虎杖种质资源的分子标记研究

本文应用RAPD、ISSR和SRAP标记对26份虎杖种质资源遗传多样性进行检测.在22个引物中有17个引物(77.3%)扩增产物具多态性,多态性水平相对较高.22个引物共得到98条扩增DNA片段,其中90.8%具有多态性.每个多态性引物平均可扩增出5.24个多态性片段.聚类分析表明,利用BAPD、ISSR和SRAP技术相结合可将全部供试材料区分开,26份材料在Gs值0.54水平上全部聚为一类,以所有材料间的平均遗传相似遗传系数0.71为阈值,将其分为11类.虎杖种质资源在分子水平上确实存在较大遗传差异,RAPD、ISSR和SRAP标记可作为构建虎杖DNA指纹图谱的有效工具.     

应用SRAP标记绘制88份南瓜属种质资源DNA指纹图谱

为了给南瓜属种质资源鉴定和分类提供分子生物学依据,本研究采用SRAP分子标记技术与DNAMAN指纹图谱绘制软件对88份南瓜属种质资源(包含美洲南瓜、中国南瓜、印度南瓜)进行分子指纹图谱绘制。结果表明:35对SRAP多态性引物共扩增出499条清晰条带,其中多态性条带438条,多态性条带比率高达87.8%。根据扩增出的条带成功绘制出88份南瓜属种质资源的DNA指纹图谱,每一份种质都具有其独特的分子身份证,使得每份种质均可被区别开来。其中,多态性最好的引物是E5EM8,可以同时绘制72份南瓜属种质资源的指纹图谱。所有供试材料用5对多态性SRAP引物即可全部区别开来。研究表明,SRAP分子标记技术可成功地绘制南瓜属种质资源DNA指纹图谱。本研究对南瓜属种质资源鉴别、分子数据库构建及品种权保护具有较重要的意义。     

基于SSR标记的132份甘薯种质指纹图谱的构建及遗传多样性分析

利用SSR分子标记技术,构建132份甘薯种质的DNA指纹图谱,并进行遗传多样性分析,旨在为甘薯种质资源亲缘关系鉴定、分类提供理论依据。利用筛选的核心引物进行PCR扩增,通过聚丙烯酰胺凝胶电泳检测显示,19对引物共扩增出232个条带,其中多态性条带165条,多态性比率为71.1%,平均每对引物扩增出8.68个条带,多态性信息含量变化范围在0.6706~0.9331之间,平均为0.8158;其中引物SSR9和引物C33可将132份种质完全区分开,并构建供试材料的DNA指纹图谱,供试材料遗传距离在0.0363~0.5939之间,平均为0.4087,表明种质资源间遗传多样性丰富。基于SSR标记对供试材料进行聚类分析,将供试材料分为2个类群,第Ⅰ类群分为两个亚类,第Ⅰ-1亚类包括济薯25和3份日本引进品种日本金千贯、安納芋、日本薯;第Ⅰ-2亚类包括济徐薯23、苏丹、济薯09281。第Ⅱ类群分为两个亚类,第Ⅱ-1亚类由S07甘薯品系和与其亲缘关系较近的20份甘薯种质组成;第Ⅱ-2亚类由剩余的70份甘薯种质组成,为甘薯分子辅助育种中亲本的选择提供理论依据。     

基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立

利用TP-M13-SSR分子标记方法,构建27份中国原产苹果属植物在12个SSR位点的指纹图谱,运用条码技术生成其分子身份证。12对引物共获得251个等位基因,平均21个。引物多态性好,仅用引物CH05b06即可区分全部供试材料。27份苹果材料在12个SSR位点遗传多样性、多态性信息含量和位点杂合度的变化范围为0.6620～0.9455、0.6327～0.9211和0.6538～0.9319。基于CH05b06位点处获得的指纹谱图即可得到每份供试材料独有的分子身份证。TP-M13-SSR分子标记技术适用于苹果属植物种质资源的指纹图谱构建,利于分子基础数据库的积累。基于苹果种质资源TP-M13-SSR指纹图谱可获得每份苹果种质资源独有的分子身份证。     

基于EST-SSR标记的甘薯种质资源DNA指纹图谱构建

采用EST-SSR标记构建了国家种质广州甘薯圃中52份甘薯种质资源的DNA指纹图谱。利用52份供试资源,从早期筛选出的342对候选多态性引物中筛选出16对核心引物,16对引物在52份资源中共扩增出159个等位位点,每对引物的等位位点数为5~19个不等,平均为9.94个,多态性信息含量变化范围为0.6235~0.9593,平均为0.7991。选择带型清晰、重复性好、易于统计且能将52份资源完全区分开的2对引物组合构建供试资源的DNA指纹图谱。NTSYS软件聚类分析表明,EST-SSR标记能正确反映出不同资源间的亲缘关系,为构建甘薯大型DNA指纹图谱数据库奠定了基础。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究

利用ISSR分子标记对东北地区黑木耳生产菌株进行了分子鉴别,结果表明在选用的20个UBC-ISSR引物中,有10个引物能对供试的27个黑木耳菌株基因组DNA进行扩增,获得的指纹图谱清晰稳定、多态性强。用NTSYS软件进行聚类分析,相似水平在0.75时,可将27个供试黑木耳菌株分为3个组群。研究结果说明ISSR分子标记,可以有效地用于黑木耳生产菌株快速准确鉴别,是黑木耳指纹图谱分析的理想手段。     

绿豆种质资源的ISSR遗传多样性分析

利用ISSR标记对33份绿豆种质进行了遗传差异和亲缘关系分析。 结果表明,从46条ISSR引物中筛选出10条扩增条带清晰、多态性高的引物,利用这10条引物从33份绿豆种质中共扩增出118条条带,其中多态性条带115条,多态性位点比例98.18%。遗传相似性与聚类分析结果表明,供试绿豆种质间的遗传相似系数范围为0.50～0.98之间,平均0.68。当遗传相似性系数为0.682时,供试33份绿豆种质资源分为4个ISSR类群( ISSR Groups,IGs),第Ⅰ类群包括产地为黑龙江、吉林共9份和河北的1份绿豆种质资源,第二类群包括河南、山西和陕西的所有和河北的4份绿豆种质资源,第三类群为来自泰国的5份绿豆抗虫资源,第四类群包括山东和内蒙各2份绿豆资源;当遗传相似系数为0.98时,供试的33份绿豆种质资源可被全部区分开。 ISSR分析结果表明, ISSR类群划分与绿豆的地理来源存在一定相关性;而同一类群中,地理来源相同的绿豆种质间也存在一定的遗传差异。     

应用SRAP标记构建山药种质资源DNA指纹图谱

利用30对多态性良好的SRAP引物对90份山药种质资源进行PCR扩增,构建扩增图谱,共扩增到722个位点,多态性位点581个,多态性比例为80.47%,每对引物组合检测多态性位点3~30个,每对引物能鉴别6~51份山药种质资源;采用DNA数据分析软件对扩增出的多态性位点进行分析,构建山药种质资源方框指纹图谱,该图谱清晰地反映出每对引物能扩增的多态位点数、鉴别资源份数及资源具体编号,资源在该引物组合下所能检测得到的多态位点数及所处的具体位置等信息;从10对SRAP引物组合中挑选出的21个多态性位点,根据谱带的有无转化成的1/0字符串编码,形成山药种质资源DNA数字指纹图谱,该图谱可鉴别区分90份山药种质资源中的82份资源。同时这些指纹图谱可为下一步的山药品种鉴定,种质资源评价、利用,分子标记辅助育种及品种权保护提供技术支撑。     

Evaluation of genetic diversity and population structure of polygonati rhizoma germplasms: implications for better crop development and conservation of a traditional Chinese medicine

Polygonati rhizoma (PR) is an important and widely used product in Chinese traditional medicine and edible goods. The time-consuming nature of breaking dormancy in both rhizomes and seeds means that improving variety selection is limited to collection, identification, and selection of germplasms. In this study, we used two DNA-based molecular marker techniques—inter simple sequence repeat (ISSR) and start codon targeted (SCoT)—to assess the genetic diversity and population structure among PR source plants collected from 47 different regions and belonging to 12 populations (P1–P12). For molecular markers analysis, 15 ISSR and 10 SCoT markers were tested. Total number of 159 fragments (150–4000 bp) were amplified based on ISSR analysis with a range from 6 to 17 bands, 153 of them were polymorphic, ranging from 97.27 to 100%. For SCoT analysis, 164 polymorphic bands (150–5000 bp) were observed, varying from 14 to 19 bands for each primer. Nei’s genetic diversity analysis showed that the highest value was found in P11 for ISSR and P4 for SCoT markers. The highest Nei’s genetic diversity value was observed in P5 for combined markers and the low in P2. Nei’s dendrogram constructed with combined markers indicated a 75–89% of genetic similarity coefficient among populations. Population structure analysis revealed an optimum number of three groups, the same as their geographical distribution. This knowledge on PR genetic diversity can be used in future breeding programs, genetic improvement, product enhancement, and germplasms conservation.     

Genetic diversity of mango cultivars estimated using SCoT and ISSR markers

Two molecular marker systems, SCoT and ISSR were used for identification and genetic comparison analysis of 23 mango germplasm accessions collected within Guangxi province of China. Using 18 selected SCoT primers 158 bands were generated, of which 104 (65.82%) were polymorphic. Eighteen selected ISSR primers amplified 156 bands with 87 (55.77%) being polymorphic. The cultivars of Xiang Ya Mango type and their progeny have high genetic similarity with each other. The 23 cultivars were clustered into two major groups based on the SCoT analysis and three major groups based on the ISSR analysis with UPGMA. These clusters are in accordance with their known origins and main phenotypic characteristics. Our results indicated that the SCoT analysis better represents the actual relationships than ISSR analysis, although both analyses give similar results. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

落叶松种间及无性系间ISSR鉴别技术的研究

采用ISSR分子标记技术对兴安、长白和日本落叶松种间以及不同无性系进行了鉴别。从49条引物中筛选出13条ISSR引物可以对落叶松种间和无性系间进行鉴别,特异条带个体的百分率为100%,该项技术为落叶松新品种以及良种的准确鉴别提供了新的途径和手段：其中5条引物在日本落叶松、兴安落叶松和长白落叶松不同位置扩增出特异谱带,作为种的鉴定的标准,有9条引物可以对落叶松种内不同无性系分别扩增出特异片段,进行无性系的鉴别。     

中国黑木耳主要栽培菌株ISSR指纹分析及SCAR标记

采用ISSR标记技术对来自全国的黑木耳34个主要栽培菌株进行DNA指纹分析,初步构建其标准化DNA指纹图谱;在ISSR指纹分析基础上进行聚类分析,并将黑木耳两个栽培菌株173和186中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR标记.研究表明,我国黑木耳栽培菌株遗传背景差异不大,存在同物异名现象,而采用ISSR指纹及其SCAR标记鉴定黑木耳栽培菌株具有重要意义.     

中国黑木耳主要栽培菌株ISSR指纹分析及SCAR标记

采用ISSR标记技术对来自全国的黑木耳34个主要栽培菌株进行DNA指纹分析,初步构建其标准化DNA 指纹图谱;在ISSR指纹分析基础上进行聚类分析,并将黑木耳两个栽培菌株173和186中扩增获得的ISSR特异性DNA带转化为可以直接用于菌株快速鉴定的SCAR标记。研究表明,我国黑木耳栽培菌株遗传背景差异不大,存在同物异名现象,而采用ISSR指纹及其SCAR标记鉴定黑木耳栽培菌株具有重要意义。     

福建省枳椇种质资源遗传多样性的ISSR分析

通过建立和优化枳椇Hovenia acerba的ISSR反应体系,并利用ISSR分子标记方法,对福建省12个种源地35份枳椇材料进行遗传多样性及亲缘关系研究。枳椇遗传多样性分析的ISSR最佳反应体系为反应总体积25 μL,10×Ex Taq Buffer(Mg2+ plus) 2.5 μL,dNTP Mixture 375 μmol·L-1,引物0.4 μmol·L-1,DNA模板75 ng,Ex Taq酶1 U,ddH2O 14.55 μL。程序：预变性94 ℃ 5 min;变性 94 ℃ 30 s,退火50 ℃ 1 min,延伸72 ℃ 90 s,共 35 个循环;最后延伸72 ℃ 7 min。利用筛选出的10个ISSR引物共检测了基因组DNA中175个位点,其中多态性位点155个,占总位点数的88.57%。供试材料观测等位基因数(Na) 1.8857、有效等位基因数(Ne) 1.4300,Nei’s基因多样性指数(H) 0.2572,Shannon多样性指数(I) 0.3959。UPGMA聚类分析表明,以遗传相似系数0.74为阈值,可将35份枳椇分成四大组。ISSR标记有效地揭示福建省枳椇种质资源丰富的遗传多样性,具较大开发利用价值。     

红皮云杉与嫩江云杉RAPD和ISSR分子标记反应体系优化和特异性检测

以红皮云杉和嫩江云杉为材料,采用现代分子标记技术-RAPD和ISSR标记研究红皮云杉与嫩江云杉间在基因组水平上的遗传差异。通过正交试验设计筛选RAPD和ISSR反应程序和反应体系。从近100个引物中筛选出3个RAPD引物和一个ISSR引物用来区分红皮云杉和嫩江云杉,它们分别是OPN07、OPA17、S25和ISSR67。用这4个引物扩增两种云杉基因组DNA,分别在1 000,950,1 500,2 000 bp处嫩江云杉有特异性谱带而红皮云杉没有。研究表明,采用RAPD和ISSR分子标记可以将红皮云杉和嫩江云杉在分子水平上加以区分,为今后进一步阐明云杉的系统进化,物种鉴定和种间杂交育种提供了理论依据。     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

DEVELOPMENT OF SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS FROM INTERSIMPLE SEQUENCE REPEAT FINGERPRINTS FOR THE MOLECULAR DETECTION OF TOXIC PHYTOPLANKTON ALEXANDRIUM CATENELLA (DINOPHYCEAE) AND PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA (BACILLARIOPHYCEAE) FROM FRENCH COASTAL WATERS1

Harmful algal blooms are a serious threat to shellfish farming and human health all over the world. The monitoring of harmful algae in coastal waters originally involved morphological identification through microscopic examinations, which was often difficult unless performed by specialists and even then often did not permit identification of toxic species. More recently, specific molecular markers have been used to identify specific phytoplankton species or strains. Here we report on the use of the intersimple sequence repeat (ISSR) technique to develop specific sequence characterized amplified region markers (SCAR) and to identify with these tools two toxic species in French coastal waters, the diatom Pseudo‐nitzschia pseudodelicatissima (Hasle) Hasle and the dinoflagellate Alexandrium catenella (Whedon and Kofoid 1936), Balech 1985. Six polymorphic ISSR regions were selected among amplified fingerprints of a representative sample of phytoplankton species. After cloning and sequencing the selected polymorphic ISSR regions, pairs of internal primers were designed to amplify a unique and specific sequence designed as a SCAR marker. Of the six selected SCAR markers, three were specific to P. pseudodelicatissima and one for A. catenella. The SCAR marker specificity was confirmed by using basic local alignment search tool comparison, by experimental assays on different strains from 11 countries, and by checking that the sequence amplified was the expected one. When tested on water samples collected along the French shores, the four specific SCAR markers proved to be efficient tools for fast and low‐cost detection of toxic phytoplankton species.