【作者】 周彤；

【导师】 范永坚；

【作者基本信息】 南京农业大学 ， 植物病理学， 2013， 博士

【摘要】 水稻黑条矮缩病(Rice black-streaked dwarf disease)是一种主要由灰飞虱(Laodephax striatellus Fallen)以持久性不经卵方式传播的恶性水稻病毒病。发掘抗性基因和培育抗性品种是解决这类病毒病害的根本策略,但受制于品种抗性人工接种鉴定方法的缺乏,水稻黑条矮缩病品种抗性的遗传学研究和育种实践一直进展较慢。为此,本研究在破解了南方水稻黑条矮缩病毒假阳性毒源干扰、病毒毒源保存和灰飞虱携带水稻条纹病毒干扰等技术难点的基础上,构建了高效引发水稻黑条矮缩病的人工接种鉴定方法。同时,采用多年多点方法在重病区抗鉴圃中对水稻品种和资源进行水稻黑条矮缩病的抗性鉴定,利用上述人工接种鉴定方法对筛选出的抗性品种进行进一步的抗性确认,并分析其对水稻黑条矮缩病的抗性特征。将抗性品种与感病品种构建分离群体,采用人工接种鉴定方法分析其遗传模式,同时利用分子标记分析方法构建遗传连锁图谱,对抗性基因进行了定位研究。主要结论如下。1.建立一种快速、灵敏检测的水稻黑条矮缩病毒的逆转录环介导等温扩增方法(Reverse Transcription Loop-Mediated Isothermal Amplification, RT-LAMP)。该方法可排除南方水稻黑条矮病毒的干扰而特异的检测植物和飞虱体内的水稻黑条矮缩病毒,其灵敏性与RT-PCR基本一致,但检测结果易于判定。这一方法的建立成功破解了水稻黑条矮缩病品种抗性的人工接种鉴定方法中南方水稻黑条矮缩病毒假阳性毒源干扰。2.发明了一种利用灰飞虱从冻存病叶中获得水稻黑条矮缩病毒的方法。以冷冻保存的水稻黑条矮缩病罹病植株叶片对灰飞虱进行饲毒,随后接种感病水稻品种,结果发现灰飞虱可从冻存病叶上获得并传播水稻黑条矮缩病毒,且获毒能力和传毒能力与未冻存病叶处理相比无显著性差异。这表明从冻存病叶上获得水稻黑条矮缩病毒的灰飞虱可以应用于品种抗性人工接种鉴定。3.利用梯度试验分别研究了病毒在介体体内的循回时间、接种时间、接种强度、水稻接种苗龄4个因素对鉴定效果的影响。结果显示,病毒在介体体内的循回时间为12-15d或21-24d条件下,鉴定效果优于8-11d和16-17d处理；接种48-72h条件下,鉴定效果优于12-24h处理；有效接种强度4-20虫/苗条件下,鉴定效果优于1-3虫／/苗处理；水稻接种苗龄0.5-2.5叶龄条件下,鉴定效果优于2.5-3.5叶龄处理。由此构建了水稻抗黑条矮缩病人工接种鉴定方法：循回时间为12-15d、接种时间48-72h、有效接种强度4-20虫/苗和水稻接种苗龄0.5-2.5叶。在此条件下对不同抗性表现的水稻品种进行鉴定,其鉴定效果与重病区田间鉴定效果无显著性差异,表明所构建的水稻黑条矮缩病品种抗性人工接种鉴定方法能客观地反映水稻品种对水稻黑条矮缩病的抗性水平。4.在常年水稻黑条矮缩病重发的地区江苏省建湖县、盐都县和灌云县分别建立抗性鉴定圃,对主栽品种和资源材料进行了多年多点田间抗性鉴定,发现水稻品种对水稻黑条矮缩病的田间抗性在不同地点和不同年份间并不相同,尽管不同品种对水稻黑条矮缩病表现出一定差异,但没有发现免疫或高抗的品种。一份来自越南的籼稻资源材料特特勃在两年三点的田间试验中表现出抗病和中抗的抗性水平,可望作为抗性资源运用于针对水稻黑条矮缩病的抗性育种。5.利用人工接种鉴定、非嗜性测验及抗生性测验分析了特特勃对水稻黑条矮缩病和传毒介体灰飞虱的抗性特征,结果表明特特勃对水稻黑条矮缩病表现为抗病,抗虫性鉴定发现特特勃仅表现出弱非嗜性,而无抗生性。综合抗病性和抗虫性表现,特特勃对水稻黑条矮缩病的抗性主要来自于对病毒本身的抗性,而不是对传毒介体灰飞虱的抗性。采用人工接种鉴定方法对淮稻5号/特特勃构建的F2：3家系进行水稻黑条矮缩病抗性遗传分析,发现其对水稻黑条矮缩病的抗性为数量性状,可能由1-2个主效基因控制。6.在842对微卫星标记中筛选获得在淮稻5号和特特勃间存在多态性标记160个(多态性频率19%),从中选用127个标记对淮稻5号/特特勃F2群体的138个株系进行分析,构建覆盖基因组2179.6cM的水稻遗传连锁图谱,标记间平均间距为17.16cM。采用基于复合区间作图法的软件Windows QTL Cartographer V2.5对构建的遗传图谱和之前获得表型数据进行分析,从淮稻5号/特特勃的F2群体检测出2个水稻黑条矮缩病抗性QTL,分别命名为qRBSDV-3和qRBSDV-11.其中qRBSDV-3位于第3染色体的RM5626-RM7097之间,LOD值为4.07,贡献率为17.5%。qRBSDV-11位于第11染色体的RM202-RM7120之间,LOD值为2.21,贡献率为12.4%,两抗性QTL均来自于抗病品种特特勃。通过图谱比对表明本研究获得的两个抗性QTL是新的水稻黑条矮缩病抗性位点。更多还原

【Abstract】 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.更多还原