兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

兴安、长白及华北落叶松RAPD 分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松(Larix gmelini)、长白落叶松(L. olgensis)和华北落叶松(L. principisrupprechtii)的针叶及种子胚乳为研究材料, 采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明, 通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物, 其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1 500 bp处扩增出特异条带, 而在华北落叶松中没有; 引物OPX-14在兴安落叶松基因组DNA中1 200 bp处扩增出特异条带, 而在长白落叶松中没有; 还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

西瓜种质资源遗传亲缘关系的RAPD分析

利用RAPD技术对国内外32份西瓜主栽品种与其骨干亲本及野生类型的遗传亲缘关系进行了研究。从720个随机引物（10bp)中筛选出15个能产生稳定多态性的引物用于RAPD反应,共扩增出104条DNA带,其中多态性DNA条带43条,占41．35％,平均每个引物扩增的DNA条带的数目为7．0条。聚类分析将供试材料分为6个类群：1个东亚生态型类群、1个美国生态型类群、2个中间生态型类群和2个非洲野生型类群,与传统的西瓜生态型分类基本吻合。每个生态型类群都有其特有的扩增（缺失）条带,同时分析了同一生态型中各个品种之间的亲缘关系及其品种的特异条带。本实验结果不仅从分子水平验证了西瓜是遗传基础狭窄的作用,而且在分子水平对西瓜传统分类与地理生态型分类进行了分析。     

西瓜种质资源遗传亲缘关系的RAPD分析

利用 RAPD 技术对国内外32份西瓜主栽品种与其骨干亲本及野生类型的遗传亲缘关系进行了研究。从720个随机引物(10bp)中筛选出15个能产生稳定多态性的引物用于 RAPD 反应,共扩增出104条 DNA 带,其中多态性 DNA 条带43条,占41.35%,平均每个引物扩增的 DNA 条带的数目为7.0条。聚类分析将供试材料分为6个类群:1个东亚生态型类群、1个美国生态型类群、2个中间生态型类群和2个非洲野生型类群,与传统的西瓜生态型分类基本吻合。每个生态型类群都有其特有的扩增(缺失)条带,同时分析了同一生态型中各个品种之间的亲缘关系及其品种的特异条带。本实验结果不仅从分子水平验证了西瓜是遗传基础狭窄的作物,而且在分子水平对西瓜传统分类与地理生态型分类进行了分析。     

甜樱桃品种及其砧木的RAPD分析

利用RAPD技术,从130个随机引物中筛选出46个引物,对欧洲甜樱桃、欧洲酸樱桃、马哈利樱桃和野生中国樱桃4个类型樱桃种,以及欧洲甜樱桃与中国樱桃的种间杂交种共15个品种的基因组遗传变异进行分析。结果表明,46个随机引物均得到了稳定可重复的RAPD图谱,扩增出的DNA条带大小在100~2625bp之间,多态性位点数517个,多态性位点百分率为98.85%,每个随机引物扩增出的多态性DNA条带数在4~23条。品种间Nei遗传距离在0.166~0.479之间,平均遗传距离0.329;甜樱桃新品种‘秦樱1号’与‘秦岭玛瑙’、‘CDR-1’等10个樱桃砧木之间的遗传距离在0.248~0.376,并且根据遗传距离可以相互区分,所分析的15个樱桃品种均扩增出了特有的DNA条带,每个樱桃特有标记带在2~17个之间,共扩增出149个特有标记,据此可以进行樱桃品种及砧木的RAPD鉴定。研究认为利用RAPD技术可以在分子水平上对甜樱桃品种及其砧木进行快速鉴定。     

满天星试管苗与其玻璃化苗的RAPD指纹图谱分析

采用分离群体分组分析法(BSA),用100个随机引物对满天星的正常苗和玻璃化苗进行RAPD分析的结果表明,7个随机引物扩增出多态性差异条带。再用上述7个引物分别对试管苗及其玻璃化苗个体进行DNA的PCR扩增的结果显示,引物J20在2种苗中出现差异条带。     

鸢尾属部分植物种质资源的RAPD分析

采用RAPD分子标记技术,从100个随机引物中筛选出多态性强、重复性好且稳定性高的引物18个,对38份野生鸢尾属材料进行扩增,共扩增出409条带,其中多态性带405条,多态性比率为99.0%,表明野生鸢尾属植物种间有丰富的遗传多态性;根据DNA谱带计算物种间遗传距离,聚类分析结果将鸢尾属38份材料划分为6组,其结果与传统生物学特性划分的6个亚属的分类结果基本一致;物种特有RAPD标记分析表明,利用18个引物可以较好地将鸢尾属38种植物区分开,其中9个材料得到了单一标记的扩增带,表明运用RAPD分子标记对研究鸢尾属植物特异性基因及标记的筛选等有一定的理论和实际应用价值。     

金银花五个品系的RAPD分析及DNA指纹图谱的建立

运用RAPD技术,对5个金银花品系进行遗传多样性研究并构建这5个金银花品系的DNA指纹图谱。从80个引物中筛选出25个带纹清晰,多态性好的引物用于实验。其中,引物SBSD06的扩增条带可以清楚明确区分5个品系,建立其DNA指纹图谱。在清晰、稳定出现的170条带中,153条带具有多态性。按UPGMA法进行聚类分析,计算其遗传相似系数,结果显示,金银花5个品系聚为两类,与其形态学分类结果相符。     

香果树组织培养过程中遗传变异的RAPD分析

用RAPD分子标记方法,从DNA水平上分析野生型香果树以及通过器官发生途径和体细胞胚胎发生途径得到的香果树再生植株以及体细胞胚胎发生过程中不同继代次数的培养物之间的遗传变异。筛选了100个随机引物,其中有75条随机引物能够扩增出条带,从中选取11个引物进行PCR扩增的结果显示：香果树体细胞胚胎无性系中有RAPD多态性位点,在胚性愈伤组织中也检测到少数RAPD变异位点。表明RAPD分子标记方法可以鉴定香果树组织培养过程中的遗传变异。     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

观赏南瓜及葫芦种质资源遗传多样性分子评价

利用RAPD和ISSR标记对28份观赏南瓜及葫芦种质资源进行遗传多样性分子评价。结果表明:12个RAPD引物和13个ISSR引物分别扩增出89条和93条清晰谱带,平均每个引物分别扩增出6.1条和6.2条多态性谱带,多态性比率分别为82%和86%。RAPD和ISSR标记检测供试材料的遗传相似性系数(GS)范围分别为0.31～0.99和0.33～0.99,ISSR(平均GS值0.68)检测多态性效果高于RAPD(平均GS值0.73)。利用UPGMA法基于RAPD与ISSR混合聚类,将28份观赏南瓜及葫芦种质分为3类,类群的划分与果实形状明显相关:第Ⅰ类群包括15份种质,为扁圆形、卵圆形、圆球形或圆筒状的早熟或晚熟果实;第Ⅱ类群包括11份种质,为汤匙形、梨形、扁球形或皇冠形的早中熟果实;第Ⅲ类群包括2份种质,为葫芦形的晚熟果实。     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Detection of Polymorphic DNA and Taxonomic Relationships Among 10 Wild Perennial Soybean Species Using Specific and Arbitrary Nucleotide Primers

Polymorphic DNA in complex genomes of agronomic crops can be detected using specific nucleotide and arbitrary primers and the polymerase chain reaction (PCR). Nineteen accessions representing 10 species of the wild perennial soybean were evaluated using 4 sets of specific primers and 3 sets of random amplified polymorphic DNAs (RAPD) primers. The potential of the RAPD assays was further increased by combining two primers in a single PCR. The fragments generated by the two assays discriminated 10 wild species by banding profiles. The size of the amplified DNA fragments ranged from 100 to 2100 base pairs. The resolved PCR products yielded highly characteristic and homogeneous DNA fingerprints. The fingerprints were useful not only for investigating genetic variability but also for further characterizing the wild soybean species by detecting inter- and intra-specific polymorphisms, constructing dendrograms defining the phylogenetic relationships among these species, and identifying molecular markers for the construction of genetic linkage maps. Furthermore, unique markers distinguishing particular species were also identified. Thus, it is expected that PCR will have great relevance for taxonomic studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic and geographic polymorphism of cultivated tobaccos (Nicotiana tabacum) in Turkey

Nicotiana tabacum (2n = 48) is a natural amphidiploid and shows a distribution over a geographical area in eastern Anatolia. Random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 21 primitive tobacco accessions comparing flue cure virginia genotype (FCV) and their geographical polymorphism as a source of genetic variations for breeding programs. Only 13 of all the 60 random primers used in RAPD showed polymorphism acceptable for characterization of these accessions. Totally 118 RAPD fragments were generated from 13 decamer primer and 64 of them were found polymorphic (54.2%). Mus and FCV showed the smallest genetic distance among accessions cultivated in the eastern Anatolia. These results shows that the RAPD assay is a powerful approach for identifying genetic and geographic polymorphism. This article was submitted by the authors in English.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Genetic and geographic polymorphism of cultivated tobaccos (Nicotiana tabacum) in Turkey

Nicotiana tabacum (2n = 48) is a natural amphidiploid and shows a distribution over a geographical area in eastern anatolia. Random amplified polymorphic DNA (RAPD) technique was used to evaluate both genetic diversity among 21 primitive tobacco accessions comparing flue cure virginia genotype (FCV) and their geographical polymorphism as a source of genetic variations for breeding programs. Only 13 of all the 60 random primers used in RAPD showed polymorphism acceptable for characterization of these accessions. Totally 118 RAPD fragments were generated from thirteen decamer primer and sixtyfour of them were found polymorphic (54.2%). Mus and FCV showed the smallest genetic distance among accessions cultivated in the eastern anatolia. These results shows that the RAPD assay is a powerful approach for identifying genetic and geographic polymorphism.     

Confirmation of relationships among Boesenbergia (Zingiberaceae) and related genera by RAPD

The relationships among 19 accessions of Zingiberaceae belonging to 11 species of Boesenbergia, six species of Kaempferia, and two species of Scaphochlamys from Southern Thailand were studied using random amplified polymorphic DNA (RAPD) profiles from leaf tissue samples. The RAPD was carried out using 10 random decamer arbitrary primers. Amplification occurred in five out of 10 tested primers (OPAM-01, OPAM-03, OPAM-12, OPB-14, OPZ-03). Total of 53 amplified bands were observed. Data obtained from the RAPD fingerprints from the samples clarified some doubts in morphological classification. The data were analyzed for the Nei and Li's Dice similarity coefficient for pair-wise comparison between individual samples and the distance matrix. The dendrogram resulting from cluster analysis, UPGMA and a principal component analysis of the RAPD result confirms a higher degree of relationship between Boesenbergia and Scaphochlamys than between Boesenbergia and Kaempferia.