大血藤DNA提取及RAPD研究初探

以浙江天台山大血藤为材料,对其DNA提取和RAPD分子标记方法进行了研究。结果表明:所采用的经改进的SDS提取法可获得高质量的大血藤DNA,分子量大于23kb,可满足RPAD扩增;用15个不同的随机引物对所提取的12个个体的大血藤DNA进行了RAPD分子标记分析,其中14个引物扩增产物具有多态性。建立了大血藤DNA提取和RAPD标记的分析程序,为RAPD分析应用于大血藤遗传多样性研究打下了良好的基础。     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

两个地区东方田鼠基因组RAPD分析比较研究

目的　从DNA的水平分析比较两个地区东方田鼠的分子遗传特征,探讨以RAPD标记鉴别两个地区的东方田鼠。方法　筛选6条10bp的随机引物对洞庭湖和青铜峡地区的东方田鼠基因组进行了随机扩增多态DNA(RAPD)分析,并对这两个地区的东方田鼠的基因组DNA进行了比较。结果　①两个地区东方田鼠的所有受试个体中共有的片段数为20条,这是两个地区东方田鼠的共性所在;②两个地区东方田鼠各有其特异性扩增片段;③引物S17和S80可作为鉴别两个地区东方田鼠的特异性引物;④不同地区的东方田鼠其不同个体之间的共享度较低,且存在较大差异;两个地区东方田鼠的遗传背景均呈非均一性。结论　运用RAPD方法可以作为鉴别不同地区东方田鼠的基因多态性的标记。     

RAPD技术及其在动物遗传育种中的应用

RAPD技术是在PCR基础上发展起来的一种DNA多态性检测技术,已广泛应用于基因组研究的各个领域。本文概述了RAPD反应的原理、特点,总结了其在遗传多样性检测、亲缘关系鉴定、遗传连锁分析和数量性状的辅助标记选择等方面的应用,并肯定了RAPD在动物遗传育种领域的应用前景。     

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.     

Identification of strain specific markers in Bacillus anthracis by random amplification of polymorphic DNA

Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in anthrax research. We used a PCR based method--Random Amplification of Polymorphic DNA (RAPD)--to identify genetic markers in B. anthracis strains. Twenty-five differential genetic markers were identified which divided the strains into five different groups. Three selected RAPD-markers were cloned and sequenced. The five RAPD-derived genotypes could be defined by integration of these three markers. This system offers a simple non-expensive method to classify B. anthracis strains in laboratories involved in the research of this bacterium.     

DNA分子标记技术及其在水产动物遗传上的应用研究

随着DNA分子标记技术的发展,其在动物遗传上发挥了重大作用,使用DNA分子标记可以观察到整个基因组的遗传多样性。目前,在水产养殖种类中使用的遗传标记主要包括线粒体DNA、RFLP、RAPD、AFLP、微卫星、SNP和EST标记。DNA分子标记的应用使得人们对水产养殖动物的遗传多样性、近亲繁殖、种类和品系鉴定以及遗传连锁图谱建立的研究都取得了很大进展,也加快了数量性状位点(QTL)基因的鉴定作为分子标记辅助选择(MAS)的研究。将这些标记技术在水产动物上的应用进行了论述,以及如何从人类基因组工程和斑马鱼这种模式鱼的研究中得到启发,更好的应用于水产动物基因组学和遗传学研究做一讨论。     

RAPD分子标记及其在作物遗传育种中的应用

ＲＡＰＤ分子标记是一种新型的分了遗传标记,其原理是采用１０个碱基的随机引物,以基因组ＤＮＡ为模板进行ＰＣＲ随机扩增。其优越性在于其方法简单;分析中的花费少,用时短;分析所需的ＤＮＡ用量也不多等。本文着重介绍以电泳技术和ＰＣＲ扩增技术为核心的分子标记以及在农作物遗传育种中的应用。     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.     

两个不同地区东方田鼠杂交子代RAPD标记分析

目的研究不同地区东方田鼠杂交后产下的子代的遗传特性.方法筛选4条10bp随机引物对宁夏和洞庭湖地区东方田鼠杂交子代的基因组进行随机扩增多态DNA(RAPD)分析,并对不同地区亲代以及子代相互之间的基因组DNA进行相似性分析.结果①所有东方田鼠均有相同的扩增片段出现;②两个不同地区的亲代分别有特异性片般;③亲代的DNA带型均能在子代中找到;④同一胎次东方田鼠之间基因共享度大约在72%～96%之间.结论RAPD分析能在一定程度上反映出东方田鼠种的特性以及亚种的特异性,而且同一胎次之间基因共享度较高.     

Regression association analysis of fruit traits with molecular markers in cherries

The use of molecular markers supports the study of genetic marker–trait association of biological and agronomic interest in diverse genetic material. In this research, association between simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers with fruit traits were investigated in two collections of cherries by applying multiple regression analysis (MRA). Thirty-eight SSR alleles and 135 RAPD fragments were found associated with 14 of affecting fruit traits. Some of SSR and RAPD markers were associated with more than one fruit trait in MRA. Such an association may arise due to pleiotropic effect of the linked quantitative trait locus on different traits. For example, some SSR and RAPD markers were associated with all four traits including fruit cracking, fruit firmness, total soluble solid (TSS) and fruit shape. Also, some markers had correlations with all four characters of TSS, anthocyanin, fruit skin color and fruit flesh color, indicating a significant correlation among these traits. Therefore, it is possible to use these markers along with morphological traits in cherry breeding programs for identification of suitable parents to produce mapping populations and hybrid cultivars. Also, these results could be useful in marker-assisted breeding programs when no other genetic information is available.     

DNA polymorphism in various goose lines by RAPD-PCR

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

大豆灰斑病菌生理小种的RAPD标记*

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对发生于中国东北的大豆发斑病菌（Cercosporidiumsojinum）的10个生理小种进行基因组DNA多态性分析。用13个10-核苷酸随机引物共计获得了111个RAPD标记,其中86.5％具有多态性,通过聚类分析确定了供试小种间的亲缘关系。试验证明,RAPD技术分析大豆灰斑病菌遗传变异可提供大量分子标记,综合分析13个随机引物的扩增谱带可将供试菌株清楚分开。RAPD技术是一项操作简单、快速和灵敏的方法,极具对病菌群体遗传分析的潜力。     

大豆灰斑病菌生理小种的RAPD标记

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对发生于中国东北的大豆发斑病菌（Cercosporidiumsojinum）的10个生理小种进行基因组DNA多态性分析。用13个10-核苷酸随机引物共计获得了111个RAPD标记,其中86.5％具有多态性,通过聚类分析确定了供试小种间的亲缘关系。试验证明,RAPD技术分析大豆灰斑病菌遗传变异可提供大量分子标记,综合分析13个随机引物的扩增谱带可将供试菌株清楚分开。RAPD技术是一项操作简单、快速和灵敏的方法,极具对病菌群体遗传分析的潜力。     

Genetic variation in monoploids of diploid potatoes and detection of clone-specific random amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers have been used to study the genetic variation among androgenetic monoploids of diploid Solanum species. Cluster analysis of pairwise genetic distances was used to construct a genetic relationship among anther donor and anther-derived potato plants. The clustering based on Rogers' distances resembled classifications based on parental origins and hybrid combinations. Six of the 32 RAPD primers used resulted in the selective amplification of DNA fragments which were polymorphic between the two S. phureja parental clones, 1.22 and A95. It should be possible to construct a genetic linkage map, without making crosses, using monoploids derived from a single heterozygous diploid clone and RAPD markers.     

Patterns of genetic variation in native grape phylloxera on two sympatric host species

Random amplified polymorphic DNA (RAPD) markers were used to examine population genetic structure in populations of native grape phylloxera. This research asked: (i) do RAPD markers distinguish two groups corresponding to the two host plant species; and (ii) do RAPD markers distinguish groups according to spatial location, independent of host plant association? Forty‐nine phylloxera clones were collected from five pairs of adjacent individuals of two sympatric grape species in five sites along a 145 km transect in Missouri, USA. A high level of polymorphism was observed, with some evidence for structuring between host plant species and no evidence for spatial structuring. An analysis of molecular variance (amova ) found that 6.52% of the variance in RAPD banding patterns was attributable to host species and 7.96% of the variance was attributable to spatial location. A cluster analysis did not result in two groups corresponding to the two hosts, or to five groups corresponding to the geographical sites sampled. A Mantel test showed a low correlation between genetic similarity and spatial location. Two of the 93 RAPD markers were nonrandomly associated between the hosts. It is suggested that there may be a small host‐mediated effect on genetic variation but stochastic dispersal and a highly heterogeneous environment may be the primary influences on the observed polymorphism.     

RAPD标记与作物改良

建立于ＤＮＡ基础之上的分子标记对于作物改良和多态性研究具有重要作用。     

Applications of random amplified polymorphic DNA (RAPD) in molecular ecology

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.