| Rice black-streaked dwarf disease is a very serious virus disease in rice currently in China, which is mainly transmitted by small brown planthopper (SBPH, Laodephax striatellus Fallen) in a persistent manner but could not be transmitted to offspring of SBPH through ovary. The identification of the resistance gene against Rice black-streaked dwarf disease and the development of the resistance cultivars are the basic strategy to get this disease under control. The genetic study of resistance to Rice black-streaked dwarf disease in rice varieties and the breeding process against the disease were pretty slowly because of the lack of the artificial inoculation identification. An artificial inoculation identification method of rice varieties against Rice black-streaked dwarf disease was developed after the three bottlenecks, including the interference from southern rice black-streaked dwarf virus(SRBSDV), the preserve of the source with rice black-streaked dwarf virus and the interference from rice stripe virus, were broken. Identification the resistance lever among the cultivars and varieties were taken by multi-point test for several years in evaluation nursery. After confirm the resistance lever by artificial inoculation identification method, the resistance mechanism of the resistance variety against rice black-streaked dwarf virus was analyzed. The genetic mode of resistance variety was analyzed in the population cross by the resistance variety and susceptible variety. Meanwhile the genetic linkage map was built by molecular marker analysis and the resistance loci against Rice black-streaked dwarf disease were mapped. The main results are as follows.1. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid and sensitive detection of Rice black-streaked dwarf virus (RBSDV) from host plants and insect vector which demonstrated a high degree of specificity for RBSDV, which can distinguish RBSDV from SRBSDV. The method was also proved to be extremely sensitive, which was as much as the RT-PCR for RBSDV detection. The detection of amplified products was easily monitored. The development of this method could break the bottleneck of the artificial inoculation identification against Rice black-streaked dwarf disease which came from the interference by SRBSDV.2. A simple and reliable method which can get the rice stripe virus free SBPH acquired RBSDV from frozen infected leaves and transmitted the virus to healthy rice plants was developed. SBPHs acquired RBSDV from the thaw frozen infected leaves were put in the healthy plants to transmit the virus. The result showed that there is no different between the SBPHs acquired the virus from thaw frozen infected leaves and these acquired the virus from the normal infected plants. It demonstrated that the new method can be used in artificial inoculation identification against rice black-streaked dwarf disease and keep the virus source live for SBPH acquiring the virus for a longer time.3. The effects of four factors to the artificial inoculation identification against rice black-streaked dwarf disease, including virus latent period in vectors, inoculation time, inoculation intensity and stage of the seedlings for inoculating, were evaluated in this study. The results revealed that the effects of artificial identification for12-15days’or21-24days’ virus latent period in vectors treatment were better than that for8-11days’or16-17days’, the effects for48-72hours’inoculation time treatment were better than that of12-24hours’, the effects of the treatment inoculated by4-20viruliferous insects per seedling were better than by1-3viruliferous insects per seedling, and the effects of the treatment inoculated in0.5-2.5leaf age were better than in3.5leaf age. Thus the optimal conditions for artificial identification of resistance to RBSDV in rice varieties were12-15days’virus latent period in vectors,48-72hours’inoculation time,4-20viruliferous insects per seedling and0.5-2.5leaf age’s seedlings. The artificial inoculation identification showed the same results as field identification in further research, which fully proved this method can reflect the RBSDV resistance levels of rice varieties.4. Several evaluation nurseries against rice black-streaked dwarf disease were set in Jianhu county, Yandu county and Guanyun county in Jiangsu province around the area where the disease was very seriously recently. Although it showed some difference among the cultivars and varieties, there was no cultivar or variety showing immune or highly resistance to Rice black-streaked dwarf disease in the multi-point test for two years in evaluation nursery. A variety, Tetep, from Vietnam represented resistance or medium resistance to the disease in the entire field test, which can be widely used in the breeding program against the disease.5. The resistance to RBSDV and the vector, SBPH were analyzed in Tetep by using the method of artificial inoculation identification, non-preference test and antibiosis test. The result showed that Tetep was resistant to RBSDV and weakly resistant to vector, i.e. the Rice black-streaked dwarf disease resistance in Tetep was mainly derived from the resistance to virus. The inheritance of Rice black-streaked dwarf disease resistance in F2·3lines from the cross Huaidao No.5/Tetep was studied by using artificial inoculation identification method. The result demonstrated that the resistance in Tetep was inherited as quantitative trait, which might control by one or two major quantitative trait locus (QTLs).6.160markers with polymorphism between two parents were acquired by screeningyin842Simple Sequence Repeat (SSR) markers, which the ratio of polymorphisms was19%.129SSR markers were analyzed in138lines from the cross between Huaidao No.5and Tetep. A molecular linkage map covered2179.6cM in rice genome was acquired by these markers analysis with an average interval size of17.16cM. Resistance QTL against Rice black-streaked dwarf disease was analyzed by software of Windows QTL Cartographer2.5. Two major resistance QTLs were identified. One was mapped between RM5626%nd RM7079on chromosome3was named qRBSDV-3, which can explain17.5%of total phenotypic variation with LOD scores of4.07. The other was mapped between RM202and RM7120on chromosome11was named qRBSDV-3, which can explain12.4%of total phenotypic variation with LOD scores of2.21. Both of resistance QTLs were derived from Tetep and were newly identified by comparing the loci with these published before. |
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