红花草莓红花基因RAPD标记转化为SCAR标记

红花草莓不但具有经济价值，还具有很高的观赏价值。本研究将3个与红花基因连锁的RAPD标记即AW65679（1031bp）、S484（620bp）与S1383（500bp）进行了克隆与核苷酸测序，并根据测序结果设计4对SCAR引物，将这4对引物对红花草莓品种粉红熊猫、白花草莓品种鬼怒甘及它们的杂交后代进行PCR扩增程序优化和鉴定，筛选出一对SCAR引物可扩增出与红花基因连锁的特异片段AW65679（1038bp），这就是与红花基因连锁的SCAR标记。SCAR标记因其稳定性好，重复性高将为草莓分子育种开辟一条新的有效途径。     

结球甘蓝迟抽薹基因RAPD标记转SCAR标记

本研究以与结球甘蓝迟抽薹基因连锁的N1750为引物,应用RAPD技术进行PCR扩增,检测到迟抽薹基因,对特异片段进行回收、克隆和测序,依据测序结果设计SCAR引物。在166株BC1群体(A21与P02杂交得到F1再与P02回交)中通过与RAPD标记的比较,SCAR扩增结果同RAPD扩增结果完全一致,从而证实了SCAR标记的准确性。实验结果表明,与甘蓝迟抽薹基因连锁的RAPD标记被成功转化为SCAR标记,为甘蓝分子标记辅助选择育种提供了基础。     

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。     

利用SLAF-seq技术开发黄瓜白粉病抗性分子标记

黄瓜白粉病是黄瓜主要病害之一。本研究参考黄瓜基因组信息,利用简化基因组测序技术,共在黄瓜全基因组范围内开发了73 100个测序标签,标签整体平均深度达99.11x。通过与参考基因组的比对获得多态性SNP、EPSNP、INDEL标记5 355个,其中获得与黄瓜白粉病抗性密切相关的SNP多态性标记140个,利用毛细管电泳技术对其验证,这些特异标记与黄瓜白粉病抗性紧密相关。本研究结果有助于探索黄瓜白粉病抗病机理,可用于分子标记辅助选择抗性品种。     

SCAR标记转化失败的原因和对策

SCAR标记作为一种快速、简便可以直接应用的分子标记,在标记辅助选择育种中发挥着重要的作用。但是目前SCAR标记的转化效率不是很高。本文从引物设计、序列相似或多拷贝及DNA甲基化等方面分析SCAR标记转化失败的可能原因;同时从引物设计技巧、PCR反应条件、借助CAPS和SSCP技术以及借助原始标记邻近序列等方面综述了SCAR标记转化失败的情况下获得序列特异性标记的几种有效补救手段。     

-个新的与稻瘟病菌无毒基因AVR-Pikm紧密连锁的SCAR标记

无毒基因是病原物中决定寄主抗病性表达的功能基因，其功能的丧失导致毒性小种的产生。本研究利用随机引物扩增DNA多态性技术，从稻瘟病菌菌株S1522中新筛选到1个与无毒基因AVR-Pikm紧密连锁的DNA标记OPE121400。根据OPE121400的核苷酸序列，设计了1对含有17个核苷酸的特异性SCAR引物，并利用该引物对无毒表型亲本S1522和毒性表型亲本S159及其有性杂交后代的108个菌株进行了PCR扩增。     

核桃早实性相关的SCAR标记在母本和F_1代上的分析

本文研究目的是对与新疆核桃早实性相关的SCAR标记在母本和F1代上进行分析。通过对与核桃早实性相关的SCAR标记片段的扩增并测序,获得与早实性相关的SCAR标记片段,大小为762bp。用该特异性引物对新疆五个核桃实生品种母本和F1代进行扩增,在母本和F1代上得到了以下的结果:四个母本材料出现了该标记片段,各品种实生F1代单株出现该标记的比率分别为92.68%,97.92%,96.43%,93.10%和97.73%,分析表明核桃早实性是遗传力较高的性状。本研究可为进一步研究新疆核桃早实性相关基因的克隆及功能提供一定的研究基础。     

EST辅助的甘蓝型油菜显性核不育AFLP标记转化

甘蓝型油菜显性核不育广泛应用于轮回选择和杂种优势利用,不育基因标记的开发与应用对于基因克隆和育种实践具有重要意义。基于AFLP标记SA12MG14的序列信息,从拟南芥整合数据库中,检索与标记序列同源的甘蓝型油菜EST,结合标记和EST序列设计特异引物,转化成新的SCAR标记。获得的SCAR标记S6B3,具有很高的检测稳定性,在回交群体Popu2上分析验证,结果与AFLP标记完全一致。该标记与不育基因相距0.3 cM,将其用于临保系同源的纯合型不育系选育,可有效提高育种工作效率。     

19个华蕉类栽培品种的SCAR标记鉴别

针对华蕉(Cavendish,AAA)类主栽品种在命名过程中出现的"同物异名"现象较突出的问题,本研究利用SCAR(sequence characterized amplified regions)标记对华蕉品种进行快速鉴别。通过对19个华蕉类品种进行RAPD(random amplified polymorphic DNA)多态性分析,共获得5条具有差异性的DNA片段,并对其进行测序分析,将其转化成相应的5对SCAR引物,再分别以19个华蕉类栽培品种的基因组DNA为模板进行SCAR-PCR扩增。利用这5对SCAR标记在不同华蕉类栽培品种中的差异片段,建立快速鉴别19个华蕉品种的路线图。结果表明:组合使用这5对SCAR引物可以快速、稳定的区分其中的7个华蕉类栽培品种,而其余12个品种则被划分为4个组。本研究将多个SCAR标记进行联合分析,实现快速、稳定、高效的鉴别华蕉类栽培品种,有利于在分子水平上为华蕉类栽培品种的鉴别提供分子依据。     

基于ISSR和AFLP标记开发甜菜 SSR 引物的研究

本研究以ISSR-PCR和AFLP标记原理为基础,介绍一种新的分离甜菜基因组微卫星引物的方法。首先对甜菜基因组DNA进行酶切并连接已知序列的接头,构建基因组DNA酶切文库,同时用一个或两个ISSR引物,扩增文库中两端含微卫星序列片段并进行克隆测序,根据测序结果设计微卫星序列间的IP1引物和IP1与微卫星序列间IP2 引物;再根据侧翼序列克隆原理,采用巢式PCR进行基因组步移,扩增IP2引物下游序列,根据巢式PCR产物测序结果,设计微卫星序列另一侧的引物IP3 ,IP2和IP3即为SSR标记引物,对获得的SSR引物进行PCR验证,结果表明SSR引物产率为16%,本研究获得的SSR引物具有较高的多态性,对于后续的遗传多样性检测和遗传连锁图构建具有重要意义。     

Development and application of SCAR markers for sex identification in the dioecious species <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

There is an urgent need for early sex identification to support field planting in Ginkgo biloba L., due to the different economic and medicinal values between male and female trees. An easy, rapid and reliable molecular method for sex type determination of G. biloba was reported in the paper. Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) were used to search for specific molecular markers linked to the sex locus. A total of 48 primers were used for screening of specific RAPD markers in six male and three female samples. Only one primer, S10, showed different amplification band patterns associated with sex types. Then the sex-specific bands, S10-BandA and S10-BandB, were cloned and sequenced. Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed. The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals. Finally, the SCAR primers were used to test 16 sex-unknown samples. SCAR primers developed in this paper can be used as effective, convenient and reliable molecular markers for sex identification in G. biloba.     

Genetic mapping and QTL analysis of fruit and flower related traits in cucumber (Cucumis sativus L.) using recombinant inbred lines

A set of 224 recombinant inbred lines (RILs) derived from a narrow cross between two fresh eaten types (S94 (Northern China type) × S06 (Northern European type)) (Cucumis sativus L.) was used to construct a genetic linkage map. With the RILs a 257-point genetic map was constructed including 206 SRAPs, 22 SSRs, 25 SCARs, 1 STS, and three economically important morphological markers (small spines (ss), uniform immature fruit color (u), dull fruit skin (D)). The seven linkage groups covered 1005.9 cM with a mean marker interval of 3.9 cM. The ss locus was linked to D and u, and they were all on Linkage group 6. The RIL map contained a total of 51 sequence-specific markers, which made possible the comparison of molecular linkage maps developed in different laboratories. Using the F_6:7 derived families, a total of 78 QTLs were detected with relatively high LOD scores (2.9–84.4) for nine fruit-related traits (fruit weight, length, and diameter, fruit flesh thickness, seed-cavity diameter, fruit-stalk length, fruit pedicel length, length/diameter and length/stalk ratio) and three flower-related traits (first flower node, first female flower node and female flower ratios). Several sequence-anchor markers (CSWCT25, CS30, CMBR41, CS08 etc.) were closely linked with some QTLs for fruit weight, fruit length, fruit flesh thickness and sex expression, which can be used for the future marker-assisted selection to improve the fruit traits in cucumber breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. X. J. Yuan and J. S. Pan contributed equally to this investigation.     

RAPD and SCAR markers linked to the sex expression locus M in asparagus

Bulk segregant analysis (BSA), random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) methods were used to map molecular markers to the sex locus M of asparagus. Two parents, A19 (male, Mm) and MW25 (female, mm), and 63 progeny were used for the study. Two DNA bulks, one male and one female, were made by pooling equal amounts of DNA from 10 randomly selected progeny of each sex type. A total of 760 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Primer OPC15 produced two RAPD markers, OPC15-98 and OPC15-30, both of which were linked to the M locus at a distance of 1.6 cM. Subsequently, amplified RAPD fragment OPC15-98 was cloned and sequenced. The sequence was then used to design flanking 24-mer oligonucleotide SCAR primers SCC15-1 and SCC15-2. Both of these SCAR primers amplified a single 980 bp fragment; the same size as the cloned RAPD fragment. However, the SCAR marker was dominant as was the original OPC15-98 band from which it was derived. These RAPD and SCAR markers could be used for scoring male and female progeny in the mapping population, but were not found to be applicable to other asparagus germplasm studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

DAF marker tightly linked to a major locus for Ascochyta blight resistance in chickpea (Cicer arietinum L.)

Resistance of chickpea against the disease caused by the ascomycete Ascochyta rabiei is encoded by two or three quantitative trait loci, QTL1, QTL2 and QTL3. A total of 94 recombinant inbred lines developed from a wide cross between a resistant chickpea line and a susceptible accession of Cicer reticulatum, a close relative of cultivated chickpea, was used to identify markers closely linked to QTL1 by DNA amplification fingerprinting in combination with bulked segregant analysis. Of 312 random 10mer oligonucleotides, 3 produced five polymorphic bands between the parents and bulks. Two of them were transferred to the population on which the recent genetic map of chickpea is based, and mapped to linkage group 4. These markers, OPS06-1 and OPS03-1, were linked at LOD-scores above 5 to markers UBC733B and UBC181A flanking the major ascochyta resistance locus. OPS06-1 mapped at the peak of the QTL between markers UBC733B (distance 4.1 cM) and UBC181A (distance 9.6 cM), while OPS03-1 mapped 25.1 cM away from marker UBC733B on the other flank of the resistance locus. STMS markers localised on this linkage group were transferred to the population segregating for ascochyta resistance. Three of these markers were closely linked to QTL1. Twelve of 14 STMS markers could be used in both populations. The order of STMS markers was essentially similar in both populations, with differences in map distances between them. The availability of flanking STMS markers for the major resistance locus QTL1 will help to elucidate the complex resistance against different Ascochyta pathotypes in future. This revised version was published online in August 2006 with corrections to the Cover Date.     

ET-ISJ标记的开发及陆地棉遗传图谱构建

根据植物结构基因外显子拼接位点的保守序列,设计扩增外显子的ET-ISJ (exon targeted intron-exon splice junction)标记引物。利用1 280对ET-ISJ引物组合,在陆地棉品种渝棉1号和T586中,筛选获得69对多态性引物组合,占引物组合的5.4%。用多态性ET-ISJ引物组合检测(渝棉1号×T586)F_2:7重组近交系群体,得到70个位点。以70个ET-ISJ标记位点与523个SSR、59个IT-ISJ、29个SRAP和8个形态标记进行连锁分析,构建的遗传连锁图谱包括59个连锁群和673个位点(68个ET-ISJ、510个SSR、58个IT-ISJ、29个SRAP和8个形态标记)。连锁图覆盖3 216.7 cM,占棉花基因组的72.3%,标记间平均长度为4.8 cM。68个ET-ISJ标记分布于20条染色体。研究表明ET-ISJ标记多态性较高、稳定性好,可有效用于棉花与其他植物遗传连锁图谱构建。     

Molecular mapping of a locus controlling resistance to Albugo candida in Indian mustard

Brassica juncea (L.) Czern & Coss is widely grown as an oilseed crop in the Indian subcontinent. White rust disease caused by Albugo candida (Pers.) Kuntze is a serious disease of this crop causing considerable yield loss every year. The present study was undertaken to identify molecular markers for the locus controlling white rust resistance in a mustard accession, BEC‐144, using a set of 94 recombinant inbred lines (RILs). The screening of individual RILs using an isolate highly virulent on the popular Indian cultivar ‘Varuna’ revealed the presence of a major locus for rust resistance in BEC‐144. Based on screening of 186 decamer primers employing bulked segregant analysis (BSA), 11 random amplified polymorphic DNA markers were identified, which distinguished the parental lines and the bulks. Five of these markers showed linkage with the rust resistance locus. Two markers, OPN0_l000 and OPB06₁₀₀₀, were linked in coupling and repulsion phases at 9.9 cM and 5.5 cM, respectively, on either side of the locus. The presence of only two double recombinants in a population of 94 RILs suggested that the simultaneous use of both markers would ensure efficient transfer of the target gene in mustard breeding programmes.     

Fine mapping of the uniform immature fruit color gene u in cucumber (Cucumis sativus L.)

Mottled/uniform color at the flower end of immature fruit is a highly important external quality trait that affects the market value of cucumber. Genetic analysis of different F₂ and backcross populations revealed that one single recessive gene, u (uniform immature fruit color), determines the uniform immature fruit color trait in cucumber. Based on earlier studies, the u locus is located on chromosome 5 (Chr. 5). By combining bulked segregant analysis using 60 published molecular markers on Chr. 5, we found that eight markers are polymorphic and are linked to the u locus. In addition, we developed five new relevant polymorphic simple sequence repeat (SSR) markers between markers SSR16203 and SSR15818. Subsequently, the F₂ population (477 individuals) from the cross of S06 (uniform fruit color line) × S94 (mottled fruit color line) was used for fine mapping of the u gene. The u gene was mapped to a 313.2-kb region between markers SSR10 and SSR27, at a genetic distance of 0.8 and 0.5 cM, respectively. Moreover, validity analysis of the codominant markers SSR10 and SSR27 was performed using 50 lines with mottled/uniform fruit color, demonstrating that these two SSR markers can be used for marker-assisted selection of the mottled/uniform fruit color trait in cucumber breeding. The results of this study will facilitate the cloning of the u gene.     

甜菜遗传连锁图谱初步构建

以甜菜高产低糖型JV34-2和低产高糖型2B023两材料杂交, 构建了200个单株的F₂作图群体, 利用所筛选出的56对SRAP引物组合和20对SSR引物, 对F₂作图群体进行PCR扩增和遗传连锁分析, 初步构建了一张包含9个连锁群、141个(123个SRAP和18个SSR)标记位点的甜菜遗传连锁图谱。该图谱覆盖长度为1399.88 cM, 平均图距9.92 cM。未进入连锁群的有4个标记。9个连锁群包含3~26个标记不等, 连锁群遗传距离15.69~237.21 cM。连锁群上有20.56%的标记出现偏分离, 主要集中在Ch3连锁群上, 其余分散在Ch1、Ch2、Ch8和Ch9中。该图谱是我国甜菜领域利用SRAP和SSR相结合方法, 构建的第一个较精密的分子遗传图谱, 为重要性状的基因定位和优良基因的克隆奠定了基础。     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

绿豆遗传连锁图谱的整合

利用绿豆及其近缘种的701对SSR引物,对现有绿豆遗传连锁图谱进行补充,结果在高感豆象绿豆栽培种Berken和高抗豆象绿豆野生种ACC41两亲本间筛选到多态性SSR引物104对。群体分析后,结合其他分子数据,使用作图软件Mapmaker/Exp 3.0b,获得一张含有179个遗传标记和12个连锁群,总长1831.8cM、平均图距10.2cM的新遗传连锁图谱,包括97个SSR标记,91个来自绿豆近缘种;RFLP标记76个;RAPD标记4个;STS标记2个。对32个绿豆、小豆共用SSR标记在遗传连锁图谱的分布分析发现,二个基因组间有一定程度的同源性,共用标记在连锁群上的排列顺序基本上一致,只有部分标记显示绿豆和小豆基因组在进化过程中发生了染色体重排;利用新图谱对ACC41的抗绿豆象主效基因重新定位,仍定位于I(9)连锁群,与其相邻分子标记的距离均小于8cM,其中与右翼SSR标记C220的距离约2.7cM。与原图谱比较,新定位的抗性基因与其相邻标记的连锁更加紧密。