Effect of DNA preparation from laver (Porphyra yezoensis) thalli on reproducibility of RAPD (random amplified polymorphic DNA) patterns

Laver (Porphyra yezoensis) DNAs were extracted from thalli with five different procedures and used for RAPD (random amplified polymorphic DNA) analysis as templates. Restriction enzyme-digestive DNAs were obtained with all procedures examined. However, RAPD patterns generated with these DNAs appeared highly irreproducible and were considerably different from each other. When DNAs purified with CsCl gradient centrifugation were used for RAPD analysis as templates, highly reproducible RAPD patterns were obtained, suggesting that unpurified DNAs extracted from thalli with all five extraction procedures contained an excess of RNA, polysaccharides and/or other materials which affected the RAPD reproducibility. Thus, results indicated that purification of DNA is essential to produce reproducible RAPD patterns of Porphyra DNA. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Nuclear and genomic changes in floating and submerged buds and leaves of heterophyllous waterchestnut (Trapa natans)

The heterophyllous macrophyte waterchestnut ( Trapa natans L.) produces floating and submerged leaves which are strikingly different in morphology. In the present work, we compared, through cytological, cytochemical and immunocytochemical analyses, the nuclear features of the shoot meristem in floating and submerged buds that produce the different leaves. The occurrence of DNA polymorphism in the two types of leaves was studied by using the random amplified polymorphic DNA (RAPD) approach. The light microscope study of chromatin organization revealed an articulate structure of the nuclei, with prominent chromocentres in both floating and submerged buds. However, the meristematic nuclei of floating buds, as compared to those of submerged buds, displayed peculiar features which seem to be related to a higher proportion of A-T-ricn sequences and a higher level of DNA methylation of the nuclear genome. Moreover, RAPD analysis showed polymorphism between the genomic DNAs of floating and submerged leaves. These results show that organs of an individual plant living in different environmental conditions undergo changes in genome organization which may predispose those organs to function more effectively in different environments.     

随机扩增杂交微卫星方法在大仓鼠种群遗传多态性研究中的应用

随机扩增杂交微卫星(Random amplified hybridization microsatellites,RAHM)是一种复合RAPD扩增和寡核苷酸扫描的方法。该方法能够从RAPD产物凝胶上获得更多的信息,具有分析方法快速,高敏感性,能检测到高水平的多态性等优点。RAHM方法通过对RAPD扩增的DNA片段进行微卫星杂交来替代限制性内切酶对基因组DNA的消化,有助于揭示微卫星基因组克隆,进行微卫星引物的筛选。本文采用随机扩增杂交微卫星方法来检测小型哺乳动物大仓鼠(Techerskia triton)种群的遗传多态性,结果表明RAHM方法能够检测到大仓鼠种群中较高的多态性以及种群间的差异,这些条带模式可能代表真核基因组中另一种多态性标记的来源,可用于检测小型哺乳动物种群的遗传多态性。     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

Mutagenesis of Embryogenic Cultures of Soybean and Detecting Polymorphisms Using RAPD Markers

Embryogenic suspension cultures of soybean (Glycine max L. cv. Iroquois) were subjected to mutagenesis using varying concentrations (1, 3, 10, and 30 mM) of ethyl methanesulfonate (EMS). Depending on the concentration of EMS used, the mean survival rate of embryogenic cultures decreased from 74 % (1 mM EMS) to 43 % after 30 mM EMS treatment. Random amplified polymorphic DNA (RAPD) analysis was used to determine whether induction of genetic variability in embryogenic cultures in response to the different EMS treatments may result in identification of polymorphic markers. Two of 35 ‘core’ primers tested revealed polymorphisms. One of the primers, OPO-01/1150, revealed polymorphism in tissue treated with 10 mM EMS, while the other primer, OPO-05/1200, revealed polymorphism in tissue treated with either 1 or 30 mM EMS. These results suggest that RAPD markers are useful in detecting mutations in embryogenic cultures of soybean. This revised version was published online in July 2006 with corrections to the Cover Date.     

不同经济类型高粱的RAPD分析

对四种不同经济类型高粱采用ＤＮＡ随机扩增多态技术（ＲＡＰＤ）进行了比较研究。１８种引物扩增后共得到５４个ＲＡＰＤ标记,其中２３个标记在进行４种高粱成对比较时呈多态。根据计算出的遗传距离值可以发现虽然不同种高粱之间差异虽然较小,但仍具有丰富的遗传多样性。     

10种冬青属植物遗传多样性RAPD和AFLPs分析

采用RAPD和AFLP技术,对10种冬青属植物基因组进行DNA片段扩增,以研究该属种间遗传多样性.结果表明:在RAPD分析中,通过对100种10个碱基随机引物的筛选,发现11种引物能得到多态性较高扩增产物,11种引物共扩增出301条多态性条带,多态率为98.63%.在AFLP分析中,3对选择性引物组合均扩增出了丰富的多态性片段.利用RAPD和AFLP技术分析,结果按UPGMA类平均法进行聚类,聚类结果显示冬青和代茶冬青,木姜冬青和浙江冬青以及光枝刺缘冬青与毛枝三花冬青之间的亲缘关系最近.     

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.     

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     

阴道分离因肯毛孢子菌的分子鉴定和种内分型

分别采用rRNA基因内转录间隔区(ITS)和基因间隔区(IGS1)测序,ITS和IGS1区PCR限制性片段长度多态性分析(PCR-RFLP)和基因组DNA的随机扩增多态性DNA(RAPD)等方法,对三株因肯毛孢子菌Trichosporon inkin进行分子特征及种内分型研究。结果显示,不同菌株的rRNA基因ITS区和IGS1区的序列相似性均高达100%,RFLP酶切图谱具有较理想的种内一致性,而不同菌株的RAPD图谱不尽相同。研究表明:rRNA基因IGS1区测序及RFLP酶切可考虑用于因肯毛孢子菌的菌种分子鉴定,而基因组DNA的RAPD则较适合于菌种的种内分型。     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

Modified AFLP technique for rapid genetic characterization in plants

The standard amplified fragment-length polymorphism (AFLP) technique was modified to develop a convenient and reliable technique for rapid genetic characterization of plants. Modifications included (i) using one restriction enzyme, one adapter molecule and primer, (ii) incorporating formamide to generate more intense and uniform bands and (iii) using agarose gel electrophoresis. Sea oats (Uniola paniculata L.), pickerel-weed (Pontederia cordata L.), Bermudagrass (Cynodon dactylon L.) and Penstemon heterophyllus Lindl. were used to determine the ability to generate adequate resolution power with both self- and cross-pollinated plant species including cultivars, ecotypes and individuals within populations. Reproducibility of bands was higher in all the AFLP experiments compared to random amplified polymorphic DNA (RAPD). Formamide with or without bovine serum albumin improved band intensities compared to dimethyl sulfoxide and the standard reaction mixture with no organic solvents. Comparison between RAPD and modified AFLP using sea-oats population samples proved that modified AFLP exhibits (i) a low number of faint bands with increased specificity of amplified bands, (ii) a significantly higher number of polymorphic loci per primer, (iii) less primer screening time, (iv) easy scoring associated with fewer faint bands and (v) greatly enhanced reproducibility. The technique described here can be applied with a high degree of accuracy for plant genetic characterization.     

Rapid and high quality DNA isolation from Origanum onites for RAPD and ISSR analysis

Origanum onites is an economically important medicinal plant with high essential oil content. Lack of an appropriate DNA isolation procedure is a limiting factor for any molecular study of this plant. We have used a protocol for genomic DNA isolation based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method involves mortar grinding of leaf tissue, modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone, and successive isoamyl alcohol/chloroform extractions. The yield was approx. 20 microg DNA per 200 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in restriction digests, inter simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) reactions. This extraction method should facilitate the molecular analysis of Origanum chemotypes.     

Avirulence gene and insertion element-based RFLP as well as RAPD markers reveal high levels of genomic polymorphism in the rice pathogen Xanthomonas oryzae pv. oryzae

Genetic polymorphism within the genomes of bacterial pathogens determines their evolutionary potential during long-term interaction with their hosts. To investigate the level of genetic variation in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of rice bacterial blight disease, three DNA marker systems, including (i) restriction fragment length polymorphism (RFLP) of the avrBs3/PthA family genes (avrXa27), (ii) RFLP of insertion (IS) elements and (iii) random amplified polymorphic DNA (RAPD) markers, were used to detect polymorphism among 32 Xoo strains that differed in their virulence patterns. All these strains contained multiple avrXa27 homologs that were variable in copy number and genomic location. RFLP of six IS elements revealed that these mobile sequences were abundant in Xoo genomes, with 150 of the total of 165 discernable markers being variable. Thirty-eight decamer primers of RAPD amplified a total of 691 bands, with 100% of them being variable. In addition, analysis of molecular variance (AMOVA) of data from RFLP analysis of IS elements and from RAPD analysis showed that most of the genetic variation residues were within Xoo populations, rather than between populations. Although all three DNA marker systems supported that substantial variation was maintained in Xoo genomes, Mantel tests did not identify significant correlation between the similarity coefficients calculated from them. The results of the present study indicated that Xoo genomes contain a high level of genetic polymorphism, which greatly facilitates the evolution of this important pathogen during interaction with its host rice plant.     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

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.     

AFLP fingerprinting reveals pattern differences between template DNA extracted from different plant organs.

AFLP (amplified fragment length polymorphism) fingerprinting of cultivars of bread wheat (Triticum aestivum) and some of its wild relatives has allowed the efficient detection of large numbers of polymorphic amplified fragments. While the reproducibility of fingerprints in repeated experiments is high, pattern differences were observed between fingerprints obtained from seed and leaf DNA template from the same wheat accession. These distinct organ specific amplified DNA fragments were shown to be due neither to genotypic mixtures nor to pathogen contamination. They are likely a result of differences in DNA methylation between organs. Even greater numbers of organ specific amplified fragments were observed when fingerprints obtained from the root and shoot of individual seedlings of the wheat relatives Aegilops mutica and Aegilops speltoides were compared. This phenomenon underlines the importance of ensuring that DNA is extracted from physiologically uniform tissue in phylogenetic studies based on AFLP fingerprints. For this purpose, mature seed is a convenient source.