Rice Breeding Of Pyramiding Multiple Genes And Molecular Mapping Of Cry1Ab Gene In Transgenic Rice

Rice is one of the most important crops in the world, providing staple food for more than half of the world's population, but also one crop tends to be infested by many kinds of diseases and pests. Bacterial blight caused by Xanthomonas oryzae pv. oryzae, a bacterial disease of rice worldwide, affects the yield and quality of rice seriously. The losses in rice yield caused by lepidopteran insects (Chilo suppressalis walker, Tryporyza incertulas, Cnaphalocrocis medinalis Guenee, Naranga aenescens Moore, Borbo cinnara Wallace) can account for 10-30% of total yield. In addition, purity of hybrid rice and field weeds affect the yield and quality of rice,the use of herbicide to control field weeds increases the production cost, these have been the main problems in rice production. So breeding of transgenic rice with multiple resistance to diseases, insects and herbicide can ensure the quantity and quality of rice, decrease the use of pesticides and be beneficial to the environment. Many studies had indicated that pyramiding multiple resistant genes into one rice variety can significantly enhance the resistance and keep durable resistance of rice. Molecular marker-assisted selection (MAS) has been advocated as a highly efficient breeding method for pyramiding multiple resistant genes, because it can offer rapid and precise selection of the target gene.Molecular mapping of transgene cry1Ab and genetic linkage map construction of transgenic cry1Ab rice increases molecular markers used for marker-assisted selection in transgenic cry1Ab rice breeding. Furthermore, this research establishes the foundation to investigate the transgene location affection, and provides the theory theoretic support for transgenic rice penalty evaluation.The objective of this paper was to study from three aspects, respectively: Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide; Marker-assisted breeding of pyramiding rice fragrance gene and multiple bacterial blight resistance genes; Molecular mapping of transgene cry1Ab in transgenic rice. Main research results are as follows: 1. Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide(1) Transgenic rice line Zhongguo91 (carrying cry1Ab and bar gene) and elite restorer line Hui773 were crossed, then backcross was carried out three times using Hui773 as recurrent parent and self-cross was carried out one time. All plants were subjected to Basta painting and PCR analysis to verify presence of the cry1Ab and the bar genes. Through selection we obtained transgenic restorer line T773-1 with good agronomic traits and obvious resistance to striped stem borer and herbicide.(2) Transgenic rice line Yujing6 (carrying Xa21 gene) and elite restorer line Hui773, then backcross was carried out three times using Hui773 as recurrent parent and self-cross was carried out one time. All plants were subjected to GUS activity assay and PCR analysis to verify presence of the Xa21 gene. In BC3F2 population we obtained transgenic restorer line T773-2 with good agronomic traits, showing high resistance to bacterial blight disease under field condition.(3) Transgenic rice line Zhongguo91 (carrying cry1Ab and bar gene) and transgenic rice line Yujing6 (carrying Xa21 gene) were crossed. Through marker-assisted selection in self-cross populations we obtained transgenic restorer line T773 carrying three genes (cry1Ab, bar and Xa21) with resistance to bacterial blight, striped stem borer and herbicide. Transgenic restorer line T773 showed high resistance to bacterial blight disease, striped stem borer and herbicide under field condition. The hybrid F1 generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis.2. Marker-assisted breeding of pyramiding rice fragrance gene and resistance gene to bacterial blightThrough the combination of marker-assisted selection and conventional cross breeding technology, we crossed Xiangjing9407 and transgenic pyramiding rice line IRBB60 (carrying Xa4, xa5, xa13, Xa21), screened the self-cross population using inoculation appraisal technology and fragrance identification to obtained the multiple resistance gene and fragrance gene pyramiding plant, supplemented by investigation of agronomic traits. We obtained 1 individual plant of (2R+fgr) pyramiding in F2 self-cross population. And we obtained 1 individual plant of (4R+fgr) pyramiding, 2 individual plant of (3R+fgr) pyramiding, 7 individual plant of (2R+fgr) pyramiding, 3 individual plant of (1R+fgr) pyramiding in F3 self-cross population. Finally, we obtained 10 individual plant of (4R+fgr) pyramiding, 28 individual plant of (3R+fgr) pyramiding, 20 individual plant of (2R+fgr) pyramiding in F4 self-cross population. All the plants selected had rich aroma and high resistance to bacterial blight, with excellent agronomic traits, can be used to breed aroma rice with resistance to bacterial blight. This study has important significance of rice resistance breeding and quality improvement practice.3. Molecular mapping of transgene cry1Ab in transgenic rice(1) First, the F2 segregation population plants of transgenic Zhongguo91 (cry1Ab)×IRBB60 were subjected to herbicide Basta resistance detection. The result demonstrated that the segregation of cry1Ab gene was the ratio of 3:1, following Mendelian segregation law. The result proved that the cry1Ab gene was single dominant locus integrated in transgenic rice.(2) The flanking sequences of cry1Ab gene insertions in transgenic rice genome were rescued by Tail-PCR. Then using sequence blast analysis on NCBI website we confirmed the cry1Ab gene insertions location in chromosome 12 of rice. Using software"simple sequence repeats identification tool"and"Primer Premier 5.0", 24 pairs of primers were designed on the basis of the rice genome sequence close to the transgene cry1Ab insertions location. (3) Polymorphism screen using parent Zhongguo91 (cry1Ab) and IRBB60 we obtained 6 pairs of primers with polymorphism, then using the 6 pairs of primers and primer RM1261 we analyze the genotype of 240 plants in F2 segregation population. By"MAPMAKER/EXP Version 3.0"software we construct the molecular genetic linkage map of exogenous cry1Ab gene in 12 chromosome of rice genome according to their genotype and phenotype. The result indicated that exogenous cry1Ab gene is located between the SSR markers CM22 and RM1261 in 12 chromosome of rice genome. SSR marker CM22, CM01, CM12, CM13 were found to be linked to cry1Ab gene with genetics distances of 4.6, 4.2, 3.1 and 2.7 respectively, close to the short arm side; SSR marker CM24, CM10, RM1261 were found to be linked to cry1Ab gene with genetics distances of 3.1, 3.5 and 5.2 respectively, close to the other side.