【作者】 郑翠明；

【导师】 常汝镇；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2000， 博士

【摘要】 大豆花叶病毒（SMV）病是世界性大豆病害，在我国各大豆产区普遍发生，对大豆生产危害严重，利用抗病品种是控制SMV最经济而有效的措施。东北是我国大豆主产区，SMV3号株系是强毒株系，抗源较少。筛选鉴定和研究新抗源具有重要的理论价值和实践意义。本研究利用SMV3号株系对大豆资源进行了抗性鉴定，筛选出抗性种质，并利用农艺性状和RAPD标记分析了部分抗源的遗传关系。通过对抗病×感病杂交组合后代分析，明确了高抗SMV的大豆品系95-5383的抗性遗传规律。利用BSA法筛选出与SMV抗性基因紧密连锁的RAPD标记，并将其转化成SCAR标记。利用作图群体将RAPD标记定位于大豆遗传连锁图上。具体研究结果如下： 1．利用人工汁液摩擦法对不同来源的347份大豆种质资源接种SMV3号株系进行抗性鉴定。筛选出113份高抗资源，占32.56％；112份材料中抗SMV3，占32.27％；122份感病，占35.16％。抗源主要来自于东北春作大豆区（辽宁）和黄淮海夏作大豆区（山东、山西、北京）以及美国和韩国，南方大豆产区抗源较少。不同品种接种后的症状类型不同，表明品种和株系间存在互作。国外引进资源接种后顶枯症状较多，表明症状反应和地理来源有一定关系。本文还分析了美国一些抗源对SMV3的抗性反应及抗性基因的关系。 2．利用筛选出的高抗SMV3号株系的大豆品系95-5383与4个感病品种（品系）HBI、铁丰21、Amsoy、Williams和抗病品种PI486355配制5个杂交组合，对各组合的F1、F2代接种SMV鉴定抗性。结果表明，95-5383与各感病品种杂交组合的F1代表现为感病，F2群体分离比例为3感（花叶+顶枯）:1抗，表明95-5383对SMV3号株系的抗性受一对隐性基因控制。95-5383×PI486355的F2代接种后有感病植株分离，表明二者对SMV3的抗性基因是不等位的。利用BSA法对95-5383×HBI的F2代进行鉴定，筛选出与SMV抗病基因紧密连锁的共显性RAPD标记OPNII_980/1070。RAPD引物OPNII在95-5383和抗池扩增出OPNII₉₈₀片段，在HBI和感池扩增出OPNII₁₀₇₀片段，在F1同时扩增出OPNII₉₈₀和OPNII₁₀₇₀。用该引物分析95-5383×HBI的F2个体，OPNII_980/1070与95-5383抗病基因的遗传距离为2.1cM。 3．从95-5383和HBI中分别回收OPNII₉₈₀和OPNII₁₀₇₀特异片段，克隆在PGEM-T easy vector中，并进行序列分析。结果表明OPNII₉₈₀和OPNII₁₀₇₀的实际长度分别为986bp和1084bp，同源性为93.4％，主要差别是在两个不同区段插入（缺失）54bp和35bp的碱基。用克隆的RAPD片段OPNII₉₈₀作探针（SOPNII）对亲本基因组DNA 中国农业科学陨博十学位论义进行 Southern 65交，结果表明 OPN。。和 OPN川。。在人豆基因组中是以低拷贝形式存在。用n PD引物OPN和y卜于探针9口PN！1分析科丰1。南农1！3吕毛重组自交系作图群体，将 Opp和 SOPN定位于 F迹锁群的抗病基冈簇上．根据克隆片段OPN。羽IOPNll；。。的序列设计合成了一对SCAR引物SCNll，SCNll在95·5383中扩增出与 OPN。人小相同的片段 SCN。，在 HB中扩增出与 OPN。大小相同的片段SCN。，。，在FI同时扩增出SCN！l＿、。SCNn对95·5383 X HBI的FZ群体扩增结果与RAPD引物OPNll分析结果完全吻台。SCAR标记SCNll。。，。对于大豆抗SMV标记辅助选抒育种和抗病性鉴定有很)“泛的应用前景。 4．选用已经鉴定出的68份高抗SMV3的大亘资源，利用农艺性状和nPD分析品种间的遗传关系。从 60个 nPD引物中筛选出 11个多态性引物，共扩增出刃个片段，多态性片段为33条。扩增产物的有无分别以1和0记录，选用J＿。rd公式计算品种之间的相似系数，利用UPGMA法构建了树状图。RAPD聚类结果和农艺性状聚类结果具有一定的吻合性。聚类结果表明亲源关系相同、地理来额相同的品种多数聚在一类。分聚在不同组间的人豆品种迢传背景存在差异，可能具有不同的抗病基因。用OPN分析了68份抗性资源，其中有25份扩增出OPN。，衰明这些品种可能携带与95－5383相同的抗性基因，而其它扩增出OPN！l。；。。和OPNll。。的抗性品种可能携带不同于95．5383的抗性基因。更多还原

【Abstract】 Soybean mosaic virus (SMV) occurs worldwide and is a major soybean disease in China, resulting in yield losses and seed-quality deterioration . Using resistant varieties is the most economic and effective method to control SMV. Northeast China is the main soybean production region in China and the virulence of SMV 3 is the strongest. It抯 very important to identify and study new resistant germplasms. We have screened resistant soybean germplasm by inoculation of SMV3 and investigated the genetic diversity of part of the resistant varieties with RAPD and morphological characters. We have demostrated the inheritance of resistence of 95-5383 by studying the progenies of the crosses of resisantxsusceptible . We have identified one RAPD marker closely linked to SMV resistance gene by BSA and converted it into SCAR marker. The RAPD marker have been mapped in soybean molecular linkage group by using a mapping populations. The main results are summarized as followings: 1. 347 soybean accessions have been identified for the resistance to SMV3 by artificially inoculation with SMV 3, a strong virulent strain in Northeast China. The results showed that 113 accessions were highly resistant to SMV3. account for 32.56%; 112 accessions were moderately resistant to SMV3. account for 32.27%; 122 accessions were susceptible to SMV3, account for 35.16%. Resistant accessions were mainly originated from Northeast China (Liaoning), Huang Huaihai areas (Shandong, Shanxi province and Beijing), U.S. and South Korea. There were few resistant accessions from South China in our evaluation. Symptoms are different in different varieties when inoculated with the same SMV strain, showing there are interaction between soybean varieties and SMV strains. Top necrosis symptoms were observed in introduced varieties from abroad, indicating symptoms may relate to geographical origin of the accessions. Some varieties with known resistance gene introduced from U.S. were identified for the resistance to SMV3 and the relationship of resistance gene between Chinese accessions and U.S. accessions was discussed. 2 One highly resistant soybean line (95-5383) was crossed with four susceptible soybean varieties/line (HB 1, Tiefeng2 1, Amsoy, Williams) and one resistant variety (P1486355) Their Fl and F2 individuals were identified for SMV resistance by inoculation with SMV3. The results showed that in the four crosses of resistantxsusceptible, Fl were susceptible and F2 populations segregated in a 1 resistant: 3 susceptible (mosaic and necrosis) ratio, indicating that 95-53 83 carries one recessive gene that confer resistance to SMV. There are segregations of susceptibility in F2 progenies from the cross of 95-5383x P1486355, indicating that the SMV resistance gene in 95-5383 is located at different loci from that in P14863 55. By bulked segregant analysis (BSA) in F2 progenies of 95-5383xHB1, we have identified one codominant RAPD marker OPNI 1980/1070 closely linked to SMV resistance gene. DNA fragment OPN1 1980 was amplified in 95-5383 and resistant bulk, OPN1 1)070 was amplified in HBI and susceptible bulk. OPN1 1980,10,0 was amplified in Fl. Identification of the markers in F2 plants of 95-5383xHB1 showed that the codominant marker OPN1 1930,10,0 is closely linked to the resistance gene in 95-5383, with genetic distance of 2.1cM. 3 Both fragments of OPNI ~ and OPNI 110,0?amplified from 95-5383 and HB 1. respectively, were purified and cloned into PGEM-T easy vector and sequenced.更多还原