葡萄感霜霉病基因的分子标记(英文)

 在葡萄抗病育种中 ,幼苗期排除感霜霉病的后代具有特别重要的意义 .用 BSA,RAPD和SCAR方法研究了葡萄感霜霉病基因的分子标记 .分析了两个种间杂交组合 [毛葡萄 (抗病 )×欧洲葡萄 (感病 ) ]88- 1 1 0和 88- 84与 88- 1 1 0的 F1代自交或互交所得的 3个 F2 代 ,以及欧洲葡萄品种和中国野生葡萄种 .共筛选了 2 80个随机引物 .引物 OPO1 0产生了一个 RAPD标记 OPO1 0 - 80 0与葡萄感霜霉病主效基因紧密联锁 .将该 DNA片段克隆并测序 .OPO1 0 - 80 0的实际长度为 835bp,所以 OPO1 0 - 80 0应为 OPO1 0 - 835.据其两端序列 ,设计了一对长度为 2 6bp和 2 8bp的特异引物分别扩增上述试材 ,获得了与该 RAPD标记相同大小的一条带 ,将 RAPD标记转化为 SCAR标记SCO1 0 - 835.并证实了此 SCAR标记的通用性 ,该 SCAR标记可用于葡萄抗病育种中杂种后代对霜霉病的抗病与感病性鉴定 .     

5种杂交粳稻亲本SCAR标记的建立及其在真伪纯度鉴定中的应用

选用36个随机引物对"寒丰A"、"寒丰B"、"8204A"、"8204B"、"R161"等5份杂交粳稻亲本材料进行RAPD扩增,对其中特异RAPD标记片段进行克隆和测序.根据获得的特异DNA序列设计序列特征扩增区(SCAR)特异的引物,将18个RAPD标记转化成6个稳定的SCAR标记.用这些SCAR标记对亲本和杂种F1代单株进行检测,实验室检测种子纯度的结果与海南田间种植的结果基本一致.此外,应用水稻细胞质雄性不育特异的1对PCR引物,分辨出2对不育系/保持系亲本:"寒丰A"与"寒丰B"、"8204A"与"8204B".     

水稻苯达松敏感致死基因的RAPD标记和SCAR标记

利用RAPD技术对水稻品种农林8号(含苯达松抗性基因Ben)和其突变体农林8号m (含苯达松敏感致死基因ben)进行标记,从360个10 bp寡核苷酸随机引物中筛选出5个引物产生的7个RAPD标记.经对多态性标记的克隆和序列分析,再设计PCR引物,将其中4个RAPD标记OPG18/943、OPG18/972、OPD10/1248和OPF03/1198转化成SCAR标记SCAR/G18/883、SCAR/G18/890、SCAR/G18/919/948、SCAR/D10/1237、SCAR/F03/1186.通过对农林8号×农林8号m F2分离群体320个单株的连锁分析及在1对含ben基因的近等基因系H121和Hben121中验证,标记SCAR/G18/883、SCAR/G18/890、SCAR/G18/919/948与Ben 或ben基因共分离,SCAR/D10/1237与Ben基因的遗传距离为(14.8±2.1) cM.经Southern blotting分析并结合F2代分离比例表明,标记OPG18/943、OPG18/972及其转化的SCAR标记在基因组中为单拷贝序列,且OPG18/943和OPG18/972为一对等位STS位点.这是首次报道与ben或Ben基因相连锁的分子标记.本研究为利用分子标记辅助ben基因的转育及利用图位克隆技术分离ben基因提供了有用的分子标记.     

水稻苯达松敏感致死基因的RAPD标记和SCAR标记

利用RAPD技术对水稻品种农林 8号 (含苯达松抗性基因Ben)和其突变体农林 8号m (含苯达松敏感致死基因ben)进行标记 ,从 36 0个 10bp寡核苷酸随机引物中筛选出 5个引物产生的 7个RAPD标记。经对多态性标记的克隆和序列分析 ,再设计PCR引物 ,将其中 4个RAPD标记OPG18/ 94 3、OPG18/ 972、OPD10 / 12 4 8和OPF0 3/ 1198转化成SCAR标记SCAR/G18/ 883、SCAR/G18/ 890、SCAR/G18/ 919/ 94 8、SCAR/D10 / 12 37、SCAR/F0 3/ 1186。通过对农林 8号×农林 8号mF2 分离群体 32 0个单株的连锁分析及在 1对含ben基因的近等基因系H12 1和Hben12 1中验证 ,标记SCAR/G18/ 883、SCAR/G18/ 890、SCAR/G18/ 919/ 94 8与Ben或ben基因共分离 ,SCAR/D10 / 12 37与Ben基因的遗传距离为 (14 .8± 2 .1)cM。经Southernblotting分析并结合F2 代分离比例表明 ,标记OPG18/ 94 3、OPG18/ 972及其转化的SCAR标记在基因组中为单拷贝序列 ,且OPG18/ 94 3和OPG18/ 972为一对等位STS位点。这是首次报道与ben或Ben基因相连锁的分子标记。本研究为利用分子标记辅助ben基因的转育及利用图位克隆技术分离ben基因提供了有用的分子标记。     

耐盐绒毛白蜡特异性SCAR分子标记的鉴定

该研究以耐盐型和盐敏感型绒毛白蜡及其F1代为材料,采用混合品系分析法进行RAPD分析。结果显示:在随机选取的150个10碱基随机引物中,仅有引物S20在耐盐基因池和盐敏感基因池间扩增出特异而可重复的592bp的多态性片段,命名为S20-592。获得的RAPD标记S20-592经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记。通过F1代个体验证,耐盐型个体均能扩增出此差异条带而盐敏感型个体中不能扩增出此差异条带,证明该SCAR标记的特异引物可用于耐盐绒毛白蜡物种的快速分子鉴定。     

甜瓜抗白粉病基因SRAP分子标记筛选

以感白粉病甜瓜A120和抗白粉病甜瓜A119为亲本构建F2代分离群体,采用BSA和SRAP相结合的方法筛选与甜瓜抗白粉病基因相连锁的分子标记.结果显示,294条SRAP引物中有6条引物在抗病与感病池间表现出多态性;对8个高抗和8个高感单株进行扩增,引物me46em51和me3em6分别在高感单株中扩增出210 bp和205 bp的多态性条带,而高抗单株无此扩增带,与抗病、感病池结果一致.采用JoinMap3.0软件进行连锁分析,两标记与抗白粉病基因的连锁距离分别为18.2 cM和23.4 cM,初步推测本实验中甜瓜白粉病抗性为隐性多基因控制.     

与苹果Co基因紧密连锁的RAPD标记的筛选及其SCAR标记转换

以短枝富士(Spur Fuji)X舞姿(Telamon)的105株F1群体为试材,利用RAPD技术,结合集群分类分析法(BSA)进行了苹果柱型基因(Co)分子标记的研究。通过对300条随机引物的筛选,获得一个与Co基因紧密连锁的RAPD标记S1142682,连锁距离为2．86cM。对该标记片段进行序列测定,然后根据序列特点设计了4条特异引物(其中正向引物与反向引物各两条)。PCR结果显示,这4条引物的4种组合都可以扩增出柱型性状的特征带。选其中之一进行群体上的分析,结果表明该SCAR标记特征带与柱型性状的共分离行为与原RAPD标记表现一致。可见,此组合的引物可以作为该SCAR标记的特异引物。通过对S1142682标记片段序列分析发现,在 45～ 251区域含有一个可编码68个氨基酸残基的ORF。     

水稻抗褐飞虱基因SCAR标记的获得

采用282个随机引物对药用野生稻1665和栽培稻桂99远缘杂交的抗褐飞虱近等基因系B3F4分离群体的不抗池DNA和抗池DNA进行了特异性RAPD标记筛选,从中筛选到一个具有明显的特异性扩增带谱的RAPD标记S1159,序列分析表明,S1159序列长度为1408bp,与基因库中已报道的水稻第四号染色体的BAC克隆(编号OSJNBa0070O11)序列(67114-69100)有51.86%的同源性。为了提高所找到的RAPD标记S1159在应用上的稳定性,将RAPD标记转化为SCAR标记检测近等基因系群体,结果表明与RAPD标记结果一致,说明该研究得到的RAPD标记具有较好的稳定性和重复性,为进一步的研究打下了良好的基础。     

亚麻抗锈病基因M4的特异分子标记

用520个10碱基随机引物对含有亚麻抗锈病基因M4的近等基因系材料NM4及其轮回亲本Bison进行RAPD分析,其中OPA18引物在NM4材料中稳定地扩增出特异的DNA片段。用Bison与NM4杂交产生的F2分离群体进行的遗传连锁性分析表明,RAPD标记OPA18432与M4基因紧密连锁,二者之间的遗传距离为2.1cM。将OPA18432片段回收,克隆和测序,成功地将其转化为SCAR标记。对不同抗源材料的扩增分析表明,该标记是M4基因的特异标记。目前这一标记已成功地应用于亚麻抗锈病基因M4的分子标记辅助选择育种。     

滨麦抗条锈病基因的染色体定位和分子标记

从滨麦与普通小麦杂交后代中筛选到一条抗条锈病的小滨麦品系93784。以滨麦基因组DNA为探针的荧光原位杂交结果表明,93784是小麦与滨麦的小片段易位系,易位的滨麦染色体片段位于一对小麦染色体的短臂端部,利用该易位系构建了F2分离群体,进行F2单株成株期抗条锈鉴定,抗性分析证明,小滨麦93784中的抗条锈病基因是单基因控制的,位于滨麦染色体的易位片段上,命名为YrLm。进一步采用24对TaqⅠ（T1-T4）/PstⅠd(P1-P6)引物组合对抗感亲本及F2分离群体进行AFLP分析,筛选出一个与抗条锈病基因YrLm连锁的AFLP分子标记,经克隆和测序,该标记片段长度为205bp,定名为P1T3205。     

黄瓜霜霉病抗性相关基因的AFLP标记

运用AFLP技术,采用集群分析法研究与黄瓜霜霉病抗性基因相关的分子标记.结果表明,在F2群体中E25M63-103标记与霜霉病病情指数的相关系数(0.337)和回归分析的F值(20.98)都达到了极显著水平.用E25M63-103标记对国内外的其它27份黄瓜材料进行检测,该标记与霜霉病病情指数的相关系数为0.555,也达到极显著相关水平,进一步证明该标记与控制黄瓜霜霉病感病的相关基因是连锁的.E25M63-103片段长度为103 bp,通过BLAST查询,该片段的同源性较小,表明E25M63-103标记可能是黄瓜基因组特有的一段DNA序列.     

RAPD和SSR两种标记构建的中国对虾遗传连锁图谱

利用RAPD和SSR分子标记结合拟测交策略,对中国对虾(Fenneropenaeuschinensis)“黄海1号”雌虾与野生雄虾作为亲本进行单对杂交产生的F1代,采用RAPD和SSR两种分子标记技术初步构建了中国对虾雌、雄遗传连锁图谱。对460个RAPD引物和44对SSR引物进行筛选,共选出61个RAPD引物和20对SSR引物,用于对父母本和82个F1个体进行遗传分析。共得到母本分离标记146个(RAPD标记128个,微卫星标记18个)和父本分离标记127个(RAPD标记109个,微卫星标记18个)。雌性图谱包括8个连锁群、9个三联体和14个连锁对,标记间平均间隔为11·28cM,图谱共覆盖1173cM,覆盖率为59·36%;雄性图谱包括10个连锁群、12个三联体和7个连锁对,标记间平均间隔为12·05cM,图谱共覆盖1144·6cM,覆盖率为62·01%。中国对虾遗传图谱的构建为其分子标记辅助育种、比较基因组作图及数量性状位点的定位与克隆奠定了基础。     

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Linkage analysis of er-1, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi D.C.)

Linkage analysis was used to determine the genetic map location of er-1, a recessive gene conditioning resistance to powdery mildew, on the Pisum sativum genome. Genetic linkage was demonstrated between er-1 and linkage group 6 markers after analyzing the progeny of two crosses, an F₂ population and a set of recombinant inbred lines. The classes of genetic markers surrounding er-1 include RFLP, RAPD and allozyme markers as well as the morphological marker Gty. A RAPD marker tightly linked to er-1 was identified by bulked segregant analysis. After DNA sequence characterization, specific PCR primers were designed to convert this RAPD marker into a sequence characterized amplified region (SCAR).     

Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis) and development of linked molecular markers

Inheritance of resistance to downy mildew (Hyaloperonospora parasitica) in Chinese cabbage (Brassica rapa ssp. pekinensis) was studied using inbred parental lines RS1 and SS1 that display strong resistance and severe susceptibility, respectively. F(1), F(2), and BC(1)F(1) populations were evaluated for their responses to downy mildew infection. Resistance to downy mildew was conditioned by a single dominant locus designated BrRHP1. A random amplified polymorphic DNA (RAPD) marker linked to BrRHP1 was identified using bulked segregant analysis and two molecular markers designated BrPERK15A and BrPERK15B were developed. BrPERK15B was polymorphic between the parental lines used to construct the reference linkage map of B. rapa, allowing the mapping of the BrRHP1 locus to the A1 linkage group. Using bacterial artificial chromosome clone sequences anchored to the A1 linkage group, six simple polymerase chain reaction (PCR) markers were developed for use in marker-assisted breeding of downy mildew resistance in Chinese cabbage. Four simple PCR markers flanking the BrRHP1 locus were shown to be collinear with the long-arm region of Arabidopsis chromosome 3. The two closely linked flanking markers delimit the BrRHP1 locus within a 2.2-Mb interval of this Arabidopsis syntenic region.     

Identification, mapping, and application of polymorphic DNA associated with resistance gene Pm21 of wheat.

A new powdery mildew resistance gene designated Pm21, from Haynaldia villosa, a relative of wheat, has been identified and incorporated into wheat through an alien translocation line. Cytogenetic and biochemical analyses showed that chromosome arms 6VS and 6AL were involved in this translocation. Random amplified polymorphic DNA (RAPD) analysis was performed on recipient wheat cultivar Yangmai 5, the translocation line, and H. villosa with 180 random primers. Eight of the 180 primers amplified polymorphic DNA in the translocation line, and the same results were obtained in four replications. Furthermore, RAPD analysis was reported for substitution line 6V, seven addition lines (1V-7V), and the F1, as well as F2 plants of (translocation line x 'Yangmai 5'), using two of the eight random primers. One RAPD marker, specific to chromosome arm 6VS, OPH17-1900, could be used as a molecular marker for the detection of gene Pm21 in breeding materials with powdery mildew resistance introduced from H. villosa. Key words : RAPD analysis, 6VS-specific marker, Pm21, Erysiphe graminis f.sp. tritici, Triticum aestivum - Haynaldia villosa translocation.