玉米抗甘蔗花叶病毒SCAR分子标记开发

 【目的】玉米矮花叶病（由甘蔗花叶病毒引起）是中国和欧洲玉米产区的重要病害,开展分子标记辅助育种可以明显提高抗病育种效率。【方法】本文采用改进的BSA法（bulked segregant analysis,BSA）构建玉米抗感自交系DNA池,结合AFLP技术筛选多态性标记,转化后获得实用性强的SCAR标记,并借助100份自交系抗性鉴定结果对SCAR标记进行相关验证。【结果】获得了2个多态性稳定的AFLP标记P66M38-220和P55M51-240,其中将P66M38-220转化为SCAR112标记,此标记与甘蔗花叶病毒抗性高度相关。【结论】改良BSA法是一种发掘性状基因紧密连锁标记的有效方法;开发的SCAR112 标记可以用于玉米抗甘蔗花叶病毒的分子标记辅助选择。     

苦瓜抗白粉病SCAR分子标记的开发

[目的]获得与苦瓜抗白粉病基因紧密连锁的分子标记,为加快苦瓜抗白粉病新品种的选育奠定基础.[方法]以高抗白粉病野生苦瓜MC18为父本、高感白粉病苦瓜栽培种MC1-2为母本创建F2代分离群体;经单株抗病性鉴定后,以BSA法构建F2代单株的高抗和高感白粉病苦瓜DNA近等基因池;利用SRAP技术筛选多态扩增片段,对仅在抗白粉病近等基因池和父本中出现的差异片段进行同收、测序、比对和转化成SCAR标记,并利用已知抗病性的单株DNA分析标记与苦瓜抗白粉病的相关性.[结果]从1188对SRAP引物组合中筛选到稳定阳性差异条带的引物组合5对,其中ME20EM5引物对扩增的差异条带长度为332bp,与葡萄抗体蛋白基因（抗性基因）的DNA序列有较高相似性,并将其成功转化成与苦瓜白粉病抗性相关、大小为320 bp的SCAR标记.[结论]开发的SCAR-ME20EM5分子标记可用于苦瓜抗白粉病分子标记辅助选择.     

玉米品种抗感玉米粗缩病毒与过氧化物酶关系的研究

 研究选取对玉米粗缩病毒（MRDV）表现抗病、中感及高感3个不同类型的代表性品种,分析了三叶期、五叶期室内盆栽及抽穗期田间采取的病叶及健康玉米叶片的过氧化物酶（POD）的变化,旨在研究玉米品种抗感病性与过氧化物酶的关系及感染病毒后的诱导抗病性机理。结果表明:玉米三叶期、五叶期及抽穗期,感病品种的POD酶活性大于抗病品种,显示玉米叶片内的POD含量与品种的感病性有相关性。发病后,各品种酶活性均上升,抗病品种上升幅度最大,说明防御酶POD的应激表达与品种的抗病性相关;同工酶研究结果表明:玉米抽穗期,抗病品种健叶具有一条Rf 0.261差异酶带,而感病品种则没有,这一特异蛋白带的组成型表达显示出与品种抗病的相关性,可作为评价品种抗病性的生理生化指标之一。发病后,抗病品种及中感品种又新增一条酶带,Rf为0.471;三叶期及五叶期,抗病品种同样都比感病品种多出现一酶带区,进一步证明了在玉米植株体内存在与抗玉米粗缩病毒相关的POD同工酶,这一结果将为抗病育种提供极有价值的生理生化基础。     

利用SSR-BSA技术筛选玉米粗缩病抗性基因分子标记

采用网棚集团接种法,对感病自交系苏951和抗病自交系87-1的P1、P2、F1和F2群体进行了玉米粗缩病抗病性鉴定.并用168对引物进行了亲本间SSR-PCR扩增,其中48对引物在亲本间表现多态性.然后利用这些多态性引物检测F2的抗池和感池,只有6个SSR标记在F2的抗池和感池之间表现出多态性.这6个标记为: 5号染色体上的Blng1237(Bin5.05-5.06)、umc1941(Bin5.06)、phi087 (Bin5.06)、umc1155 (Bin5.05) 和9号染色体的phi065(Bin9.03)、umc1505(Bin9.07).进一步利用这6个SSR标记检测了465个经抗病性鉴定的F2单株的基因型,其中Blng1237和phi087明显表现为偏分离.其余4个标记用于分析表型鉴定表现为抗的181个F2单株的基因型,并对每一标记的分离数值与其相应的孟德尔理论比例(共显性1∶2∶1)相比较,进行χ2适合度检验.结果表明,umc1155、umc1505这2个标记可能与抗性基因有关.     

玉米自交系粗缩病毒抗性鉴定研究

采用网棚集团接种的方法,通过带毒灰飞虱在玉米幼苗上传毒接种玉米粗缩病毒试验,对29份国内常用玉米自交系和部分杂交组合进行了抗病性鉴定研究。以病情指数为指标,筛选出高抗自交系5份,抗病系2份,中抗系9份,感病系8份和高感系5份。结合系谱分析,筛选出的5份高抗系和2份抗病系均属于PB亚群,表明PB亚群抗病性较好,可以作为抗粗缩病育种的基础材料。通过对杂交组合鉴定结果表明,自交系抗病性对后代具有一定遗传力,且抗性遗传中显性效应占有相当大的作用。因此,选育抗病组合时最好亲本之一为高抗材料。     

玉米粗缩病抗性基因SSR标记初步研究

以高抗粗缩病玉米自交系齐319和高感粗缩病玉米白交系掖478的189个F<,2>群体单株作为标记群体材料,结合BSA法,用88对SSR引物进行筛选研究,结果表明:引物Bnlg125和Bnlg1064在抗、感DNA池呈多态性,其中Bnlg125在抗、感池之间扩增出1条约410 bp的多态性片段(记Bnlg125-410);通过Mapmaker/Exp(Version3.0)软件分析F<,2>单株,结果Bnlg125-410标记与玉米粗缩病抗性位点连锁,遗传距离为5.8 cM.     

感染玉米粗缩病毒后玉米植株的超微结构病变研究

 对接种后感染玉米粗缩病毒 (MRDV)的玉米植株的叶片及侧根超微结构进行了电镜观察。研究结果表明 ,受侵染的玉米叶肉细胞中叶绿体的数量有所减少 ,细胞质丰富 ,细胞器发生了不同程度的病变。液泡膜发生明显内陷 ,随着病情的严重液泡膜内陷加剧 ,呈极度松弛状态 ,局部破裂 ;叶绿体被膜破裂 ,轻者成为一松弛的单膜结构 ,严重者被膜完全消失 ,叶绿体中的片层膜系统消失 ,取而代之的是大量淀粉粒。线粒体及细胞核形态异常 ,随着病害的加重 ,线粒体逐渐肿大 ,基粒缩小 ,膜破裂 ,类囊物流入细胞中 ,细胞膜破裂。在玉米植株的根部细胞中观察到了大量的病毒粒体 ,这些粒体大多集中在细胞壁处形成病毒质体。感病细胞的叶绿体、线粒体、核、质膜及胞间联丝中均未见病毒状颗粒     

甘蓝抗枯萎病SCAR标记的开发

 【目的】开发与甘蓝枯萎病抗性基因紧密连锁的SCAR标记,利用该标记对甘蓝抗枯萎病基因跟踪、鉴定,为甘蓝抗枯萎病分子标记辅助选择奠定基础。【方法】以高抗枯萎病的甘蓝自交系8024与感病自交系6A为亲本构建F2代分离群体和相应F3代家系,通过F3代家系抗病性分离表现确认F2代单株的基因型,选择10株纯合基因型显性抗病单株和10株纯合基因型隐性感病单株,利用BSA法构建甘蓝抗感基因池,筛选出与甘蓝枯萎病抗性基因紧密连锁的AFLP标记,克隆测序后根据序列差异将其转化为SCAR标记,通过142株F2代分离群体连锁验证该标记与甘蓝枯萎病抗性基因的连锁关系,并利用两个不同的抗感分离F2群体共100株对SCAR标记的通用性进行验证。【结果】获得了1个以相斥相连锁的SCAR标记S46M48199,该标记在感病亲本中扩增出199 bp的单一条带,而在抗病亲本中无扩增条带,142株F2代分离群体连锁分析表明,其遗传距离为2.78 cM,在F66和C1两个F2群体中的通用性验证结果,与抗性鉴定结果的吻合率分别为81%和83%。【结论】开发的SCAR标记可用于甘蓝抗枯萎病的分子标记辅助育种。     

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize (Zea mays L.), which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one (BC3Q) derived from the cross Qi319 (resistance)x Huangzao 4 (susceptible),the other (BC3M) from Mol 7 (resistance)x Huangzao 4 (susceptible), were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA (bulked segregant analysis) with AFLP (amplified fragment length polymorphism) method was applied to map the genes involving the resistance to S. Reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of P13M61-152 was converted into SCAR (sequence charactered amplified fragment) marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. Reiliana resistance.Furthermore, S130 was highly associated with resistance to S. Reiliana, and could be useful for marker-assisted selection and facilitate map-based cloning of resistance genes.     

Development of Sequence Characterized Amplified Region （SCAR） Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.     

Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice （Oryza sativa L.） Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.     

SSR标记技术在玉米品种“先玉335”真伪性鉴定中的应用

利用20对SSR基本核心引物和20对SSR扩展核心引物进行扩增,以鉴定待测玉米样品是否与“先玉335”标准样品相符.通过分析待测样品与“先玉335”标准样品在DNA水平上的差异,结果显示:待测样品Ⅰ与标准样品有明显差异,即该样品不是“先玉335”;待测样品Ⅱ与标准样品无明显差异,说明该样品是“先玉335”.SSR标记技...     

Inheritance of Powdery Mildew Resistance in Cucumber （Cucumis sativus L.） and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.     

分子标记在优质蛋白玉米育种中的应用

通过与opaque-2（o2）基因紧密连锁的SSR分子标记phi057对X178和CA335回交群体回交一代BC1F1和回交二代BC2F1 o2基因的前景选择,发现可以在苗期对回交群体进行o2基因的检测和追踪,可提高选择的准确性.通过利用AFLP分子标记进行轮回亲本的背景选择,表明利用AFLP分子标记辅助选择可以使回交二代与轮回亲本的相似性比回交一代与轮回亲本的相似性显著增加,使位点进一步纯合,选择出与轮回亲本具有较高相似性单株个体,从而加快优质蛋白玉米育种进程, 为分子标记辅助选择QPM提供了重要理论依据.     

玉米种子中矮花叶病毒分布部位的研究

植物种传病毒在农业生产中占有重要地位。病毒经种子传播不仅起到初侵染源的重要作用,而且也是植物病毒病害远距离传播的主要途径之一。本研究首次采用 RT-PCR 法对感染玉米矮花叶病毒(MD-MV)的玉米自交系 Mo17和掖107种子的种皮、胚乳和胚等不同部位进行了检测,结果显示,在供试两个自交系种子的种皮和胚乳中均检测到了 MDMV,而在胚里没有检测到病毒,说明玉米矮花叶病毒在由玉米种子传播过程中,主要是通过受侵染的种皮和胚乳来完成的。本文对胚不受病毒侵染的原因也进行了分析,认为可能是由于玉米花粉不带毒以及胚作为一种特殊的分生组织不受病毒侵染所致。     

玉米雄穗分枝性状的数量遗传分析

以2个株型不同的玉米自交系自330和PH4CV构成的6世代群体为材料,目测计数玉米不同群体雄穗分枝数量。通过P1、P2、F1、F2、B1和B2共6个世代联合分析法,以玉米雄穗分枝数为性状,研究控制玉米雄穗分枝性状的基因遗传分离规律。结果表明:该性状在F2分离世代群体呈双峰分布,B1和B2群体分离世代呈多峰分布,说明玉米雄穗分枝性状遗传为多基因数量性状控制,且符合加性-显性-上位性多基因遗传模型(即C模型)。显性效应起主要作用,多基因遗传力较高,在74.83%～80.42%之间,若选择雄穗分枝较少的自交系必须从基础材料入手。这一研究结果为玉米雄穗分枝选择提供方法和理论依据。     

玉米GY220×1145组合粗缩病抗性的QTL定位分析

玉米粗缩病是近年严重影响中国玉米生产的病害,研究其QTL定位有助于利用分子标记辅助选择提高玉米粗缩病抗性育种效率。该研究调查了GY220×1145杂交衍生的重组自交系(RIL)群体109个家系(F10∶11)在2个环境下粗缩病的抗感表型值,结合该组合由272个DNA分子标记构建的遗传连锁图谱,分别采用基于多元回归模型的Win QTL Cartographer 2.5软件的复合区间作图法(CIM)和基于混合线性模型的QTL Network 2.0软件中CIM方法,检测了玉米粗缩病的抗性位点。结果表明:(1)运用Win QTL Cartographer 2.5软件中CIM法,检测到5个抗玉米粗缩病的QTL,解释表型变异的6.9%～17.6%,其中有3个QTL在2个环境下都检测到。5个QTL的加性效应变异幅度为-8.57～11.94。(2)用QTL Network 2.0软件中CIM法,检测到1个控制玉米粗缩病的位点MRDD2-22,解释表型变异的9.0%,加性效应为6.93。在第5连锁群与13连锁群之间存在1对非主效QTL间的互作,解释表型变异的7.4%,互作效应为-7.70。运用多元回归模型和混合线性模型都检测到的位点是MRDD2-22,位于第4染色体长臂g7M7806～n142标记区间,抗性等位基因来自自交系1145,平均加性效应为9.3。MRDD2-22位点可用于分子标记辅助选择进行玉米自交系粗缩病抗性的改良。