RNAi介导SMV-P3基因沉默增强大豆对花叶病毒病的抗性

大豆花叶病毒(soybean mosaic virus, SMV)病是我国各大豆主产区最重要的病害之一,严重影响大豆产量和籽粒外观品质。培育抗病品种是防治该病最经济有效的措施。本研究基于植物介导RNA干扰(RNA interference, RNAi)技术,将编码参与SMV运动和影响宿主域范围的P3蛋白基因RNAi片段导入栽培大豆品种,研究RNAi介导SMV-P3基因沉默对大豆抗SMV的影响。Southern杂交检测结果表明,外源RNAi片段以低拷贝的形式(1~4个)整合至大豆基因组中。对T1~T3代转基因大豆喷施除草剂和PCR鉴定结果表明,外源T-DNA插入片段在转基因大豆不同代际间能够稳定遗传。对T2和T3代转基因大豆接种SMV鉴定结果表明,转基因大豆对我国大豆产区主要流行SMV株系SC-3较非转基因对照受体品种Williams 82和SN9的抗性水平显著提高,病情指数降低至4.37%~18.51%,且抗性能够稳定遗传。综上所述,RNAi介导SM-P3基因沉默能够显著提高转基因大豆对SMV的抗性水平。     

与大豆SMV3号株系抗性相关的分子标记的鉴定

对大豆花叶病毒SMV抗性的遗传研究一直是大豆抗病遗传研究的热点之一。本研究以哈91R3-301×黑农41组合构建了遗传群体,其F2分离单株的SSR标记基因型基本符合1:2:1的比例,说明这个群体没有偏分离。根据F3株系的病情指数分布推测SMV3的F2成株抗性似乎由多基因控制。根据SSR分子标记的基因型和F2:3株系对SMV3抗病性表型结果连锁分析,推测Satt296是与大豆花叶病毒(SMV)3号株系抗性主基因连锁的分子标记,应用Joinmap作图软件将该标记定位在D1b连锁群上,这一结果与部分文献报道的研究结果一致。本研究获得的与抗性基因连锁的分子标记在其他的RIL群体中的验证得到了初步证实,推测定位在D1b连锁群上的抗性座位可能是控制SMV3的主基因之一,该标记可望应用于大豆抗SMV3的分子标记辅助选择。     

农杆菌介导的RNAi CP基因在大豆中的转化

花叶病毒(soybean mosaic virus, SMV)病是大豆主要病害之一,生产上常采用种植抗性品种方法来防治。本研究以RNA干扰花叶病毒衣壳蛋白(coat protein, CP)基因为表达载体,Bar基因作为筛选标记基因,成熟子叶节为外植体,采用农杆菌介导法获得了22株T₀代转基因大豆生根苗,经草丁膦涂抹、Bar试纸条和PCR法鉴定,获得RNAi CP转基因植株18株;对转基因植株T₁代的遗传分析表明,外源基因能够稳定遗传到下一代且符合孟德尔遗传规律;T₁代Southern杂交表明,导入的干扰片段为单拷贝;花叶病毒摩擦接种表明RNAi CP转基因大豆植株具有抗花叶病毒特性;摩擦接种后3周,DAS-ELISA检测进一步表明,RNAi CP转基因植株花叶病毒检出率仅为7.69%,而非转基因植株为100%。这表明RNAi花叶病毒CP基因可用于抗大豆花叶病毒的研究。     

大豆抗SC3候选基因的克隆及分析

大豆花叶病毒(Soybean mosaic virus, SMV)病是大豆主要的病害之一,给我国大豆生产带来了巨大的损失。大豆抗病育种是目前防治大豆花叶病毒病最为经济有效的措施,发掘抗病基因是抗病育种的基础。本文在前期对大豆抗SMV株系SC3基因精细定位的基础上,克隆了2个具有TIR-NBS-LRR典型抗病结构域的基因(GmR47和GmR51)。生物信息学分析表明, GmR47和GmR51基因均在抗感品种中存在氨基酸位点的突变,而且突变位点都位于保守结构域内,这2个基因编码的蛋白质预测为烟草花叶病毒(TMV)抗性N蛋白;物种间同源比对结果显示, GmR47和GmR51基因与野生大豆亲缘较近。qRT-PCR结果表明, GmR47和GmR51能够响应SMV的侵染增加表达量,且在抗病品种中的表达量高于感病品种。2个基因存在IN1、IN2和IN3不同的剪接体,所有的剪接体都能够响应病毒的诱导增加表达量,且在抗病品种中的表达量高于感病品种, IN1和IN2的表达量随时间的变化较为明显, IN3的表达量则相对稳定,说明这些剪接体可能参与大豆对SMV的抗病过程。本研究为后续基因功能的研究奠定了基础。     

冀豆系列品种抗病基因型分子推断

大豆花叶病毒病(SMV)和大豆孢囊线虫病(SCN)是危害大豆生产的重要病害。本试验以冀豆系列14个品种为材料,利用RAPD和SCAR标记技术对其进行了大豆花叶病毒病和孢囊线虫病的抗病基因型分析,以寻找可供在大豆生产和育种中利用的抗源。所用引物为重复性较好的OPL_07,OPAO_19和SCW_05。通过分析,我们初步推断冀豆4号和五星一号同时具有大豆花叶病毒SC和Sa两种株系的抗性基因,其中五星一号既具有大豆花叶病毒病抗性基因同时还具有大豆孢囊线虫病抗性基因,可推荐作为大豆生产和育种中优先选用的抗源材料。     

重组型大豆花叶病毒河北分离物序列特征及侵染性

重组型大豆花叶病毒(recombined soybean mosaic virus,SMV-R)是一种新SMV类型,在我国多个大豆产区广泛流行。本研究对一个重组型SMV河北分离物(HB-RS)进行全基因组测序,比较与非重组型SMV在侵染4个大豆品种后病毒浓度积累的差异。结果显示,除poly-A尾巴外,HB-RS(NCBI登录号为KR065437)由9993个核苷酸组成,包含一个开放阅读框(open reading frame,ORF),翻译后形成3202个氨基酸,系统进化分析结果显示HB-RS分离物与另外两个重组型SMV分离物聚在一组。抗性鉴定结果显示,4个品种对HB-RS和Sc6平均病情指数分别为59.5和60.5,相同大豆品种对不同的株系(分离物)可能呈现不同的症状和抗性表现,其中冀豆17对Sc6和HB-RS分别表现高抗和中抗,表明大豆对SMV的抗性存在一定的株系(分离物)专化性。此外,HB-RS在4个品种中的浓度积累均高于Sc6,在南农1138-2病毒浓度最高,达522 U,其次为五星1号(471 U)和冀黄13(199 U),最低为冀豆17,仅90 U。说明HB-RS在寄主体内更具有生存适应性,不同品种对SMV存在抗性差异。冀豆17可作为抗性品种和亲本进一步推广。     

大豆广谱抗源对SMV优势株系的抗性遗传和等位性分析

为了研究大豆广谱抗源对我国大豆花叶病毒优势株系SC3和SC7的遗传方式及抗源材料对SMV抗性基因间的等位性关系,利用广谱抗源科丰1号和齐黄1号与感病材料南农1138-2配制抗感及抗抗杂交组合,通过人工摩擦接种法进行鉴定。结果发现,接种株系SC3和SC7后,科丰1号和齐黄1号与南农1138-2配制抗感组合的F1均表现抗病,经卡方测验,F2抗感分离比例符合3∶1,F2∶3家系分离比例为1(抗)∶2(分离)∶1(感),说明这2个广谱抗源均有1对显性基因控制株系SC3和SC7的抗性;等位性测验结果表明2个抗抗组合的F1对SC3和SC7优势株系均表现抗病,F2分离比符合15(抗)∶1(感),说明科丰1号和齐黄1号对株系SC3和SC7的抗性基因不等位且独立遗传。进一步分析2个广谱抗源携带的抗性基因可以发现,科丰1号对株系SC3的抗性基因RSC3和齐黄1号对SC7株系的抗性基因RSC7Q可能位于大豆的2号和13号染色体上,为利用大豆广谱抗源进行抗SMV育种奠定了很好的基础。     

4种主要黄瓜病害的遗传分析

番木瓜环斑病毒西瓜株系(PRSV-W)、小西葫芦黄花叶病毒(ZYMV)、西瓜花叶病毒(WMV)和黑星病(Cladosporiumcucumerinum)是为害黄瓜的主要病害。以欧洲八号×秋棚为亲本构建的RIL群体为材料,以病情指数为指标,对群体和亲本进行了小西葫芦黄化花叶病毒(ZYMV)、番木瓜环斑病毒西瓜株系(PRSV-W)、西瓜花叶病毒(WMV)株系和黑星病的抗病性鉴定。结果表明:上述抗性基因的病情指数在黄瓜RIL群体呈双峰分布,表明对ZYMV,PRSV和WMV的抗性是受主基因控制的性状,但也存在微效基因的修饰作用。连锁分析表明,ZYMV,PRSV与WMV连锁,3个抗病基因的排列顺序和遗传距离是ZYMV-10cM-PRSV-22cM-WMV。抗黑星病基因与上述三种抗病毒基因不连锁。     

大豆对大豆花叶病毒抗侵染与抗扩展育种应用的讨论

大豆既可抗SMV的侵染,又可抗扩展。抗侵染由一或两对基因控制,具有明显的株系专化性,存在因株系变化而丧失抗性的可能,但抗侵染品种不受SMV的影响,且抗性基因鉴定、转育方便,在品种更替速度不断加快以及注意SMV动态变化的情况下,针对主要流行株系的抗侵染已被广泛利用并将继续发挥重要作用。抗扩展由一对加性主基因和加性-显性多基因共同控制,以主基因作用为主,多基因起修饰作用。这种抗性虽不能抵抗SMV的侵染,但大豆感染后病情较轻,产量损失一般在5%以下,且抗谱广、抗性稳定,抗源丰富。因此,对抗扩展育种应予以重视。随着分子标记辅助选择技术的发展,期望能通过分子标记辅助抗性选择,把对多个株系的抗侵染基因聚合到同一品种,甚至把两类抗性聚合到同一大豆品种。     

河北省推广大豆品种对六个SMV株系的抗性鉴定

为调查河北省推广大豆对大豆花叶病毒的抗性情况,本研究对9份大豆品种,包括高蛋白品种:冀豆12号,冀豆7号;高油品种:冀黄13号,nf37,nf58;兼性品种:冀豆15号,鉴15;以及无腥大豆品种:五星1号,五星2号,均采用人工汁液摩擦法分别接种6个SMV株系进行抗性鉴定。鉴定结果表明,五星1号、冀豆12号和五星2号是3个较理想的抗SMV品种,适合推广种植。     

Transfer of the Rsv3 locus from ‘Harosoy’ for resistance to soybean mosaic virus strains C and D in Japan

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety ‘Harosoy’ has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as ‘Fukuibuki.’ Because ‘Harosoy’ harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from ‘Fukuibuki’ into a leading variety ‘Ohsuzu’ by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, ‘Tohoku 169,’ was equivalent to ‘Ohsuzu’ with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.     

Characterization of broad‐spectrum resistance to Soybean mosaic virus in soybean [Glycine max (L.) Merr.] cultivar ‘RN‐9’

Soybean mosaic virus (SMV) can cause serious yield losses in soybean. Soybean cultivar ‘RN‐9’ is resistant to 15 of 21 SMV strains. To well‐characterize this invaluable broad‐spectrum SMV‐resistance, populations (F₁, F₂ and F_2:3) derived from resistant (R) × susceptible (S) and R × R crosses were tested for SMV‐SC18 resistance. Genetic analysis revealed that SC18 resistance in ‘RN‐9’ plus two elite SMV‐resistant genotypes (‘Qihuang No.1’ and ‘Kefeng No.1’) are controlled by independently single dominant genes. Linkage analysis showed that the resistance of ‘RN‐9’ to SMV strains SC10, SC14, SC15 and SC18 is controlled by more than one gene(s). Moreover, Rsc10‐r and Rsc18‐r were both positioned between the two simple sequence repeats markers Satt286 and Satt277, while Rsc14‐r was fine‐mapped in 136.8‐kb genomic region containing sixteen genes, flanked by BARCSOYSSR_06_0786 and BARCSOYSSR_06_0790 at genetic distances of 3.79 and 4.14 cM, respectively. Allelic sequence comparison showed that Cytochrome P450‐encoding genes (Glyma.06g176000 and Glyma.06g176100) likely confer the resistance to SC14 in ‘RN‐9’. Our results would facilitate the breeding of broad‐spectrum and durable SMV resistance in soybeans.     

大豆花叶病毒(SMV)株系SC4和SC8的抗性遗传分析

选用我国黄淮和长江流域大豆产区发生频繁的SMV株系SC4和SC8,利用大豆抗病材料和感病材料配制抗感和抗抗杂交组合,研究抗病材料对SC4和SC8株系的遗传方式以及不同大豆材料对SMV抗性基因位点间的等位性关系。结果表明, 接种SC4株系后,由冀LD42、徐豆1号、跃进4号、科丰1号、PI96983、晋大74、汾豆56、大白麻和齐黄22为抗源配制的9个抗感组合的F₁均表现抗病,经卡方测验, F₂抗感分离比例3∶1,F_2:3家系分离比例为1(抗)∶2(分离)∶1(感),表明这些抗源均有1对基因控制对SC4株系的抗性,且抗病表现为显性;5个抗抗组合的F₁均表现抗病,F₂群体分离比例15(抗)∶1(感),表明大白麻与汾豆56、科丰1号和齐黄1号携带抗SC4的基因是不等位的,冀LD42与汾豆56,晋大74与中作229是不等位的。接种SC8株系后,用齐黄1号、中作229、NY58、科丰1号、PI96983、晋大74、汾豆56、大白麻和齐黄22为抗源配制的抗感组合杂交后代分离符合1对基因的分离比例且F₁均表现抗病,说明这些品种对SC8株系的抗性也均由1对显性基因控制。抗抗组合晋大74×汾豆56接种SC8株系后的F₂群体全部表现抗病,F_2:3家系没有抗感分离,表明抗病品种晋大74与汾豆56携带的抗病基因是等位的;齐黄1号×科丰1号、大白麻×汾豆56的F₂群体分离比例15(抗)∶1(感),表明抗病亲本齐黄1号与科丰1号、大白麻与汾豆56携带抗SC8的基因是不等位的,而且独立遗传。     

Marker-assisted identification of resistance genes to soybean mosaic virus in soybean lines

Soybean plants react differentially to soybean mosaic virus (SMV) strains because of interactions among different resistant genes in the soybean genome. Three independent genes resistant to SMV have been identified by inheritance studies and linkage analyses. To develop durable SMV-resistant soybean cultivars, it is necessary to determine which soybean SMV resistance genes can be readily transferred from resistant to susceptible cultivars in a breeding system. Here, we report the type and number of resistance gene(s) in four Korean elite soybean cultivars using a combination of disease reaction symptoms, inheritance studies, and molecular marker mappings. The disease reactions of Sowonkong and Keunolkong soybean varietals in response to infection with SMV strains suggested that both cultivars most likely harbor the Rsv1 gene similar to that in York. Subsequent inheritance studies confirmed that Sowonkong has the Rsv1 gene. The inheritance studies suggested that Sinpaldalkong harbored the Rsv1 gene, which was then confirmed by molecular marker mapping. The inheritance studies also suggested that Jinpumkong 2, which is the most resistant to SMV infection among the four cultivars, contained the Rsv1 and Rsv3 genes; this was confirmed by molecular marker mapping. Our approach, which combined inheritance studies and molecular linkage analyses, allowed the efficient identification of resistance gene(s) in four Korean soybean cultivars.     

大豆对SMV抗侵染与抗扩展的遗传分析

大豆花叶病毒（SMV）抗性研究最早着重于系统病症,后来发现感病材料还存在发病程度上的遗传差异,抗侵染与抗扩展并不相同,从而鉴别出一批具不同类型抗性的抗源。本研究利用抗侵染和抗扩展品种（系）配置10个不同类型杂交组合,在分别接种Sa或SC8株系条件下,研究两类抗性的遗传模式。结果表明,大豆对大豆花叶病毒抗侵染和抗扩展分属不同遗传体系,抗侵染由一对主基因控制,抗病对感病表现为显性;抗扩展由一对加性主基因+加性-显性多基因控制,F₂代主基因和多基因遗传率分别为23.91%~74.97% 和18.43%~37.04%,F_2∶3代主基因和多基因遗传率分别为49.46%~82.42% 和17.42%~39.93%,抗性大小依亲本而异。两类抗性都有育种价值。因中抗×高感组合的遗传率明显低于高感×高抗组合,抗扩展育种应尽量选择抗性强的品种作亲本。     

Grouping patterns of rainfed winter wheat test locations and the role of climatic variables

Soybean mosaic virus (SMV) is a member of genus Potyvirus, which causes worldwide soybean [Glycine max (L.) Merr.] yield loss and seed quality deterioration. It is of great significance to find new resistance loci and genes for cultivation of soybean variety. In the present study, a recombinant inbred line (RIL) population and a genome-wide association study (GWAS) panel, which contained 193 lines and 379 germplasms, respectively, were used for QTL mapping of resistance to SMV. Linkage mapping identified a major QTL, qSMV13, on chromosome 13, conferring resistance to SMV SC3 and SC7 strains, explaining phenotypic variations 71.21 and 76.59?%, respectively. The QTL qSMV13 was located close to the known SMV resistance loci Rsv1-h. GWAS analysis revealed five single nucleotide polymorphisms (SNPs) significantly associated with resistance to SC3 on chromosomes 2, 11, 13, 14 and 16. One of the SNP markers, ss715614844, was the right flanking marker of qSMV13. Combining linkage mapping and GWAS analysis enabled us to delimit qSMV13 in a 97.2-kb genomic region containing seven genes. A LRR-RLK protein was proposed as the candidate gene of qSMV13. These results provided selection markers and candidate genes for SMV resistance in soybean molecular breeding programs.