Analysis of Genetic Diversity amongIxoraCultivars (Rubiaceae) using Random Amplified Polymorphic DNA

We have optimized the genomic DNA extraction method from freshand dry laminas, as well as fresh and dry corolla lobes ofIxoracultivars.Some woody tropical species such as these contain excessiveamounts of phenolic compounds that co-precipitate with DNA resultingin poor or no amplification during the polymerase chain reaction(PCR). Repeated precipitation with CsCl coupled with phenol:chloroformextraction yielded high quality DNA suitable for consistentPCR amplification. The DNA from fresh laminas of 22 cultivarsofIxorawas subjected to random amplified polymorphic DNA (RAPD)analysis. Individual taxa could be identified using specificDNA markers from the RAPD profiles. Cluster analysis of datafrom six primers grouped all 22 cultivars distinctly under twocultivar groups, viz.,IxoraCoccinea andI.Javanica. The percentagegenetic similarity was calculated for all the cultivars basedon the RAPD data. The two cultivar groups and the outgroup plantswere also clearly distinguishable with polar ordination usinga matrix of genetic dissimilarities (one minus similarity).Our data indicate that besides the use of RAPD markers for identificationof particularIxoracultivars within a germplasm collection, thephylogenetic relationships generated by RAPD analysis may beuseful for future breeding programmes. IxoraL. cultivars; Rubiaceae; RAPD fingerprinting; DNA extraction; woody tropical species     

The effect of shade on leaf structure and physiology of tree seedlings from a mixed dipterocarp forest

This study builds upon past work investigating seedling leaf physiology and structure among tropical trees. We seek to explain how related and unrelated species and genera co‐occur in relation to varying amounts of shade. Seedlings of eight Sri Lankan rain forest tree species in three genera (Dipterocarpus, Mesua, Shorea section Doona) were grown for 2 years in four treatments that simulated a variety of shade environments across the understorey of a rain forest. All three genera comprise major canopy tree species of mixed dipterocarp forest, a widespread and important Asian tropical forest type. Compared with the other genera, Dipterocarpus spp. had the largest leaves, the thinnest leaf blades and relatively high rates of stomatal conductivity across all shade treatments, making them water‐loving species sensitive to droughty soils. Mesua spp. had intermediate sized leaves, with the thickest leaf blades and palisade mesophyll layers, the highest stomatal densities, the smallest aperture sizes and the lowest rates of stomatal conductance, making them the most water conservative. Shorea spp. were generally intermediate in blade and palisade mesophyll dimensions between Dipterocarpus spp. and Mesua spp., but they had the smallest leaves. Greater differences among genera than among species within genera were apparent, but species differences within genera were also apparent. Differences among genera and species conform to their known successional status and topographical affinities and provide a more comprehensive understanding of species site adaptation. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 332–343.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

AN EVALUATION OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) FOR THE IDENTIFICATION AND PHYLOGENY OF FRESHWATER SNAILS OF THE GENUS BULINUS (GASTROPODA: PLANORBIDAE)

The Random Amplified Polymorphic DNA (RAPD) assay was used tostudy genetic variation within and between 9 species of thegenus Bulinus and to determine whether RAPD profiles could beused as markers for identification purposes. RAPDs were generatedwith 8 primers of two different sizes (l0mers & 15mers)and were visualised using both polyacrylamide gel electrophoresis(PAGE) with silver staining and agarose gel electrophoresiswith ethidium bromide staining. The species groups of Bulinushad few similarities in their RAPD profiles and there was interspecificvariation within groups. Intrapopulation variation was observed,with all primers, for B globosus collected from a single sitein Zimbabwe PAGE/silver staining methods visualised a greaternumber of RAPDs in comparison with agarose/ethidium bromidemethods. Phenetic analysis indicated that distance estimatesbetween taxa were sometimes non-additive and the phylo-geneticanalysis of such non-metnc data is discussed. The resultantphenograms, constructed using a least squares method, were constrainedalmost into a polytomy with topologies often differing betweendata sets. It was concluded that this phenomenon was most likelyattributable to large nucleotide divergences between the speciesgroups which go beyond the phylogenetic scope of RAPD analysis.RAPD profiles, when used in conjunction with other taxonomicmethods, may contribute to the identification of species ofBulnus on a regional basis, but the observed variability ina natural population suggests that a diagnostic RAPD profilefor each species throughout its geographic range is unlikely. (Received 19 April 1995; accepted 1 September 1995)     

Molecular phylogeny of Dipterocarpaceae in Southeast Asia using RFLP of PCR-amplified chloroplast genes

Dipterocarpaceae is the dominant family of Southeast Asia's climax tropical rain forest region, and it contains the region's most important commercial timber species. A molecular phylogeny of the Dipterocarpaceae subfamily Dipterocapoideae was constructed using restriction fragment length polymorphisms of polymerase chain reaction-amplified specific genes in chloroplast DNA. A total of 141 site changes were detected among ten genera and 30 species in 11 different genes: rbcL, psbA, psbD, rpoB, rpoC, petB, atpH, 16S, psaA, petA and trnK. Phylogenetic trees constructed by Wanger parsimony and neighbor-joining methods, using Upuna as the outgroup, displayed five monophytelic groups that included Upuna: HopeaShorea-Parashorea-Neobalanocarpus; Dryobalanops; Dipterocarpus; Anisoptera-Vatica-Cotylelobium; and Upuna. The phylogenetic trees clearly separate species with two different base chromosome numbers: the first group is x=7, and the other is x=11. The x=7 group is thought to be in a synapomorphic character state. Parashorea lucida is a sister to most Shorea species. Neobalanocarpus heimii and Hopea from a clade of a sister to two Shorea species, and Cotylelobium and Vatica are closely related species. Our conclusions agree with a phylogeny derived from wood anatomy data analysis, and with Symington's and Ashton's taxonomic classifications.The raw data of the PCR-RFLP analysis can be obtained from the authors     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

用RAPD技术对特化等级裂腹鱼类亲缘关系的探讨

为确定特化等级裂腹鱼类的遗传分化关系 ,对 7种 2 2个裂腹鱼个体进行随机扩增多态DNA(RAPD)研究。从所使用的 40个随机引物中 ,选择了 37个扩增带谱清晰的引物进行分析。根据构建的分子系统树显示 ,特化等级裂腹鱼类划分为 3个属级分类单元较为合适 ,这与采用形态学特征进行系统分析所得出的结论一致。在 2 2个个体之间遗传距离矩阵中 ,最大的遗传距离指数达到了 90 38% ,此外 ,还有较多的遗传距离指数也在 6 0 %～ 90 %之间。通过分析 ,认为用RAPD标记技术来分析属级或属级以上分类单元的亲缘关系 ,具有一定的适用性。对于一些遗传差异较大的类群 (如裂腹鱼类 ) ,应用属级或属级以上分类单元的亲缘关系 ,很可能会出现个体之间遗传距离接近或达到 10 0 %的情况 ,从而无法探讨其相互之间的亲缘关系。     

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.     

五种索科线虫RAPD亲缘关系分析

采用RAPD技术构建了索科线虫4属5种的指纹图谱。从47个引物中筛选出12个稳定性好、多态性高的引物,共扩增出161条谱带,其中150条谱带具遗传多态性,占93·17%。所获片段长度大小为200~3200bp,单个引物扩增的条带数在11~16之间,平均为13·42条。采用RAPDistance软件及MEGA程序,计算Nei氏相似系数和遗传距离,建立UPGMA和NJ聚类图,两个聚类图拓扑结构相同,将5种索科线虫分为两大分支:同属于蚊幼寄生罗索属线虫的食蚊罗索线虫(Romanomermisculicivorax)与武昌罗索线虫(R.wuchangensis)亲缘关系最近,先聚在一起,再与同翅目(Homoptera)寄生长沙多索线虫(Agamermischang-shaensis)聚为一支;鳞翅目(Lepidoptera)寄生中华卵索线虫(Ovomermissinensis)和同翅目寄生两索属线虫(Amphimermissp·)亲缘关系较近,两者聚为一支。5种索线虫属内种间的遗传距离较小,食蚊罗索线虫与武昌罗索线虫之间遗传距离仅为0·1789;而属间遗传距离较大,在0·4471~0·5488之间。上述结果表明:RAPD技术可以应用于索科线虫亲缘关系的分析,能够反映出不同线虫间的遗传差距,从而成功地进行属、种的分类及进化问题研究。     

Construction of phylogenetic tree forNicotiana species based on RAPD markers

To apply random amplified polymorphic DNA for analysis of phylogenetic relationships, we used 34 synthetic oligonucleotides as primers to examine interspecific and intraspecific variations among 18 genotypes, nine species ofNicotiana. The nine species used in this study belong to sectionsTomentosae andAlatae. In addition, we attempted to clarify the taxonomic position ofN. sylvestris. A total of 354 distinct DNA fragments were obtained by polymerase chain reaction. Pair-wise comparisons of unique and shared amplification products were used to generate Jaccard's similarity coefficients and Nei and Li's similarity coefficients with the computer software of numerical taxonomy and multivariate analysis system. On the basis of the dendrogram constructed with the similarity coefficients, the 18Nicotiana genotypes were divided into two clusters. The classification analyzed by RAPD markers is in accordance with the classification of Goodspeed thatN. sylvestris is a member of sectionAlatae.     

Genetic Relationships within Tripsacum as Detected by RAPD Variation

Genetic diversity within species of Tripsacum was surveyed basedon randomly amplified polymorphic DNA (RAPD) variation, as detectedwith the polymerase chain reaction (PCR). Thirteen of the 16Tripsacum species, including both temperate and tropical species,were included in this study using 56 decamer oligonucleotideprimers. All of the 56 primers generated repeatable RAPD profilesand 53 of them detected polymorphic bands among the Tripsacumspecies. These 53 primers generated 350 repeatable bands rangingin size from 150–1600 bp, each primer generating an averageof seven scoreable bands. Cluster analysis of polymorphic RAPDsindicated four major clusters. Cluster 1 consists of North AmericanTripsacum species, cluster 2 consists of South American Tripsacumspecies, cluster 3 includes T. zopilotense and T. latifoliumfrom Mexico, and cluster 4 consists of Mesoamerican Tripsacumspecies. Cluster analysis does not reveal the division of twotaxonomic sections (Fasciculata and Tripsacum). Copyright 1999Annals of Botany Company Tripsacum, RAPD, genetic relationships, genetic diversity.     

Seed Surface Architecture and Random Amplified Polymorphic DNA Profiles of Paulownia fortunei, P. tomentosa and their Hybrid

The surface patterns of winged seeds of Paulownia fortunei,P. tomentosa and P. fortuneixP. tomentosa were examined by scanningelectron microscopy. The pattern of reticulation on the wingsand seed coat of P. fortunei and the hybrid are comparable,while that on P. tomentosa is different and more elongated.Also, the wings are more extended at the oblong ends of theseeds in the former when compared to the wings of P. tomentosa.Distinct random amplified polymorphic DNA (RAPD) patterns wereobtained for the three taxa and P. kawakamii with five differentrandom oligonucleotide primers, suggesting that the method canyield genetic markers for differentiating the taxa. Also, Southernblot analyses of the RAPD products of the hybrid and the twoparent species revealed shared (inherited) genetic polymorphisms.Copyright 1999 Annals of Botany Company Paulownia species and hybrid, seed surface architecture, reticulated thickening, RAPD markers, Scrophulariaceae.     

Identification and relationships of cultivated accessions fromLolium-Festuca complex based on RAPD fingerprinting

Randomly Amplified Polymorphic DNA (RAPD) technique with 15 arbitrary primers was used to identify and reveal relationships of the accessions comprising 4 species ofLolium-Festuca complex. Altogether 252 RAPD markers were considered in statistical treatment, 60 of which could be identified as potentially taxon-specific. All analyzed taxa were fully distinguishable using RAPD markers.Lolium-Festuca relationships based on RAPD data were evaluated using cluster analysis (UPGMA) and principle coordinate analysis (PCO). The results of UPGMA as well as PCO performed on data pooled from all RAPD profiles support separation of the two generaLolium andFestuca.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

用RAPD技术探讨抗病草鱼F_7(83-2系鱼F_2)与原代双亲的亲缘关系

用RAPD技术对抗病草鱼F7(83-2系鱼F2)与原代亲本草鱼、兴国红鲤进行亲缘关系的研究.在事先优化的反应条件下,在使用的60个随机引物中,有10个引物扩增出清晰稳定的片段,共计161条,大小在200 bp～2 500 bp之间.3种鱼均有其特异性扩增片段,可作为种类鉴别的依据.根据BAPD data analyzer 1.3v软件所得数据,用UPGMA和NJ程序构建的分子系统树显示:抗病草鱼F2与草鱼的亲缘关系较近、与兴国红鲤的亲缘关系较远.两种聚类的结果相一致.研究结果与借助形态学和生化特征进行的传统系统分析具有一致性.在3个群体之间遗传距离矩阵中,抗病草鱼F,与草鱼的平均遗传相似度为0.6801,与兴国红鲤的平均遗传相似度为0.5961.通过分析,认为RAPD技术对分析杂交选育鱼的亲缘关系具有一定的适用性.     

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.     

Determination of RAPD Markers in Rice and their Conversion into Sequence Tagged Sites (STSs) and STS-Specific Primers

We produced 102 randomly amplified polymorphic DNA (RAPD) markersmapped on all 12 chromosomes of rice using DNAs of cultivarsNipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers10 nucleotides long were used both singly and in random pairsand about 1,400 primer-pairs were tested. Using both agarosegel and polyacrylamide gel electrophoresis enabled us to detectpolymorphisms appearing in the range from <100 bp to 2 kb.The loci of the RAPD markers were determined onto the frameworkof our RFLP linkage map and some of these markers were mappedto regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs weredetermined by cloning, identifying and sequencing of the mappedpolymorphic fragments.     

RAPD Analysis of Pathogenic Variability in Ascochyta rabiei

Thirty Italian isolates of the phytopathogenic fungus Ascochyta rabiei (Pass.) Labr., the causal organism of Ascochyta blight on chickpea (Cicer arietinum L.), were analysed by a random oligonucleotide primer dependent polymerase chain, reaction (PCR) technique called random amplified polymorphic DNA analysis (RAPD) using three decamer primers. In previous investigations these isolates had been differentiated in six pathogenic groups. RAPD results were summarized in an analysis using the program PAUP. With each of the primers several amplification products were observed which were common to all isolates. The results of the RAPD analyses also showed that all isolates could be identified by a unique RAPD pattern. No correlation between RAPD patterns and the division of the isolates in pathogenic groups could be established. The application of the RAPD technique for cataloguing isolates and to obtain specific genetic markers for all isolates of the species Ascochyta rabiei is discussed.