Genetic Analysis And Molecular Mapping Of Two Brown Planthopper Resistance Genes Derived From Wild Rice

The brown planthopper (BPH), Nilaparvata lugens Stal (Homoptera: Delphacidae), is one of the most destructive and widespread insect pests throughout the rice-growing areas in Asia. Planting the resistant variety can efficiently restrain the breaking-out of BPH and its damage to rice. Mapping and cloning the BPH resistance genes will be propitious to the development of resistant rice variety and understanding biological basis for BPH-resistance in rice.The wild species of rice are valuable resources of genes for resistance to major diseases and insects and for tolerance of abiotic, stresses. Our laboratory has developed a series of novel breeding materials showing resistance to BPH, via wide hybridization between wild species and cultivated rice. Two rice lines B14 and B5, showing high resistance to BPH, were introgressed from the highly resistant wild rice, Oryza latifolia and Oryza officinalis Wall ex Watt, respectively.An F2 population (containing 184 plants) and an F7 recombinant inbreeding lines (RIL) population (containing 209 lines), derived from the cross between B14 and susceptible variety TN1 were used to investigate inheritance pattern of the BPH resistance gene in B14. The tiller test technique and standard seedbox screening technique (SSST) test were conducted to evaluate the resistance phenotype of the F₂ plants and RILs, respectively. Analysis the BPH resistance scoring data revealed that resistant and susceptible plants fit a 3:1 ratio in the F₂ population and segregation of resistant and susceptible plants fits a 1:1 ratio in the RIL population. These indicated that a single dominant gene controlled the resistance to BPH in B14.In order to determine the chromosome location] of the BPH resistance gene in B14, 20 highly resistant and 20 highly susceptible; lines of RIL population were pooled to form two bulks. 305 SSR markers distributed along the whole rice genomewere used to survey polymorphism between the resistant and'susceptible bulks. Three SSR markers RM185, RM261, RM335 on rice chromosome 4 showed polymorphism between them. This indicated that the BPH resistance gene located on rice chromosome 4. RFLP markers on chromosome 4 were selected to analyze the whole RIL population and the chromosome location of the BPH resistance gene was further confirmed. According to the denomination rule of plant gene, we designated this BPH resistance gene as Bphl2(t).We have previously identified two dominant BPH resistance genes Bphl4 and BphlS (previously named Qbpl and Qbp2) in B5. Bphl4 was mapped on the long arm of chromosome 3 flanked by RFLP markers R1925 and G1318. BphlS located on the short arm of chromosome 4 flanked by RFLP markers C820 and S11182. Genetic analysis revealed that BphlS showed more constant and steady contribution to the BPH resistance in B5. That Bphl5 and Bphl2(t) derived from different wild rice species were mapped on the vicinal chromosome'region is interesting.Knowledge about insect resistance gene in plants is very limited at present, it's therefore not feasible to adopt a candidate gene approach to clone BPH resistance gene in rice. Map-based cloning represents the most promising approach to isolating the BPH resistance genes. According to the publicly available genomic sequence data of rice, C820 and SI 1182 locate around the centromeric region of chromosome 4 and the physical distance between them is longer than 2 Mb. The large physical distance and the repetitive sequence-rich property of the centromeric region are the main problems hindering cloning BphlS by chromosome walking. Thus, construction of a high-resolution linkage map is a crucial step in map-based cloning ofBphlS. !To avoid the interference of Bphl4, we developed an F2 segregation population of BphlS derived from a cross between RI93, a selected recombinant inbred line of MH63/B5 RIL population bearing the resistance gene BphlS, and a susceptible variety TNI. Using two markers RM261 and MSi, which are much farer from BphlS than C820 and SI 1182, we screened recombinant individuals from the F2 populations of BphlS. Forty-eight recombinant individuals were selected out from 9,472 F2 plants. We employed the tiller testing technique to evaluate the BPH resistance of each recombinant F2 plant. Accordingly, the standard seedbox screening technique was performed on the F2.3 families to examine their phenotype.Statistical results from both methods showed that the BPH resistance phenotype exhibited a segregation ratio of 3:1 in the 48 recombinant F2 plants. This indicatedthat single dominant gene-control resistance was observed and Bphl 4 and Bphl 5had been separated in the F2 population.Efforts were undertaken to enrich the molecular marker density of the Bphl 5 chromosome region. AFLP technique was employed and six polymorphic AFLP markers were obtained. Three new SSR markers and eight novel RFLP markers were successfully developed based on the publicly available sequence information of the Bphl 5 chromosome region. These newly developed markers were assayed in the 48 recombinants and a local linkage map was constructed. Subsequently, Bphl 5 was located in the interval between RGl and RG2, co-segregating with Ml. Screening the genomic library of BS by these markers, five positive BAC clones were obtained. The physical map of Bphl5 indicated that 20M14 and 6409 covered the Bphl5 locus. â– Sequencing results of 20M14 and 6409 showed that Bphl5 was physical located in a 47 kb genomic segment. Gene prediction analysis revealed that the region between RGl and RG2 could encode 12 open reading frames, but none of them could produce the corresponding EST hit by1 BLAST search. RT-PCR was carried out to analyze the expression of five genes among them. RT-PCR result showed that none of the 5 genes can be induced by BPH feeding. Among the five genes, 0RF5 and 0RF9 were selected for further study. We studied the expression pattern of 0RF5 and 0RF9 in rice leaf and sheam by mRNA in situ hybridization technique. In order to conduct complementary test, genomic DNA fragments containing the complete 0RF5 and 0RF9 gene werd cloned and ligated into vector pCAMBIA1301, respectively. The japonica rice variety H1493, which shows more strong susceptibility to BPH than TNI, was used for transformation. TO independent transgenic lines of 0RF5 and 0RF9 were produced.