与大白菜抗霜霉病基因连锁的分子标记研究

【研究目的】 研究与大白菜抗霜霉病基因紧密连锁的DNA分子标记。【方法】利用高抗自交系‘660’和高感自交系‘654B’及其杂交F2群体162个单株为材料,采用构建抗、感病池,利用BSA法筛选了87对SSR引物,35对拟南芥抗霜霉病相关基因的特异引物,其中特异引物RPP13P2和RPP131-2R在抗感病亲本和抗感病池中扩增出一条多态性片段RPP13MK,利用这对引物对F2代单株构建的分享群体进行扩增,验证标记RPP13MK与目的基因的连锁关系,Mapmaker 3.0软件计算遗传距离。【结果】通过F2单株验证后证明RPP13MK与抗霜霉病基因紧密连锁,其遗传距离为5.6 cM。【结论】获得了一个与大白菜抗霜霉病基因紧密连锁的分子标记RPP13MK。     

苜蓿耐盐基因分子标记的筛选及鉴定

以耐盐苜蓿×敏盐苜蓿组合的F2群体为试验材料,利用改良BSA法筛选与苜蓿耐盐基因紧密连锁的分子标记。在对26组520条RAPD随机引物筛选中,共有66条引物为DNA多态性引物,选出一个与苜蓿耐盐基因相连锁的分子遗传标记。通过F2代群体的遗传分析,观测到分子标记与耐盐性等位基因之间发生重组,但重组值较小,在A8×D2杂交组合     

烟草PVY抗性的遗传分析与分子标记筛选

本文以抗马铃薯Y病毒(简称PVY)的烟草品种RY5,感病品种Coker176为亲本,构建F1、正反交F2和正反交BC1群体,苗期摩擦接种PVY的抗性遗传分析结果表明,接种后第21d群体PVY抗性数据符合孟德尔单基因隐性质量性状的遗传模型。接种后第28d群体PVY抗性数据偏离孟德尔单基因隐性质量性状的遗传模型。提取F2代群体中抗病和感病单株DNA,从多条RAPD引物和一对SCAR引物中,筛选出两个紧密连锁的分子标记。RAPD标记O12V3695与RY5的抗病基因对应的显性等位基因位点(Va)间的遗传距离为2.10cM,而SCAR标记与Va间的遗传距离为2.52cM,这两个分子标记可用于抗PVY抗性育种。     

谷子显性雄性不育基因Msch的AFLP标记

利用雄性不育是实现谷子杂种优势利用最经济、有效的途径之一.为了寻找与不育基因Msch紧密连锁的分子标记,提高不育系的选育效率,本研究构建了Msch不育/可育近等基因系(NILs),通过对400对AFLP引物组合进行筛选,找到了与不育基因紧密连锁的两个AFLP标记(P17/M37224和P35/M52208),与不育基因的遗传距离分别是2.1 cM和1.4 cM,而且位于不育基因的同一侧,标记间相距0.7 cM.这两个AFLP标记可有效用于分子标记辅助选择育种.     

中国小麦LB0288中抗叶锈病基因的鉴定

明确中国小麦LB0288中所含的抗叶锈病基因,找到与其紧密连锁的DNA分子标记。将小麦LB0288和感病小麦品种Thatcher杂交,获得F1、F2代群体,用叶锈菌小FHTT分别对双亲及其杂交后代进行叶锈鉴定并进行标记分析。抗性鉴定结果表明F2代群体时呈现一对显性基因的抗感分离比例,经过亲本和抗感池间标记筛选以及F2代群体的标记检测,位于5DL的SSR标记barc144与抗病基因连锁,遗传距离为5.3 cM,同时Lr1的STS标记与之共分离,根据该基因的抗性特点和染色体位置推断为Lr1。此实验通过抗性鉴定、遗传分析和分子标记等手段确定LB0288中含有小麦抗叶锈病基因Lr1。     

辣椒CMS恢复基因的遗传分析及基因定位

本研究以辣椒胞质雄性不育系131BC5A与恢复系139D-21-3为亲本,构建了包含210个单株的F2群体,采用CAPS、SSR及SCAR等分子标记技术,对辣椒胞质雄性不育恢复基因进行遗传分析和基因定位研究。田间调查和遗传分析结果表明,F2群体的表现型中可育与不育的分离比为3:1。采用分离群体分组分析法(BSA),从F2可育群体和不育群体中各随机选取10株,构建可育和不育基因池。研究选用295对引物在亲本间进行多态性筛选,其中43对引物在亲本间表现出多态。通过进一步分析43对引物在基因池间的多态性,筛选出Rf基因连锁标记14个。然后对F2群体的210个单株进行连锁分析,最后将恢复基因定位在SSR标记pep43和pep20之间,约3.9 c M(或498.6 kb)的区间内,与两个标记的遗传距离分别为1.3 c M与2.6 c M。本研究为Rf基因的精细定位和克隆奠定了良好基础,也为辣椒雄性不育恢复系的分子标记辅助选择育种提供了理论参考。     

一个粳稻来源抗稻瘟病基因的鉴定、遗传分析和基因定位

7001S是一个广谱抗稻瘟病的粳稻两用核不育系,对来自全国不同稻区的22株稻瘟病菌系均表现为高度抗性。通过构建7001S/80-4B F2群体的遗传分析和初步定位表明,F2分离单株对稻瘟病菌的抗性呈明显的抗、感双峰分布,抗感分离符合3﹕1的理论比例,说明粳稻7001S对稻瘟病菌的抗性由1对显性核基因或一个显性QTL位点控制,并将该基因初步定位于第11染色体长臂末端。进一步通过扩大遗传群体和分子标记开发,利用基于BSA的隐性群体分析技术,将目的基因精细定位于P21-2415和RM27322之间约310 kb的范围内,并获得了可用于分子标记辅助选择的紧密连锁和共分离分子标记,同时对目标基因所在区域进行基因预测,初步确定了候选基因。为进一步开展该抗稻瘟病基因的克隆、功能验证和抗病机理研究,以及通过分子标记辅助选择技术培育抗稻瘟病水稻新品种等工作奠定了基础。     

谷子显性雄性不育基因Msch的AFLP标记

利用雄性不育是实现谷子杂种优势利用最经济、有效的途径之一。为了寻找与不育基因Ms^ch紧密连锁的分子标记，提高不育系的选育效率，本研究构建了Ms^ch不育/可育近等基因系（NILs），通过对400对AFLP引物组合进行筛选，找到了与不育基因紧密连锁的两个AFLP标记（P17/M37₂₂₄和P35/M52₂₀₈），与不育基因的遗传距离分别是2.1 cM和1.4 cM，而且位于不育基因的同一侧,标记间相距0.7 cM。这两个AFLP标记可有效用于分子标记辅助选择育种。     

水稻显性早熟基因Ehd的SSR标记定位

以籼稻品种广陆矮4和粳稻品种台中65为亲本构建高世代回交分离群体,选用分布于水稻全基因组的145个SSR标记对亲本及抽穗期早熟基因进行分析.结果表明,114个标记在亲本间具有多态性,多态率78.6%;在BC3F1群体中,检测到10个标记的基因型来源于供体亲本广陆矮4号;在BC3F2定位群体中,早熟植株数与晚熟植株数的分离比例为3:1,早熟植株平均比晚熟植株提早抽穗21 d;通过SSR标记与抽穗期共分离分析将显性早熟基因Ehd界定在分子标记RM271和RM258之间;Ehd与标记RM184和RM271紧密连锁,遗传距离分别为2.6 cM和2.1 cM,此结果为该基因分子标记辅助选择奠定了基础.     

芸芥抗菌核病相关基因的分子标记

芸芥属于十字花科芝麻菜属植物,在中国分布广泛.长期以来,国内外学者从植物学特征、经济性状和抗逆性等方面对芸芥品种资源进行了研究工作[1~4];李方球研究了芸芥的抗油菜菌核病[5],但从分子水平上的研究还较少.目前,转基因技术和分子标记辅助选择为高效育种展示了广阔的前景.但应用这2项技术的前提是必须首先获得优良基因克隆或优良基因紧密连锁的分子标记.本文利用RAPD技术,通过对芸芥抗病相关基因的遗传分析和分子标记,阐明其抗病相关基因的遗传,并提供可利用的分子标记,以便为芸芥抗菌核病种质鉴定和芸芥抗菌核病的分子标记辅助选择提供准确快速的鉴定方法.     

亚麻显性雄性核不育基因的RAPD标记

应用252条10-mer随机引物对遗传背景相似的可育株和不育株亚麻进行了RAPD分子标记,在不育株与可育株之间寻找DNA的多态性差异带,结果发现,在252条随机引物中有2条引物(即S62和S135)可分别得到1个与显性核不育的雄性基因有关的RAPD分子标记为S62-500和S135-350。     

Construction of a linkage map for watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) using random amplified polymorphic DNA (RAPD)

Summary A linkage map for watermelon (Citrullus lanatus) was constructed on the basis of RADP, ribosomal DNA restriction fragment length polymorphism (RFLP), isozyme, and morphological markers using F₁BC1. A segregating population of 78 individuals was the result of a backcross of a cultivated inbred line (H-7; Citrullus lanatus; 2n=22) and a wild form (SA-1; C. lanatus; 2n=22), in which the latter was the recurrent (male) parent. A total of 69 RAPD, one RFLP, one isozyme, and three morphological markers was found to segregate in the BC1 population. Linkage analysis revealed that 62 loci could be mapped to 11 linkage groups that extended more than 524 centimorgans (cM), while 12 loci segregated independently of all other markers. The locus for exocarp color was linked to two RAPD markers within a region of 5 cM on linkage group 4. The locus for flesh color was linked to a RAPD marker within a region of 30 cM on linkage group 6. The isozyme marker GOT was located on the linkage group 1. Linkage group 2 contained a locus for ribosomal DNA within 5 cM of a RAPD marker. Half of the RAPD markers on the linkage group 7 displayed severely distorted segregation. The construction of linkage map using molecular markers is necessary for the breeding of watermelon to introduce useful gene of wild watermelon efficiently. However the linkage map that was constructed for the most part on the basis of RAPD markers could not cover significant parts of the genome, the linkage map provides breeders of watermelons the possibility of tagging useful agronomic traits, as well as the gene for exocarp color.Abbreviations RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - GOT glutamate oxaloacetate transaminase - MDH malate dehydrogenase - ACP acid phosphatase - 6PGH 6-phosphogluconate dehydrogenase     

对除草剂敏感致死水稻bel基因的RAPD和SCAR分子标记

以8077s与抗感的籼稻品种丰35亲本及杂交后自交所得的F2群体为材料，采用群分法（Bulked Segregant Analysis, BSA），从210个10mer随机引物，找到两个水稻苯达松敏感池和抗感池之间表现多态性的特异引物——S20和S316，分别产生的标记片段为S20-440和S316-590。它们与bel基因的连锁距离分别为12.132 cM和7.97 cM。对RAPD扩增标记的片段进行克隆、测序，根据测序结果合成两对特异性的SCAR引物，包含原有的RAPD序列。SC01引物在敏感单株中扩增出一条423 bp带；SC02引物在敏感单株中扩增出一条606 bp带，它们的SCAR标记与bel基因的连锁距离为10.66 cM和7.04 cM。应用SCAR标记对水稻恢复系进行了辅助选育。     

小麦抗病种质贵农775中抗白粉病基因的RAPD标记

运用RAPD技术,采用分离群体分组分析法（BSA）进行了小麦种质贵农775抗白粉病基因连锁的分子标记研究,其中有一个引物S2018在抗病亲本贵农775和抗病材料中扩增出了特异的DNA片段,而在感病材料和感病亲本丰产3号中没有扩增出同样的DNA片段。此片段长度约为880 bp。用F2分离群体（106株植株）进行遗传连锁性分析,引物S20188     

甘蓝型油菜隐性核不育遗传标记的初步研究

为了进一步证实P6-9紫茎性状与可育性状的连锁关系,本试验选用S45A×自1968的BC1F1群体,利用BSA分析法,筛选到两个与可育基因Ms1连锁的RAPD片段：UBCl58.580和UBC187.880。通过对可育株群体的分析,证明了P6-9紫茎基因Pur与Ms1基因以及UBC158.580和UBC187.880位于同一连锁群上;对不育株分析得出其在同一连锁群上的精确位     

甘蓝型油菜隐性核不育遗传标记的初步研究 Ⅱ.P6-9紫茎基因与可育基因连锁的分子证据

为了进一步证实P6-9紫茎性状与可育性状的连锁关系,本试验选用S45A×自1968的BC_1F_1群体,利用BSA分析法,筛选到两个与可育基因Ms_1连锁的RAPD片段:UBC_(158.580)和UBC_(187.880).通过对可育株群体的分析,证明了P6-9紫茎基因Pur与Ms_1基因以及UBC_(158.580)和UBC_(187.880)位于同一连锁群上;对不育株分析得出其在同一连锁群上的精确位置,Pur(3.25cM)—Ms_1(8.67cM)—UBC_(158.580)(8.78.M)—UBL_(18,880).     

Genetics of self-compatibility in a self-incompatible wild diploid potato species Solanum chacoense. 2. Localization of an S locus inhibitor (Sli) gene on the potato genome using DNA markers

A self-compatible (SC) hybrid plant F₁-1 was obtained from a cross between a SC variant of a wild diploid potato species, Solanum chacoense, and a self-incompatible (SI) cultivated diploid species, S. phureja. The clone F₁-1 has previously been proposed to have a dominant S locus inhibitor gene (Sli) in a heterozygous condition. It was crossed as a male parent with a selected clone from a S. stenotomum-S. phureja population, resulting in a segregating population consisting of 116 hybrid plants. Self-compatibility was assessed by selfing each of the hybrids. Sixty-six of them were SC, while 35 were SI, showing a significant distortion from an expected Mendelian ratio of 1:1. A genetic linkage map was constructed using DNA markers to localize the Sli gene. A total of 28 RAPD and 127 RFLP markers identified 109 mapping positions on 12 linkage groups. The Sli gene was mapped at a distal end of chromosome 12. Since the S locus has been localized on chromosome 1 on the potato RFLP map, it is confirmed that the Sli gene is independent of the S locus. This revised version was published online in August 2006 with corrections to the Cover Date.     

水稻温敏核雄性不育系培矮64S广亲和位点S-5的SSR紧密连锁标记

广亲和(WC)是水稻(OryzasativaL.)亚种间杂种优势利用的重要遗传工具。本研究对我国南方大面积推广应用的亚种间两系杂交稻亲本培矮64S的WC基因S-5n进行分子标记定位。从培矮64S//T8/秋光三交F1中选出由263个高育和低育极端类型组成的标记群体,选用来源于CornellSSR连锁图的20个标记和根据GenBank数据库公布的序列合成的9个SSR标记,通过BSA(分离群体分析)法对标记群体进行分析,该S-5位点精确定位于第6染色体上,距SSR标记GXR6和RM276的距离只有0.2cM。培矮64S的S-5n对育性贡献可使小穗结实率从平均43.5%提高到77.5%,贡献率达34.0%,表明它是一个很强的控制广亲和特性的主效基因。本研究所获得的这些紧密连锁标记对分子标记辅助选择培育广亲和水稻品种和基因克隆具有重要的利用价值。     

水稻核不育系柱头性状的主基因+多基因遗传分析

柱头性状是影响水稻不育系异交繁殖和杂交水稻制种产量的重要性状。为创制长柱头、高外露率的水稻温敏核不育系提供遗传信息,调查了短柱头、低外露率的粳型光温敏核不育系7001S和长柱头、高外露率的温敏核不育系紫泰S及其杂交、自交获得的F_1、F_2群体(350个株系)和F_2:3群体(320个株系)的4个柱头性状,分析了4个性状之间的相关性,并运用主基因+多基因混合遗传模型,对2个世代4个性状进行了遗传分析。结果表明,4个柱头性状间均表现出极显著正相关,相关系数介于0.262和0.895之间。柱头长度、花柱长度、柱头和花柱总长度(以下简称柱花总长度)均表现出受2对主效基因和微效基因共同控制,除F_2群体中柱花总长度的2对主基因表现为等加性效应和等显性效应外,其余均表现为加性-显性-上位性效应,3个性状均表现出以主基因间的上位性效应为主;F_2群体柱头外露率受2对加性-显性-上位性主基因+多基因控制,而F_2:3群体则表现为受1对加性-显性主基因+多基因控制,以主基因间的加性效应为主。2个世代中的4个柱头性状均以主基因遗传为主。     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.