Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA(RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China.A captive population has been established in the Beijing Zoo since 1999.In order to determine the kinship of the offsprings in 2001,randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo.To amplify the genomic DNA of each individual,53 arbitrary primers were selected.The results of amplifica tions showed that 14 primers had clear and distinct RAPD patterns.Totally,226 amplified fragments were generated by RAPD in this study.Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male).This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds.     

利用RAPD对稻蝗属昆虫亲缘关系的研究

通过20个随机引物的PCR扩增,得到了日本主要稻蝗的随机扩增多态性DNA（RAPD）图谱,根据扩增结果,计算了种间相似系数和遗传距离,建立了UPGMA系统树。结果表明,分布没有重叠、种间容易交配、能产生杂种的中华稻蝗台湾亚种与小翅稻蝗的亲缘关系最近;分布重叠的日本稻蝗与中华稻蝗台湾亚种、日本稻蝗和小翅稻蝗的亲缘关系较近。小稻蝗与其它3种稻蝗的亲缘关系较远。     

Assessment of random amplified polymorphic DNA (RAPD) for determining the origin ofYoungia koidzumiana Kitamura (compositae)

We determined the parental species ofYoungia koidzumiana (a natural interspecific hybrid) using PCR and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. These markers, generated by three primers, were sufficient to distinguishYoungia sonchifolia, Youngia denticulata, Youngia chelidoniifolia, andY. koidzumiana. The electrophoresis profiles of the amplified products from each of the four species were then compared. Three primers produced a total of 42 scorable markers; nine were specific markers forY. denticulata andY. chelidoni-ifolia. The length of the amplified DNA fragments ranged from 370 to 2500 b p. The three primers revealed polymorphic bands, which were indicators of the parental species ofY. koidzumiana. These bands showed a combination of specific profiles forY. denticulata andY. chelidoniifolia. Our results also were comparable to the data obtained for flowering times, floret numbers, and chromosome numbers of the four species. Therefore, we suggest thatY. koidzumiana is a hybrid betweenY. denticulata andY. chelidoniifolia}, and that RAPD markers are well suited for assessing the origins of plant species.     

Random amplified polymorphic DNA (RAPD) analysis in Indian mung bean (Vigna radiata (L.) Wilczek) cultivars

Greengram [Vigna radiata (L.) Wilczek], also known as mung bean, widely cultivated in a large number of countries, is an important pulse crop of Asia and is considered one of the ancestral species of the genus Vigna. Since yields of greengram have remained low across subtropical and tropical Asia, it is important to estimate genetic diversity in existing cultivars in order to see if the lack of genetic variability might be a constraining factor. In this study, 32 Indian cultivars of greengram were subjected to random amplified polymorphic DNA (RAPD) analysis using 21 decamer primers. A total of 267 amplification products were formed at an average of 12.71 per primer with an overall polymorphism of 64%. The extent of polymorphism was moderate to low. Jaccard similarity coefficient values ranged from 0.65 to 0.92. The cluster analysis resulted in mainly three clusters revealing greater homology between cultivars released from the same source. The results of principal components analysis also substantiated this conclusion. The close genetic similarity between the cultivars could be explained due to the high degree of commonness in their pedigrees. The narrow genetic base of the greengram cultivars revealed in the present analysis emphasises the need to exploit the large germplasm collections having diverse morphoagronomic traits in cultivar improvement programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

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。     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Genetic relationship of 32 cell lines of the euplotes octocarinatus species complex revealed by random amplified polymorphic DNA (RAPD) fingerprinting

Random amplified polymorphic DNA (RAPD) fingerprinting was used in this study to determine the genetic relationship of different cell lines of the hypotrichous ciliate Euplotes octocarinatus. Stocks isolated from different habitats in the USA, and from a group of genetically recombined laboratory strains, were characterized. Band-sharing indices (D) for all possible pairwise comparisons revealed a remarkable genetic diversity between the different cell lines. Investigation of the genetic structure in natural populations found diversity--although to a different extent--in all populations investigated. No clonal structure could be observed, as proposed for several protozoa and recently shown for E. daidaleos. These findings suggest frequent conjugation in the population of E. octocarinatus. No correlation between the genetic relationship of cell lines from different habitats and the distance between the corresponding sampling locations was found. Once separated geographically, the exchange of genetic material between populations appears to be nearly impossible. Therefore, these groups tend to separate into sibling species. The data generally support the occurrence of different syngens in the E. octocarinatus species complex. This finding is in accordance with our observation that the morphological 'species' of E. octocarinatus consists of several syngens or sibling species, similar to findings for the Paramecium aurelia-, Tetrahymena pyriformis- and E. vannus- species complexes.     

三种摇蚊幼虫RAPD扩增条件的优化及在昆虫系统发育分析中的应用

为了研究摇蚊科昆虫种群遗传的多样性,以促进对其资源的合理保护,以萨摩亚摇蚊Chironomus samoensisEdwards基因组DNA为模板,对摇蚊幼虫的RAPD扩增条件进行优化,建立了摇蚊幼虫RAPD扩增反应的最佳体系:按照利用优化的RAPD扩增条件进行研究,实验有着良好的重现性。用16个随机引物对3种摇蚊幼虫类群各10个个体进行RAPD扩增,其中萨摩亚摇蚊共扩增出78个条带,多态座位率为41.03%,Shannon遗传多样性指数为0.2570,群体内相似度为0.8730;红裸须摇蚊Propsilocerus akamusi(Tokunaga)共75个条带,多态座位率为44.0%,Shannon遗传多样性指数为0.2472,群体内相似度为0.8731;刺铗长足摇蚊Tanypus punctipennis(Fabricius)共67个条带,多态座位率为41.79%,Shannon遗传多样性指数为0.1943,群体内相似度为0.9066。聚类分析结果表明,刺铗长足摇蚊与红裸须摇蚊的亲缘关系较近。     

Application of randomly amplified polymorphic DNA (RAPD) analysis for typing avian Salmonella enterica subsp. enterica

Randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular genetic typing of 30 Salmonella enterica subsp. enterica strains isolated from chickens and ducks in Thailand. Six different primers were tested for their discriminatory ability. While some of the primers could only differentiate between the different serovars, the use of multiple primers showed that the RAPD method could also subdivide within a given serovar. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, we suggest that it may be a valuable new tool for studying the molecular genetic epidemiology of S. enterica ssp. enterica, both inter- and intra-serovars.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

华山新麦草自然居群沿海拔梯度的遗传分化

华山新麦草为我国特有种 ,仅分布于陕西华山 ,是国家一级珍稀濒危植物和急需保护的农作物野生亲缘种。应用 RAPD技术 ,选取 8条引物对华山新麦草自然分布区的 3个山峪(种群 ) 1 1个样方 (亚居群 ) 79个华山新麦草个体的总 DNA进行随机扩增 ,共得到 65个RAPD位点。统计分析表明 ,在华山的 3个山峪 (居群 )中 ,黄埔峪 (居群 )与其它 2个山峪 (居群 )发生较显著的遗传分化 ;华山峪 6个亚居群的个体平均 RAPD位点数有随海拔的升高而下降的趋势 ;6个亚居群间的相似性系数也有随海拔的升高而下降的趋势 ;华山峪的高海拔亚居群和低海拔亚居群间表现出明显差异。主成分分析结果进一步证明了黄埔峪居群与华山峪居群和仙峪居群间、以及华山峪的高海拔亚居群与低海拔亚居群间已发生一定程度的分化。研究结果暗示海拔差异是导致华山新麦草自然居群遗传分化的主要因素 ,海拔差异造成的有限的基因流可能才是影响居群和亚居群遗传分化的主要因子 ,而亚居群内遗传变异程度则与该亚居群的所处的特定生境有关     

RAPD analysis of natural populations of Acanthopanax brachypus

Random amplified Polymorphic DNA(RAPD) analysis is a new technology of molecular marking which has proved very powerful in detecting genetic diversity at the level of population.The genomic DNAs used in our experiment were extracted from fresh leaves taken from 59 individuals sampled from three natural populations in Yan an,Shanxi Province.Through more than 2,000 PCRs,deep-going RAPD analysis was carried out on DNA samples from 49 inviduals.The percentage of polymorphic RAPD loci found in these three populations were respectively 27.2%,18.6% and 5.4%;the average genetic distances within population,0.055,0.026 and 0.008;the average genetic distances between populations (I-II),(I-III) and (II-III),0.105,0.096 and 0.060.The genetic diversity of A.brachypus within and between populations was found,for the first time,to be rather poor,thus revealing innate factors as the cause contributing to its endangered status.In addition,our work also provides basic materials for elucidating the underlying cause of its endangerment and for its protection biology.     

The use of random amplified polymorphic DNA (RAPD) for hybrid detection in Scirpus from the river Schelde (Belgium)

Along the unique freshwater tidal zone of the river Schelde (Belgium), plants of Scirpus species occur primarily in small and fragmented populations. The majority of these are native Scirpus lacustris, S. pungens, S. triqueter, S. tabernaemontani and intermediate morphological forms. The distribution area of S. triqueter is even restricted to this tidal habitat. However, several cultivated S. tabernaemontani strains have recently been introduced. The latter species is often used to stabilize riverbanks. To determine the existing genetic diversity among these species, stems from plants from 44 different locations were subjected to random amplified polymorphic DNA (RAPD) analysis, using 22 decanucleotide primers. Data analysis of the amplified DNA fragments enabled us to unambiguously differentiate among these Scirpus taxa. Hybridization between S. triqueter and S. tabernaemontani was documented, and the studied hybrids were always genetically more similar to S. triqueter than to S. tabernaemontani . Among the introduced clones, at least two different origins could be distinguished. Several of the introduced S. tabernaemontani clones were clearly different from individuals of the same species that were native to this region. As there were different hybrid genotypes owing to different hybrid events or to introgression, this complex should be safeguarded from genetic pollution. New S. tabernaemontani genotypes with markers not previously observed in that hybrid complex of the unique tidal freshwater habitat should be avoided in replantation projects.     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars

Summary We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.Journal series No. 3692 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Molecular analysis of Helicobacter pylori populations in antral biopsies from individual patients using randomly amplified polymorphic DNA (RAPD) fingerprinting

Abstract In the present study, randomly amplified polymorphic DNA (RAPD) fingerprinting has been used to analyse multiple single colony isolates of Helicobacter pylori from antral biopsies in an attempt to ascertain whether or not multiple strains are present in individual patients using single biopsy samples. The RAPD fingerprints derived from single colonies obtained from the same biopsy specimen were in all cases indistinguishable. The previously noted heterogeneity between H. pylori strains from different individuals was confirmed. RAPD fingerprinting, combined with a simple method of template preparation, was shown to be an excellent method for H. pylori strain differentiation. The results of this study indicate that the H. pylori population is homogeneous in individual patients at a single gastric site.     

长筒石蒜居群遗传多样性RAPD分析

利用RAPD标记对长筒石蒜3个居群的遗传多样性及分化程度进行了研究。12条随机引物扩增出94个可分析位点,多态位点比率(PPB)为65.96%,表明长筒石蒜具有比较高的遗传多样性。经POP-GENE32分析表明:Nei’s基因多样性指数(h)为0.1897,香农多样性指数(Ⅰ)为0.2945,基因分化系数(GST)为0.1191,基因流(Nm)为3.6980。经WINAMOVA分析表明:居群内遗传变异占71.75%,而居群间只占28.25%。遗传多样性分析表明,各居群的遗传多样性水平由高到低为琅琊山居群>宝华山居群>盱眙居群。遗传分化表明:长筒石蒜各居群间遗传分化程度较低;大部分遗传变异存在于居群内部,表明其具有较强的进化潜力,自然情况下不会处于濒危状态,野生种质资源的破坏,主要来自于人为干扰。