Quantitative Properties of Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA analysis (RAPD) is a methodology that has been used as a tool for monitoring microbial communities. To be useful in this application RAPD, and any other methodology, must show properties that allows for the detection of quantitative changes in composition of the microbiota. Therefore, the objective of this study was to establish whether RAPD possesses such properties. The strategy was to use genomic DNA, extracted from a set of tertiary bacterial mixtures defined according to an experimental mixture design, and containing varying proportions of Escherichia coli, Bacillus subtilis, and Pseudomonas CF600. RAPD-PCR was performed on the mixed DNA extracts and the amplified DNA fragments were separated on sequencing gels to produce genomic fingerprints that were digitized and modeled by Partial Least Squares regression (PLS). Significant predictions were obtained using an external test set for validation, with Root Mean Square Error of Predictions (RMSEP) of 0.21, 0.19 and 0.20 for the proportion of E. coli, B. subtilis and Pseudomonas CF600 respectively. Taken together, the results showed that RAPD patterns quantitatively represented the initial mixture proportions. Therefore, the view that RAPD could be useful for whole microbial community monitoring was strengthened.     

用RAPD法研究火炬松愈伤组织发生的遗传特性(英文)

体外培养对于植物的快速繁殖是非常有效的。和其它一些松果类硬木植物一样,火炬松的体外培养成功率却一直很低。本工作研究了不同的基本培养基和低温条件对于火炬松Ｊ－５６, Ｓ－１００３, ａｎｄ Ｅ－４４０等三个品系的成熟合子胚形成愈伤组织、分化出芽、成苗的影响。在不同的基本培养基条件下芽分化的程度差异很大。合子胚经过９－１２周培养,开始分化,形成具有器官发生的愈伤组织（Ｆｉｇ．２ａ）。分化后３周,开始诱导出芽（Ｆｉｇ．２ｂ）,芽的生长快慢不同（Ｆｉｇ．２ｃ,ｄ）。同一个愈伤组织上会生出几个芽来（Ｆｉｇ．２ｅ）。在添加有ＩＢＡ和ＢＡ的ＴＥ培养基上芽生长最快（Ｆｉｇ．１）。低温条件持续 １５天,能增加芽的数量和分化的程度（Ｔａｂｌｅ１）。上述培养基中增加ＧＡ３时表明,ＧＡ３对于根的诱导有决定性的作用。将９８株再生苗转移到特殊的混合土壤上;成活了７５株苗（Ｆｉｇ．２ｆ）。以这三种火炬松的再生苗尖为材料制备ＤＮＡ。用２０个引物进行ＲＡＰＤ分析,结果表明：这三种火炬松苗的扩增产物是相同的（Ｆｉｇ．２ｇ,ｈ＆ｉ）。这说明：用愈伤组织克隆植株的过程中没有引起植物遗传变异。     

RAPD技术及其在哺乳动物生态学研究中的应用

1　引　言现代生态学的不断发展 ,改变了过去主要采用宏观的方法来研究生物与生物及环境的关系。在种群生态学研究中 ,越来越多的学者采用微观的分子生物学技术来研究种群的生存问题 ,新学科分子生态学得到快速的发展。目前 ,从分子水平来研究种群生态 ,较多采用限制性酶切图谱 (ＲＦＬＰ)和以ＰＣＲ为基础的ＲＡＰＤ技术以及ＤＮＡ序列分析技术。在使用这些技术时 ,很多实验的条件往往会影响实验的结果。本文根据作者在实验中的体会 ,结合一些研究者已有的工作 ,对ＲＡＰＤ的原理、影响因素以及在哺乳动物生态学研究中的应用作简单介绍。2 …     

利用RAPD技术分析实验用比格犬的遗传背景

目的　运用RAPD技术对实验用比格犬 (Beagle)进行遗传背景分析。方法　筛选的 16个RAPD引物对 4 0个样品的分析 ,共得到 93个扩增条带。结果　所有样品的相似系数 80 4 %到 10 0 %间变动 ,平均相似系数为93 2 8% ,聚类分析得到了这些个体的同源树资料。结论　本遗传背景资料可以为以后的比格犬繁育生产和生物医药学的研究应用提供技术指导     

Random Amplified Polymorphic DNA (RAPD) for differentiation between Thai and Myanmar strains of Wuchereria bancrofti

Background

Information on the physical and psychosocial disability of lymphatic filariasis in Sri Lanka is scarce. Therefore this study was carried out to describe the physical disability and psychosocial impact associated with chronic lymphoedema in patients attending filariasis clinics in the Colombo district, Sri Lanka.

Methods

Four hundred and thirteen patients with lymphoedema of limbs attending filariasis clinics in Werahera and Dehiwala in the Colombo district were enrolled in the study after obtaining informed written consent. Data were collected using a pre-tested, interviewer-administered questionnaire and analyzed using SPSS.

Results

Majority (95%) of patients had lower limbs affected and there was a significant association with difficulty in walking (p = 0.023). The swollen limb affected the work of 87 (52 %) of employed patients and 26 persons reported loss of job. Approximately 25 % and 6 % reported having problems interacting with the community and family, respectively and 8.7 % felt that they were rejected by society. The swollen limb was perceived as a major problem by 36.8 % of patients. Of the married persons, 5.7 % and 6.2 % reported sexual and marital problems respectively, due to their swollen limb/s. Of those who had marital problems, 77.3% reported sexual problems as well (p < 0.001).

Conclusion

Lymphoedema significantly affects physical, psychological and social functioning in affected individuals. Morbidity control, in addition to control of physical disability, should target the psychosocial consequences.     

我国主要地方绵羊品种随机扩增多态DNA研究

对蒙古羊、湖羊、滩羊、小尾寒羊、乌珠穆沁羊、藏绵羊、阿勒泰羊7个地方绵羊品种和无角陶赛特羊、德国美利奴羊、萨福克羊3个引入品种基因组DNA进行了RAPD分析。结果表明：（1）RAPD可作为一种有效的标记用于绵羊品种之间遗传亲缘关系的分析。（2）在所使用的43种随机引物中,有35种引物扩增出多态谱带,多态频率为66.24%,说明RAPD技术用于研究绵羊核DNA的遗传变异具有较高的检出率和灵敏度。（3）总群体平均遗传多样性指数（HSP)为0.9139,说明绵羊群体具有较为丰富的遗传多样性。（4）我国地方绵羊品种间的分子聚类关系与其所处的地理位置、考古学结果,以及细胞遗传学研究结果基本,引入品种间的分子聚类关系也与其育成史基本一致。     

中国大陆拟果蝇(Drosophila simulans)的随机扩增多态性DNA分析

近几年发现了拟果蝇(Drosophila simulans)在中国大陆的广泛分布。用随机扩增多态性DNA(RAPD)方法研究了中国大陆38个不同地理群体的拟果蝇(D．slmulam)在DNA水平上的遗传多样性,初步讨论了拟果蝇在中国大陆的起源。以40种10bp长的寡聚核苷酸随机引物进行PCR扩增,根据遗传距离利用UPGMA法作出的相关聚类图显示：(1)38个地理群体按纬度以南京(NJ)为界明显地分为南北两大支系,北方支系以北京(BJ)为界又分为明显的两个亚支,一支为东北支系包括漠河、海拉尔、黑河、佳木斯、哈尔滨、长春、沈阳、丹东、延吉、图们等10个群体;另一支包括北京、大同、呼和浩特、银川、西宁、兰州、太原、石家庄、烟台、济南、徐州、连云港等12个群体。南方支系包括郑州、武汉、上海、南京、杭州、重庆、南昌、温州、长沙、贵阳、福州、昆明、厦门、广州、南宁、海南等16个群体。(2)各地理群体之间的遗传距离与地理分布有密切关系,基本按地理位置的相关性聚类在一起。根据了解到的事实,(1)拟果蝇在中国是一个外来种;(2)拟果蝇在中国大陆的入侵是最近30年左右的事情;(3)拟果蝇在全国的广泛分布是最近10多年的事情。所以拟果蝇各群体的聚类关系可能并不是地理分化的结果,而是由于建立各地方群体的祖先群体或个体本身具有不同的遗传组成,但是某些地理上相邻近的群体可能拥有共同的祖先群体或个体,从而造成了拟果蝇各地方群体随地理分布关系而聚类。推测有两种可能的原因会产生以上结果：一是中国大陆的拟果蝇有多个不同的来源;二是拟果蝇在扩张过程中经历了可产生奠基者效应或瓶颈效应的偶然事件。     

Potential Utility of Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Assay (REA) as Typing Systems for Madurella mycetomatis

Two molecular methods were compared, random amplified polymorphic DNA (RAPD) and restriction endonuclease analysis (REA), in order to evaluate their ability to discriminate, and to characterize Madurella mycetomatis strains isolated from human mycetomas in different parts of the world. Both methods were able to cluster the Madurella mycetomatis isolates into the same number of distinct typing groups. However, RAPD, presenting several advantages over REA such as its rapidity, simplicity, and the accessibility for implementation in the laboratory, is a more sensitive and reproducible tool for the study of Madurella mycetomatis epidemiology than REA. Received: 24 March 1999 / Accepted: 24 June 1999     

Random Amplified Polymorphic DNA (RAPD) Variation among Populations of the Insular Endemic Plant Campanula microdonta(Campanulaceae)

RAPD variation was examined in nine populations of Campanulamicrodonta Koidz., endemic to the Izu Islands, Japan. Ninety-eightbands were obtained for all populations, 94% of which were polymorphicat least within a population. Shannon's H values were calculated;these have frequently been used in RAPD studies to estimategenetic diversity. The values within populations did not correlatewith the allozyme gene diversity estimated by a previous studyor with distance from the Japanese mainland. The possible reasonsfor this discrepancy are different selection regimes betweenthe two markers, higher RAPD mutation rates, and each marker'sdifferent coverage of genomes. Cluster analysis of genetic similaritiessuggested that colonization of each island probably occurredonce, except for Miyake Island, where immigration has occurredat least twice. Copyright 2001 Annals of Botany Company AMOVA, Campanula microdonta Koidz., insular endemic plant, genetic diversity, population genetic structure, RAPD     

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

Genetic variations among mulberry genotypes (Morus alba) as revealed by Random Amplified Polymorphic DNA (RAPD) markers

Turkey is one of the most important mulberry fruit producers in the world. In particular, in Eastern and Inner part of Turkey, mulberry fruits are processed into several products such as `Mulberry Pekmez', `Mulberry Pestil', `Mulberry Kome' etc. Therefore, mulberry fruits give higher economic returns thus have greater employment potentials in Turkey. In order to improve the yield and fruit quality through breeding, the genetic variations and relationships among 15 selected white mulberry genotypes (Morus alba L.) were evaluated using Random Amplified Polymorphic DNA (RAPD). A total of 101 DNA markers were generated by 16 random primers, with an average of 6.3 easily detectable markers per primer. Several RAPD markers showed unique patterns of mean frequency that differed among the white mulberry genotypes. The distance matrix showed that the highest genetic distance (0.902) was between EMS13 and EMS8 and the least (0.413) was between EMS12 and EMS15. According to the results, RAPD analysis can be used for the characterization and grouping of mulberry genotypes. The genetically divergent genotypes identified from this study would be of much use in the future breeding program.     

Molecular polymorphisms in Palestinian Figs (Ficus carica L.) as revealed by Random Amplified Polymorphic DNA (RAPD)

The genetic diversity of 13 local Palestinian fig genotypes was investigated using RAPD markers. Among the 30 tested primers, 28 revealed various banding patterns and 2 generated no polymorphic bands. In addition, 13 primers (46.4%) produced good amplification products with high intensity and pattern stability. A total of 94 DNA fragments (loci), separated by electrophoresis on agarose gel were detected, ranging in size from 190 to 1300 bp. Of these fragments, 72 (76.6%) were polymorphic and 22 (23.4%) were monomorphic. A minimum of three and a maximum of eight DNA fragments were obtained using (OPH-02 and OPT-10) as well as (OPA-13, OPA-18 and OPY-07) primers respectively. The maximum percentage of polymorphic markers was 100.0 (Z-5, Z-12, and OPT-10) and the minimum was 60.0 (OPH-02). Primers OPY-07 and OPA-13 revealed high collective resolving power (Rp) values with 4.640 and 4.760 respectively and therefore, they were the most useful RAPD primers to assess the genetic diversity in the Palestinian figs. Genetic distance matrix showed an average distance range from 0.186 to 0.559 with a mean of 0.373. Thus, the cultivars tested in this study were characterized by large divergence at the DNA level. To our knowledge, this is the first report using RAPD marker to assess genetic diversity of Palestinian figs.     

稻蝗属部分种类RAPD及其分子系统学关系

用经筛选的8个随机引物对稻蝗属(Oxya Audient-Serville)中的中华稻蝗(Oxya chinensis)、日本稻蝗(Oxyajaponica)、山稻蝗(0xya agaxisa)、短翅稻蝗(Oxya brachyptera)和小稻蝗(Oxya intricata)等5个种41个个体进行扩增,均扩增出特异性产物,在96条清晰稳定的扩增片段中,多态性片段为95条,种间共有片段1条(引物S362扩增的约850 bp的片段),所获片段的长度大小为200—2500bP,单个引物扩增的条带数在8-16之间,平均为12。根据扩增结果,应用SPSS软件包中的Between-groups linkage法进行聚类分析,得到5种稻蝗的分子系统树。结果显示：中华稻蝗与短翅稻蝗关系密切,日本稻蝗与山稻蝗关系较近,而小稻蝗与它们的亲缘关系较远。聚类图所呈现的种间关系与传统的形态及细胞分类学结果基本一致,5种稻蝗亲缘关系的远近程度得以清楚显示,说明RAPD技术在稻蝗属种间分子系统学研究中具有很好的适用性。     

Genetic Mapping of Soybean [Glycine max (L.) Merr.] Using Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. We have adapted the assay to soybeans by using Stoffel Fragment DNA polymerase and by optimizing the reaction conditions. To increase the percentage of RAPD polymorphisms, the DNA template was digested with restriction enzymes before amplification. The combination of twenty-four primers and five DNA template treatments (Undigested, DraI, EcoRI, HindIII, and TaqI digested) revealed 94 polymorphic DNA fragments differing between soybean lines PI437654 and BSR101. Many polymorphic DNA bands were found unreliable or non-scoreable after re-screening of primers and verification of marker-allele segregation with 20 recombinant inbred lines (RILs). However, 28 RAPD markers were consistently polymorphic between the parental lines and followed Mendelian expectations. The use of DNA templates digested with DraI, EcoRI, HindIII or TaqI increased three times the number of RAPD markers compared to undigested DNA template alone. The 28 RAPD markers obtained were further screened with 72 RILs and placed on an existing RFLP map.     

Use of Random Amplified Polymorphic DNA Markers for Mapping the Chickpea Genome

Three interspecific crosses were developed using Cicer arietinum (ICC 4918) as the female parent and wild Cicer species [C. reticulatum - JM 2100, JM 2106 and C. echinospermum - ICCW 44] as the male parent. Cicer arietinum (ICC 4918) × C. reticulatum (JM 2100) cross produced the largest number of F₂ plants and was chosen for linkage mapping using Random Amplified Polymorphic DNA (RAPD) primers. A partial linkage map was constructed based upon the segregation of 36 RAPD markers obtained by amplification using 35 primers. The linkage map consists of two linkage groups with 17 linked markers covering a total of 464.9 cM. Analyses also revealed association of three morphological traits with linked RAPD markers. Out of seven morphological traits tested for association with linked markers in the segregating plants, four Quantitative trait loci (QTL) were detected for the trait leaf length and three QTLs each for the traits leaf width and erect plant habit.     

Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis

Isolates of three races of Bipolaris maydis from China (races O, C and T) were compared using two techniques. Random Amplified Polymorphic DNA (RAPD) analysis using 24 primers indicated that race O and C isolates were more similar to one another than to the race T isolate. Twenty of the primers produced RAPD profiles that were similar for the race O and C isolates but differed for the race T isolate (four primers did not amplify products in any of the isolates). Four primers produced profiles which differed for all three races and two of these (A-09 and B-18) clearly differentiated the race O and race C isolates. Genetic fingerprinting of B. maydis using M13 DNA as a probe differentiated race O and C isolates from the race T with all four restriction enzymes used. Furthermore, when DNA was digested with Hind III, the hybridization profiles of the race O and C isolates differed from one another.     

Typing of Clostridium perfringens strains by use of Random Amplified Polymorphic DNA (RAPD) system in comparison with zymotyping

The definition of strain clonality postulates that strains showed identical phenotypic and genetic traits are likely to descend from a common ancestor even if they were isolated from different sources and locations. Regarding this definition, non-epidemiologically linked strains might be clonal strains. To overcome this ambiguity, the discriminatory capability of RAPD typing was assessed firstly on eight Clostridium perfringens strains proven to be chromosomally different with one being the mutant of another one. Thirteen primers were tested but only two were able to differentiate seven of the eight strains. With none of the used primers it was possible to differentiate the parental strain and its mutant harboring an insertion of 180 kb. The four most discriminant primers were retained to determine the RAPD fingerprints of a further 20 previously zymotyped strains from which seventeen were unrelated. To compare the two typing systems, the zymotype of the eight chromosomally different strains was determined. Thus, the discriminatory index was calculated on the basis of 25 unrelated C. perfringens strains. This was 0.97 with RAPD typing and 0.99 with zymotyping. From these results we conclude that the RAPD typing which is less fastidious than zymotyping can be used as an epidemiological marker for C. perfringens.     

用RAPD技术对罗非鱼遗传变异的研究及应用(英文)

应用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术检测了一个奥利亚罗非鱼（ａｕ）和湘湖（ｎｘ）、美国（ｎｍ）、沙市（ｎｐ）三个尼罗罗非鱼养殖群体（Ｔａｂｌｅ１）。在２０个引物（Ｔａｂｌｅ ２）中筛选到１２个引物,它们的扩增产物显示了罗非鱼和尼罗罗非鱼二者在群体内或群体间存在遗传差异。其中（Ｆｉｇ．１）, ＯＰＺ０６、 ＯＰＺ１６、 ＯＰＺ１２和 ＯＰＺ１９四个引物分别有一个扩增片段具有种的特异性。它们的大小分别是９００、１５００、１７００和７３０ｂｐ。可以作为鉴别罗非鱼和尼罗罗非鱼二者的分子遗传标记。湘湖（ｎｘ）、美国（ｎｍ）和沙市（ｎｐ）三个尼罗罗非鱼群体内遗传变异相似性指数Ｓ分别为 ０．７９８、 ０．７９５和０．８２４（Ｔａｂｌｅ ３）。表明：这三个尼罗罗非鱼群体都保留了较高水平的遗传变异。而奥利亚罗非鱼（ａｕ）的群体内遗传变异最小。奥利亚罗非鱼（ａｕ）与湘湖（ｎｘ）、美国（ｕｍ）、沙市（ｕｐ）三个尼罗罗非鱼群体之间的遗传距离分别是０．２８５、０．２６２和０．３４４（Ｔａｂｌｅ ３）,说明奥利亚罗非鱼（ａｕ）和沙市尼罗罗非鱼（ｎｐ）杂交将可能产生较强的杂种优势。     

Use of Random Amplified Polymorphic DNA (RAPD) to study genetic diversity within a population of blackfly, Simulium gravelyi from Palni hills, peninsular India

The genetic variation of local subpopulations (Palni hills, India) of the blackfly Simulium gravelyi was surveyed at different altitudinal locations using random amplified polymorphic DNA (RAPD) analysis. By comparing the similarity of the bands produced by RAPDs, it may be concluded that there is variation within this population. These data suggest that the heterogeneity of band pattern of S. gravelyi steadily increased from low elevation to high elevation. Site 5 (1590 m altitude) was more polymorphic and it differed most from site 1 (290 m). This is established by the ecological diversity and statistical analyses (PCA) and predicting two major ecological factors of conductivity and current velocity. The speciation hypothesis is consistent with the molecular evidence, postulates a high elevational site 5 (1590 m) and a subsequent site 4 (1250 m).     

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917^T) and L. pentosus (NCFB 363^T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.