DNA preparation method in eggs, immature stages, and diapausing females of Tetranychus spider mites (Acari: Tetranychidae) for diagnostic PCR-RFLP

A DNA preparation method for diapausing females, immature stages, and eggs of spider mites for diagnostic analyses using restriction fragment length polymorphisms after polymerase chain reaction (PCR-RFLP analysis) of the internal transcribed spacer (ITS) region including 5.8 S of the nucleic ribosomal DNA was developed using Tetranychus urticae Koch as a typical example of tetranychid mites. In diapausing females and deutonymphs, the method of crushing an individual mite with a glass rod was eminently successful and a useful method to obtain the DNA template for PCR. For protonymphs, larvae, and eggs older than 48?h after oviposition at 24?°C, the DNA preparation method for a single nematode, crushing a tiny sample with a filter paper chip, was also successful and a useful method to obtain the DNA template from mite individuals (eggs) for PCR. Consequently, we found a single distinctive band of an amplified DNA fragment after electrophoresis in all developmental stages, and digestion of the PCR product by a restriction endonuclease, DraI, resulted in identical banding patterns being exhibited in all developmental stages. Our findings indicate that PCR-RFLP analysis of the ITS region can be used for species identification of diapausing females, immature stages, and eggs of Tetranychus species.     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

Mitochondrial COI gene transfers to the nuclear genome of Dendroctonus valens and its implications

Mitochondrial gene transfer to the nuclear genome could affect the accuracy of results in population genetics and evolutionary studies using mitochondrial gene markers. In a population genetics study of the red turpentine beetle (Dendroctonus valens), an invasive species in China, we found numerous ambiguous sites existing in the Cytochrome Oxidase I (COI) gene sequences obtained directly from polymerase chain reaction (PCR) products amplified from total genomic DNA using universal primers. By comparing the profiles of restriction endonuclease digestions and the sequences of PCR products amplified from mitochondrial DNA and nuclear DNA of the same individuals, we confirmed it was a phenomenon of mitochondrial gene transfer to the nuclear genome. Large numbers of COI pseudogenes were detected in this species. According to different levels of condon position bias and phylogenetic analysis, these should have originated from independent integration events. The impact of nuclear mitochondrial DNA sequences on population genetics analyses was discussed.     

Anatomy of mitochondrial DNA from Paramecium aurelia

Summary The linear genome of mitochondrial DNA from four species of Paramecium aurelia was investigated with respect to restriction endonuclease fragments, location and number of ribosomal RNA genes, and interspecies EcoRI and HindIII fragment homologies. One copy of each of the rRNA genes was found in all four species and the 14s and 20s rRNA genes were separated by at least 3,000 bp. R-Loop analysis of the 20s rRNA gene did not reveal the presence of an intervening sequence. Interspecies homology studies showed species 1, 5, and 7 to have a high degree of homology but species 4 was less than 50% homologous to species 1 mt DNA. For all four species, rRNA genes showed good homology indicating that these DNA sequences are highly conserved, even between species having many non-homologous regions. A major region of DNA which displayed little homology between species 1 and 4 was that fragment containing sequences essential for initiation of DNA replication.     

DNA analysis methods for recognizing species invasion: the example of Codium,and generally applicable methods for algae

Analysis of DNA can help to distinguish those morphological characters indicative of species difference from those representing retained traits or parallel evolution. This can be of great value in detecting recent invaders. The choice of which DNA characters to examine not only dictates the methodology to be used but must also be appropriate for the detection level sought. Restriction endonuclease fragment comparisons of plastid DNA have been used to assess Codium species; the results show C. fragile subsp. tomentosoides from east and west coast North America to be identical while sympatric endemic Codium species each display their own unique set of fragments. For species of other algae, plastid DNA fragment patterns are not necessarily identical across a morphological species, e.g. Pandorina morum. Such repetitive element probes as M13 and the use of RAPDs are more appropriate for analysis of populations within species. DNA base sequence comparisons of nuclear rDNA genes often yield too few variant bases between closely related species for reliable identifications. Analysis of the more variable Internal Transcribed Spacer (ITS) region, lying between the small and large ribosomal subunit genes in nuclear DNA, yields more extensive base pair variation between species and relatively little within species; it may be an alternative choice for endonuclease restriction fragment analysis or for sequencing.     

Differentiation of phytoplasmas associated with sugarcane and gramineous weed white leaf disease and sugarcane grassy shoot disease by RFLP and sequencing

 Restriction fragment length polymorphism (RFLP) and sequencing were used to elucidate the genetic relationship between phytoplasmas that cause white lead disease and grassy shoot disease in sugarcane and white leaf disease in gramineous weeds found in the cane-growing areas (Crowfoot grass, Bermuda grass and Brachiaria grass). A 1.35-kb DNA fragment encoding for the 16s rRNA was amplified by PCR using universal primers and analysed by digestion with nine restriction endonucleases. A DNA fragment containing the 3′ end of the 16s rRNA and the spacer region between the 16s rRNA and the tRNA(Ile) was amplified by PCR and sequenced. Analysis of the RFLP patterns and of the sequence showed that grassy shoot and white leaf diseases in sugarcane are caused by two different phytoplasmas. Sequence analysis of phytoplasma DNA obtained from three species of weeds showing symptoms of white leaf disease failed to detect any organism that is identical to those infecting the sugarcane. Moreover the phytoplasma species that infect the three types of gramineous weeds, although closely related, are nevertheless different Received: 15 April 1997 / Accepted: 18 April 1997     

SstII polymorphic sites in the promoter region of the human cystatin C gene

Summary By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85 bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.     

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels. Accepted: 6 September 1996     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Summary Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm^–3 in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2–30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).     

Sequence-tagged-sites (STSs) of cDNA clones in Cryptomeria japonica and their evaluation as molecular markers in conifers

 We have generated 66 sequence-tagged-site (STS) markers from cDNA clones of Cryptomeria japonica, and 60% of them have already been mapped into C. japonica linkage groups. All of the STS markers showed a single fragment following polymerase chain reaction (PCR) amplification. We investigated by polymorphism of these STS markers in a mapped F₂ population and 15 plus trees by means of a restriction endonuclease analysis. Polymorphism levels were 10.6% and 22.7% in the F₂ population and the 15 plus trees, respectively. PCR amplification levels of the 66 STS markers in 14 conifer species varied depending on their genetic relationship with C. japonica. Taxodium, which is closely related to C. japonica, had the most amplifications (31.82%), followed by Sequoiadendron giganteum, which is of the same family. The average proportion of PCR amplifications in each family gradually declined in the following order: from Taxodiaceae to Cuppresaceae, Sciadopityaceae, Pinaceae, and Taxaceae. These results are in general agreement with a molecular phylogenetic relationship based on chloroplast DNA. The 66 STS markers will be useful as on anchor point for genome mapping and population genetics, and some of them will also be useful when studying other conifers. Received: 8 September 1996 / Accepted: 8 November 1996     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Variation in the psbC gene region of gymnosperms and angiosperms as detected by a single restriction site polymorphism

 This paper reports on a PCR-RFLP analysis in a chloroplast DNA region consisting of coding and intergenic spacer sequences of trnS and the adjacent psbC gene. This region was PCR-amplified in 62 woody plant species, predominantly tree species, that represent a broad systematic range in both gymnosperms and dicotyledonous angiosperms. The amplification products were digested by the restriction endonuclease HaeIII (GG↓CC). Fourteen different restriction patterns occurred, 5 of which characterised representatives of the gymnosperms, and 9 angiosperm representatives. A single restriction site polymorphism revealed most of the species to share restriction patterns. Groups formed which showed relationships to plant systematic units. This phenomenon is discussed with regard to the psbC gene and the GGCC motif for tracing species’ relationships on a high taxonomic level of gymnosperms and angiosperms. Received: 5 June 1996/Accepted: 20 September 1996     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

登革病毒NS3全基因的扩增与克隆

登革热(ＤＦ)、登革出血热及登革休克综合征(ＤＨＦ/ＤＳＳ)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区。ＤＨＦ/ＤＳＳ以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题。迄今,ＤＨＦ/ＤＳＳ的发病机制仍不清楚,亦无有效的特异性预防方法[1]。登革病毒属于黄病毒科的黄病毒属,有Ⅰ、Ⅱ、Ⅲ、Ⅳ四个血清型,基因组为单股正链ＲＮＡ,全长约11ｋｂ,编码三种结构蛋白和七种非结构蛋白。基因组顺序为5′ＣＰｒＭＥＮＳ1ＮＳ2ａＮＳ2ｂＮＳ3Ｎ…     

Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

The objective of this study was to determine the genetic relatedness among the Cercospora and Pseudocercospora species closely related to Cercospora apii by using a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the internal transcribed spacer (ITS) region. A single PCR fragment (about 550 bp) was obtained from all Cercospora species categorized as the C. apii-group, Pseudocercospora purpurea, Pseudocercospora conyzae, and Pseudocercospora cavarae. Cercospora caricis yielded a 680 bp PCR fragment. The similarity in the PCR fragment size and RFLP profiles among the C. apii-group isolates, including Pseudocercospora purpurea, and Pseudocercospora conyzae strongly suggests that these species are conspecific. Synonymy with C. apii (lectotype) at a subspecific rank has been proposed. Amplified ITS regions of genomic DNA extracted from spinach leaves showing 12 and 233% leaf spot disease symptoms caused by Cercospora beticola yielded two PCR fragments (i.e., one from the fungus and one from the host plant) and were resolved by electrophoresis of the PCR product in 3% LMP agarose. Digestion of the total PCR product with HinfI restriction enzyme yielded RFLP profiles similar to those obtained from amplified DNA from the causative agent, C. beticola. The method described in this preliminary study offers rapid detection and diagnosis of fungal infections in plants for disease prediction and management and screening of plant materials for quarantine purposes.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.