Allelic Segregation In The F₂Populations Derived From Crosses Of An Insect-Resistant Transgenic Rice Line And Its Non-transgenic Parent With Common Wild Rice

The global transgenic technology and genetically modified crop research are in a period of rapid development. The commercialization of production and large-scale cultivation of GM crops bring enormous economic benefits, while also cause widespread concern and debate of global issues about safety of GM crops. Among these, potential environmental and ecological risk caused by transgene escape from the cultivated GM crops to their wild relatives is one of the most concerned environmental biosafety problems. Transgene introgression from cultivated crops to their wild populations makes important influence to the evolutionary direction of the wild populations. Hybrid-introgression makes important influence to species and populations, hybrid-introgression can generate new combinations of genes, leads to increase and maintain genetic diversity of populations; inter-specific hybridization and gene flow may lead to genetic assimilation, demographic swamping and selective sweep, and thus make an impact on the direction of evolution of groups. After hybrid and introgression occurred, the continued ability of genes from different parents in the hybrid offspring makes great significance for the genetic structure and evolutionary direction of the hybrid populations. Under natural conditions, the hybridization process by reproductive isolation can be divided into isolation before fertilization and after fertilization, because of reproductive isolation and nature selection, non-random separation of the offspring genes or traits and genetic linkage are often observed. In population genetics, non-randon separation and linkage disequilibrium analysis are often used to explore the role of natural selection in evolution.In this study, hybrids from cultivated rice (including transgenic cultivated rice and its non-transgenic parent) and common wild rice were used as materials. We carried out the following mainly three studies:Firstly, we build the artificial hybridization segregating populations of cultivated rice-wild rice, and used micro-satellite (SSR) and Insertion/Deletion (InDel) molecular markers to detect the allele separation ratio of the different sites in the F2populations. The wild rice was selected from Suixi County, Guangdong Province, the cultivated rice included genetically modified varieties MF2and its non-transgenic parent Minghui-86. Artificial hybridization was made between the wild rice and cultivated rice, and F2segregating populations were collected from F1hybrids, each F2segregating population included more than400individuals to detect the F2populations’gene and genotype frequencies, and continuous chi-square(χ2) test was used to examine the consistency of observed versus theoretical values of the segregation ratios in the F2populations for detecting the distorted segregation. Through the above study to understand whether natural selection happened in this genetic separation, and what kind parental origin (cultivated rice or wild rice) genes in the offspring of hybrid separation will be more preserved, and we also focused on whether insect-resistant transgene makes any influence to this genetic separation. The results showed that high frequency of non-randomly segregations were observed in the two crosses (the non-transgenic cross was25.93%, while the genetically modified cross was33.33%), distorted separation did not show a consistent parental bias, only two alleles showed consistent bias in the two crosses of cultivated rice parent. These results indicated that in the wild rice and cultivated rice hybrids, high non-randomly segregation was observed, and some sites had a bias to cultivated rice, this may be caused by nature selection, this provides genetic and molecular evidence to the hybrid introgression and evolution direction of wild populations after crop gene introgression.Secondly, our genetic segregation study showed that the segregation ratio of insect-resistant transgene of F2population followed Mendelian theory. Separation of transgene is very important for the study of ecological risk caused by transgene escape, if the transgene gets positive selection in the genetic segregation, the transgene in the wild population may gradually spread into the wild population and produce lots of ecological impact. The segregation of transgene may be influenced by-pest pressure and environmental conditions, different environmental conditions may result in different segregation of the transgene. and therefore it has much more meaning to study the segregation of the transgene in the wild environment.Thirdly, in order to optimize the separation experiments, it is necessary to explore an appropriate sample size for the different generations in the hybrid self-cross generation. Ideal modeling groups for the best sample size of the F2to F5generations allele separation were build, while using the actual data collected in the experiment to test the reliability of the ideal sampling method of the F2population. The results showed that the ideal sampling strategy results are very consistent with the actural F2population sampling results. The sampling number for F2generation should be above200to make sure that the expertment reault is accurate and reliable, and with the increase of hybrid generation (F3-F5), the corresponding optimal sample size should be added at the same time.It is concluded from this study that through the analysis of allele segregation after hybridization of cultivate rice (including the transgene variety and its non-transgene parent) and wild rice, we find significant segregation distortion in both two crosses, the segregation distortion ratio of two cross is about26%(non-transgene) and33%(transgene), and there is some difference between the two crosses, some sites show same bias of cultivates rica, and200sample size is needed for the F2generation, different sampling size is needed for different generations. This thesis addressed the genetic impact of the wild population after crop genes" introgression into the wild populations caused by natural selection, and provides a scientific basis for understanding the direction of the evolution after crop gene introgression and the ecological risk after transgenic escape into the wild population.