability. Totally 77 SSRs, which comprise of 60 Nu-SSRs from nuclear DNA,10 Cp-SSRs from chloroplast DNA and 7 Mt-SSRs from mitochondrial DNA, were used to analysis 3 OsMsh6 mutants planted in different generations and the control Nipponbare.14 Nu-SSRs,5 Cp-SSRs and 2 Mt-SSRs showed polymorphism between the mutant and control, accounting for 23.33%,50.0% and 28.57% polymorphic rate of total SSR used in each group. Polymorphic forms not only included changes in band number, but also the shift of main band. SSR polymorphism rate varied with different mutants, following the order of NF9010> ND6011> NF7784. These results indicate that OsMsh6 gene mutation can not only cause the instability of nuclear DNA SSR, but also cause the variaton of extranuclear DNA SSR. This research confirms the important role of OsMsh6 gene in maintaining genome stability and provides the evendence for elucidating its function.(2) Effect of OsMsh6 gene mutation on homologous recombination. Three F2 mapping populations were established by the cross between Zaoxian B and NF9010, NF7784 or Nipponbare, repectively. Totally 40 polymorphic SSRs were mapped on chromosome 1,3,9 and 10. Total map distance of SSR markers on each chromosome was all increased with a range from 2.0% to 32.5% in the population involving the mutant NF9010 or NF7784 compared with the Nipponbare. The increased degree in map distance varied with different chromosomes and mutants. These results show that the OsMsh6 gene may restrict the recombination and its mutation leads to the increase of homologous recombination rate. This research not only confirmed the role of OsMsh6 gene in homologous recombination, but also laid the foundation for promoting gene introgression from wild genetic resources in rice breeding.(3) Response of OsMsh6 gene mutants to γ-ray radiation. Seeds of OsMsh6 mutants and Nipponbare were irradiated by γ-ray with different dosages,75,150,300 and 450Gy. Seed germination rate, seedling height, root number and length, as well as the OsMsh6 gene expression were analyzed at seedling stage. All physiological indexes under same dosages were higher in Nipponbare than that in mutants, suggesting that the radiation sensitivity of the mutants increase, especially when the dose reaches to 150Gy or above. Furthermore, the sensitivity varied with different mutants, with the order of NF9010> NF7784> ND6011. RT-PCR results showed that the expression level of OsMsh6 gene increased with increase of the radiation dose, indicating that the OsMsh6 gene is involved in the repair of DNA damage caused by y-ray radiation. This research provides a basis for the further use of MMR mutants to set up an efficient mutation breeding system.