| Remarkable increase in food production has been achieved worldwide over the past half century, but more than one seventh of globle population is under shortage of protein and energy or malnutrition situation. The application and promotion of genetically modified (GM) crops may be a promising solution to food security problem and the insect-resistance GM crops is one of the most widely used and successful transgenic crop. Till 2011, the global planting area of insect-resistance GM crop has reached 24 million hectares, about 15% of all GM crops. Many insect-resistance transgenic rice varieties have access to the stage of national biosafety assessments, promising the future of wide commercial application of GM rice. However, two environmental biosafety concerns are now the key constraints to the future successful commercial application of GM rice:(1) the resistance of target pest; (2) the ecological risk of insect-resistance gene escape.In rice planting area in China, the major target pest of insect-resistance rice is rice striped stem borer (RSSB), whose resistance to Bt toxin will directly threat the longevity of the commercial product. In the meantime, wild relatives of cultivated rice, including weedy rice and common wild rice, are also widely distributed in rice planting areas in China. With the commercialization of GM rice, once the insect-resistance transgene escapes into the wild relative populations, it will alter individual fitness and change the genome structure, which may finally cause a serious biosafety concern. Thus, this research was based on the relation and mutual influence of RSSB and cultivated rice, weedy rice and common wild rice, studying the resistance evolution potential to the Bt toxin by the pests, the impacts of insect-resistance GM rice hybridization-introgression effect to weedy rice and common wild rice.The main research contents include:(1) using fluorescent AFLP (amplified fragment length polymorphism) to estimate genetic variation and gene flow of RSSB populations collected from the Southeast China, which may predict the potential resistance development in rice striped stem borer populations; (2) using nylon cage isolation and egg massive incubation method to detect the tolerance differences between cultivated rice, weedy rice and common wild rice, which may simulate the consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population; (3) using the SSR neutral molecular markers to test the distortion segregation in hybrids of insect-resistance GM rice and weedy rice. The main results and findings were exhibited below:(1) The moderate level of genetic diversity combined with relatively frequent gene flow among RSSB populations was detected across Southeast China. The genetic diversity was estimated based on fluorescent AFLP fragments and the results showed that the striped stem borer collected from Southeast exhibited moderate level of genetic diversity (PIP:59.90%～91.58%, H:0.1651～0.2699 and H(=0.27). The limited divergence among populations was detected in RSSB populations collected from Southeast China. Evident genetic divergence was found between the JX stem borer population sampled from water-oat and RSSB populations with the FST value of 0.275. However, a relatively low level of genetic divergence was detected among the 12 RSSB populations, with the overall FST value of 0.120-about 12% of among-population genetic variation was found in the 12 RSSB populations. AMOVA test results showed that majority of the genetic variation was present within populations (>88%), and only a very small proportion (<12%) of genetic variation was found among populations and between the two regions, agreeing with the FST results. In addition, the Mantel test between genetic distance and geographical distance of the 12 RSSB populations did not show pattern of isolation by distance (r2=0.024; P=0.118; and 999 permutations).The level of gene flow among the RSSB populations collected from Southeast China was relatively high, regardless of their geographical distances. The results showed that the average Nm value of all 13 striped stem borer populations was 1.63, and the average Nm value of all 12 RSSB populations was 2.62. In assignment test, about 56% of individuals was assigned into the 12 native populations with a high confidence (P<0.01). However, about 19% of individuals were assigned to neighboring populations sampled in this study, and about 25% of individuals were assigned to other populations that were not sampled in this study. Thus, the results of assignment test also confirmed the considerable level of gene flow between the RSSB populations.(2) After the three-years experiment of nylon cage isolation and egg massive incubation of RSSB, the result show that the insect-resistance transgene escape event greatly enhance the fitness of cultivated rice and weedy rice, contrasting to the low fitness benefit to common wild rice. The artificial nylon cage isolation and egg massive incubation of RSSB were successful:From 2009 to 2011, the white-headed rate of cultivated rice were 17.9%,18.4% and 5.4% respectively in high pest pressure cage, whereas the white-headed rate of cultivated rice were 2.7%,4.1% and 0.5% respectively in low pest pressure cage. In high pest pressure treatment, the cultivated rice and weedy rice had poor performance in plant height, number of seeds per plant, number of florets per plant, grain weight and number of effective panicles compared with the low pest pressure treatment, but the common wild rice had no significant difference. These results indicate that the insect-resistance transgene escape event will greatly enhance the fitness of cultivated rice and weedy rice, contrasting to the low fitness benefit to common wild rice. The results above agree with the previous study in consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population, indicating that nylon cage method is able to effectively simulate the consequence of insect-resistance transgene escape to cultivated rice, weedy rice and common wild rice population.(3) In F3 and F4 generations of insect-resistance cultivated rice ×weedy rice, lots of loci (transgene negative combinations:88.89%, transgene positive combinations:66.67%) showed distortion segregation. The inbreed character and environment selection might be the main reason. For those loci, no apparent bias to crop allele or weedy rice allel is detected, indicating that both alleles from parents can be submitted to higher generations. X2 test for the change of gene frequency between different generations showed that lots of loci (48%) in transgene positive combinations remains unchanged while less loci (20%) in transgene negative loci remains unchanged, indicating anti-insect transgene might have influence on the gene trasmition between generations. In F3 and F4 generations, lots of loci showed the pattern of heterozygote excess, which indicates heterozygotes genotype might have advantage in natural selection.In summary, this study found moderate level of genetic diversity and high level of gene flow among RSSB populations in Southeast China, indicating the proper High dose-refuge strategy may slow down the resistance evolution of the RSSB populations. Besides, by the means of nylon cage isolation and egg massive incubation to simulate the effect of transgene, we found the anti-insect transgene escape will influence weedy rice and cultivated rice in their fitness, while common wild rice suffers less from anti-insect transgene for its compensation effect to RSSB. This cage isolation method using non-transgene material provides new means for assessment of ecological consequence of transgene escape. In the hybrid offerings of weedy rice and sensitive cultivated rice to the insect-resistance transgene, crop alleles can be successfully transmitted and fixed in high generation, in the mean time posing influence on gene transmition pattern between generations. |
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