【作者】 杨清华；

【导师】 盖钧镒；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 大豆花叶病毒（Soybean Mosaic Virus, SMV）病是一种主要的世界性大豆病毒病害。在与寄主长期互作的过程中,SMV发生了致病性分化,产生了不同的致病类型,即株系。在我国,以往的研究者各自采用了不同的鉴别寄主体系,划分的株系缺乏可比性,给抗病信息和抗性材料的交流带来困难。有的鉴别寄主体系的株系划分过细,不利于实际育种研究。因此,本研究将利用前人收集得到的SMV分离物和株系,通过在相对较一致的环境下接种鉴定,依据寄主的症状稳定性进一步精选鉴别寄主,最终构建一套更完善的鉴别寄主体系,从而调整全国大豆花叶病毒株系；比较三种不同保存方法对SMV株系的保存效果,寻找一种适合SMV长期保存的方法；从病毒分子水平,分析SMV的P3序列差异及其与致病性的关系；针对强毒株系进行抗源筛选,研究抗病品种对强毒株系的抗性遗传规律以及抗病基因的分子标记定位。主要结果如下：1.我国大豆花叶病毒株系的分化（1）根据来自全国23个省市的310份SMV分离物和4个株系在王修强-杨雅麟-战勇确定的10个鉴别寄主上的抗/感反应,归类得到24个株系。经与前人鉴定结果比较,有149份分离物在10个鉴别寄主上的抗/感反应完全一致,其余分离物在个别鉴别寄主上的抗/感反应存在差异。通过比较10个鉴别寄主的症状稳定性,最终确定将其中7个（南农1138-2、诱变30、Davis、早熟18、Kwanggyo、齐黄1号和科丰1号）组成一套简化的鉴别寄主体系,从而将我国SMV调整为16个株系,并命名为C1-C16。调整后,原SC体系的21个株系,除株系SC17因涉及另一鉴别寄主外,其他株系均归到调整后的C体系中。株系地理分布表明,弱毒株系C1和强毒株系C16分布范围最广,遍及6个大豆品种生态区,而株系C3、C12和C13分别仅分布于生态区Ⅱ、Ⅱ和Ⅴ。（2）选用4份SMV分离物在低温（-40℃）、超低温（-80℃）和液氮（-196℃）三种保存方法下,保存3个月、8个月和15个月。在南农1138-2上接种、鉴定,以发病率为指标,保存3个月时,三种保存方法下的各分离物的保存效果无明显差异；保存8个月和15个月,三种保存方法对各分离物的保存效果有差异,且株系间不同分离物的保存效果有显著差异。三种保存方法均能保持SMV的长期保存,其中液氮效果最佳。2.我国大豆花叶病毒株系P3基因的序列特征（3）从C1-C16中每株系取1-3个分离物进行P3基因测序,结果表明,P3基因均编码347个氨基酸。23个分离物间核苷酸及氨基酸的同源性分别为90.5%-100%和94.5%～100%;系统发育分析和多重序列分析表明,株系致病性的强弱与其P3基因的序列差异未发现有相关性。与SMV其他分离物P3序列同源性比较结果,中国和韩国的SMV P3序列同源性相对较高（核苷酸92.4%-98.9%,氨基酸96.0%-100%）,和美国SMV的P3序列同源性相对较低（核苷酸88.5%-97.9%,氨基酸91.4%-98.6%）；与文献报道为SMV的从半夏（Pinellia ternata）得到的分离物比较结果,同源性明显低于从大豆叶片分离得到的SMV分离物（核苷酸80.4%-85.2%,氨基酸82.1%-84.7%）,因而对该分离物是否为SMV提出异议。与其他16种Potyvirus病毒的P3序列同源性以及系统发育树分析显示,SMV与西瓜花叶病毒的同源性最高（核苷酸76.0%-81.9%,氨基酸77.5%-85.3%）,亲缘关系最近；与花生斑驳病毒、甜菜花叶病毒和落葵皱叶嵌纹病毒的同源性最低（核苷酸44.4%-54.3%,氨基酸21.4%-28.8%）,亲缘关系较远。从氨基酸多重序列分析显示,P3序列在种内高度保守,而在种间相对可变,尤其是C端区域。3.大豆对大豆花叶病毒强毒株系C16的抗源筛选、遗传和抗性基因标记定位（4）鉴于SMV强毒株系C16可侵染全部鉴别寄主,从205份来自该株系有分布的湖南、湖北、江西等17个省份（或国家）的大豆材料中鉴定、筛选到具有抗性的RN-9、89-29、大绿豆、赣豆1号、晋大53、南农87-23、通山薄皮黄豆甲、皖82-178、早16号和矮秆黄等10个抗源。（5）对RN-9（抗病）×7605（感病）杂交组合的P1、P2、F1、F2和重组自交家系（RIL）接种鉴定结果,F1表现抗病,F2和200个RIL表现抗／感分离,经卡方适合性测验,F2群体抗：感符合3:1的表型分离比例,RIL符合1:1的基因型分离比例,抗性亲本RN-9对强毒株系C16的抗性受一对显性基因控制,并命名为Rcl6。（6）选用覆盖大豆全基因组的957对SSR引物,采用分离群体组群分析法,将大豆品种RN-9携带的抗病基因Rcl6定位于C2连锁群,所获连锁片段的标记与距离为Sat₂46-（0.9 cM）-Sat₂13-（8.0 cM）-RC16-（6.6 cM）-Sat₂86-（9.4 cM）-Satt100-（2.7 cM）-Sat₂38-（0.6 cM）-Satt079-（1.0 cM）-Sat₂63-（1.7 cM）-Staga001-（13.4 cM）-Satt433。更多还原

【Abstract】 Soybean mosaic virus （SMV） disease is one of the main diseases in soybean production worldwide. The interaction between soybean mosaic virus and host plants in a long-term co-evolution induced the pathogenic differentiation, and produced various pathogenic types, namely strains. In China, many strains were classified based on the different soybean differentials systems, which was short of the comparability and difficult for the communication of resistant information and materials. Some soybean differentials systems produced too many strains, which was inappropriate for resistance breeding. Therefore, the objectives of this study were to inoculate the SMV isolates and strains collected by the previous reporters in the relatively consistent environment, carefully choose the soybean differentials with the stable symptoms to assign a more perfect system and adjust SMV strains in China. Effects of three methods for SMV preservation were compared to find a suitable method for SMV long term preservation. P3 sequences were determined to reveal the relationship between sequence differences of P3 gene and pathogenicity of SMV in soybean differentials. Resistant-source selection, genetic analysis and gene mapping of resistance to virulent strain in soybean were conducted. The main results were as follows:1. Differentiation of soybean mosaic virus strains in China（1） Based on the reactions of ten soybean differentials previously selected by Wang-Yang-Zhan to three hundred and ten isolates and four strains of SMV from twenty-three provinces （cities） in China,24 SMV strains were grouped. Comparing with the previous results, the reactions of 10 determined soybean differentials to 149 isolates were consistent, while that of some soybean differentials to the rests were different. Based on the symptom stablility, Nannong1138-2, Youbian30, Davis, Zaoshul8, Kwanggyo, Qihuangl and Kefengl were finally considered as a simplified differential system to adjust SMV strains. All of tested SMV accessions were classified into sixteen strains finally, designated C1～C16. The results of adjustment showed that the previous strains in the SC system could be included in the C system, except for SC17. The results from geographic distribution of strains indicated that the weak strain C1 and the virulent strain C16 covered over all six eco-regions of soybean, while strains C3, C12 and C13 only distributed in eco-regionⅡ,ⅡandⅤ, respectively.（2） Four SMV isolates were preserved in low temperature refrigerator （-40℃）, ultra low temperature freezer （-80℃） and liquid nitrogen （-196℃） for three, eight and fifteen months. Based on the incidence rate of inoculated cultivars Nannong1138-2, the results showed that the preservation effects of each isolate preserved in the three methods for three months had no significant differences, while the three methods had significant influences on the effects of each isolates preserved for eight and fifteen months. The preservation effects of isolates from different strains had significant differences. The three methods were all suitable for a long term preservation of SMV, in which the method of liquid nitrogen was the best.2. Sequence characteristics of P3 genes of soybean mosaic virus in China（3） P3 genes of 1～3 isolates from C1～C16 strains were determined. The results showed that P3 regions of all isolates encoded 347 amino acid residues. Pairwise comparisons of P3 gene of isolates were 90.5%～100% （nucleotide, nt） and 94.5%～100% （amino acid, aa） at nucleotides and amino acid levels, respectively. Phylogenetic tree and multiple sequence alignment showed that pathogenicity of strains was uncorrelated with genetic relationships of P3 gene.Comparing with the documented data of other SMV strains, the results showed that the P3 regions shared high identities （92.4%～98.9% nt and 96.0%～100% aa） with the reported Korean isolates, but a little lower identities （88.5%～97.9% nt and 91.4%～98.6% aa） with the reported American isolates. The identities betweem the isolates obtained from Pinellia ternate and that obtained from soybeans were obviously lower than that of the SMV isolates obtained from soybeans （80.5%～85.2% nt and 82.1%～84.7% aa）, resulting that whether the virus can be regarded as SMV needs further evidences.The results of pairwise comparisons and phylogenetic tree between SMV and the 16 potyviruses in P3 gene showed that SMV had a close relationship with watermelon mosaic virus （76.0%～81.9% nt and 77.5%～85.3% aa）, and a distant relationship with peanut mottle virus, beet mosaic virus and basella rugose mosaic virus with the lowest identities of 44.4%～54.3% nt and 21.4%～28.8% aa. Meanwhile, multiple sequence alignment indicated that P3 regions within a species were highly conserved, while that among species were relatively variable, especially in C terminal regions.3. Identification, inheritance and gene mapping of resistance to the virulent strain C16 in soybeans（4） The virulent strain C16 could infect all soybean differentials under the new system. Of 205 soybean accessions collected from seventeen province （or countries）, ten resistant accessions （RN-9,89-29, Dalvdou, Gandoul, Jinda53, Nannong87-23, Tongshanbopi-huangdoujia, Wan82-178, Zao16 and Aiganhuang） resistant to strain were screened out.（5） The P1, P2, F1, F2 and recombinant inbred line （RIL） of RN-9×7605 were inoculated with strain C16. The symptom reaction of F1 was resistant, the F2 populations segregated in a phenotypic ratio of 3 resistant:1 susceptible and the RIL exhibited a genotypic segregation ratio of 1 resistant:1 susceptible based on the test for goodness-of-fit. Therefore, a dominant gene was conferring to the resistance to C16, designated as Rc16.（6） 957 pairs of simple sequence repeat markers covering all over the soybean genome were screened. The linkage analysis using bulked segregant analysis method indicated that the resistance gene RC16 of RN-9 cultivar was located on the linkage group C2, and the order and genetic distance of linked genes on the the segment linkage map were Sat₂46-（0.9 cM）-Sat₂13-（8.0 cM）-RC16-（6.6 cM）-Sat₂86-（9.4 cM）-Satt100-（2.7 cM）-Sat₂38-（0.6 cM）-Satt079-（1.0 cM）-Sat₂63-（1.7 cM）-Staga001-（13.4 cM）-Satt433.更多还原