PCR-RFLP of the mitochondrial cytochrome oxidase (subunit I) gene provides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae)

Adult and larval identification of Diabrotica can be difficult. Some adult identifications require considerable taxonomic experience while larvae of many Diabrotica species are morphologically indistinguishable. This study was conducted to determine whether 12 pest and non-pest Diabrotica species could be separated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A 1308 bp portion of the mitochondrial cytochrome oxidase subunit I gene (COI) was amplified using PCR and digested using several restriction endonucleases. Double digests of COI amplicons with AluI and MspI resolved on polyacrylamide gels revealed several diagnostic inter- and intraspecific polymorphisms. A key to the 12 species was constructed using the PCR-RFLP patterns.     

Cytochrome P450 and actin genes expressed in Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene conversion and evolution.

Molecular phylogenetic analysis was conducted using conserved cytoplasmic actin and diversified cytochrome P450 (P450) sequences isolated from Helicoverpa zea and Helicoverpa armigera, two species thought to be closely related based on allozyme analyses. These sequences were compared in turn with published sequences from other insects to gain insight into how different gene families evolve. In Bombyx mori and these Helicoverpa species, cytoplasmic actin genes are present as a pair of tandemly duplicated paralogs with coding sequence identities as high as 95.5% (B. mori), 98.9% (H. zea) and 98.5% (H. armigera) due to recent 5'-polar gene conversions. Phylogeny and interspecies comparisons assign the six actin genes into two orthologous groups: HaA3a/HzA3a/BmA3 and HaA3b/HzA3b/BmA4, which exhibit more similarities between H. zea and H. armigera than between Helicoverpa species and B. mori. Like the actin genes in H. zea, four CYP6B genes exist as two pairs of duplicated paralogs with recent 5'-polar gene conversions. Interspecific comparisons and phylogeny analysis identified three groups of orthologous CYP6B genes: H. zea CYP6B8 or CYP6B28/H. armigera CYP6B7, H. zea CYP6B27/H. armigera CYP6B6, and H. zea CYP6B9/H. armigera CYP6B2/Heliothis virescens CYP6B10. The low degree of divergence in the first two of these groups is comparable to allelic variation within a single species. These orthologous relationships and the high degrees of similarity in both actin and P450 genes strongly indicate that these Helicoverpa species are extremely closely related.     

Using COI gene sequence to barcode two morphologically alike species: the cotton bollworm and the oriental tobacco budworm (Lepidoptera: Noctuidae)

Due to limited morphological difference, the two closely related sister species, the cotton bollworm, Helicoverpa armigera (Hübner) and the oriental tobacco budworm, H. assulta (Guenée) (Lepidoptera: Noctuidae), are very difficult to distinguish, especially at the larvae stage. Recently, DNA sequence has been widely used as a bio-barcode for species identification. In this study, we attempted to distinguish H. armigera and H. assulta using the mitochondrial cytochrome C oxidase subunit I gene (COI) gene sequence as the barcode. We determined a 658 bp segment of the COI gene for 28 individuals of H. armigera, 8 individuals of H. assulta, and 10 individuals of Mamestra brassicae (as the outgroup) in Yunnan Province, southwest of P. R. China, together with one H. assulta and two H. armigera reported sequences from GenBank. Twenty-three haplotypes were identified in all 49 samples. As expected, network analysis of the haplotypes of the three species presented a clustering pattern consistent with the respective species status. Haplotypes of the same species differed from each other by no more than three nucleotide substitutions. However, each haplotype of H. armigera differed from that of H. assulta by at least 22 nucleotide substitutions. Both species differed from M. brassicae by more than 50 nucleotide substitutions. 17 unique diagnostic nucleotides were also used to discriminate the two species. The finding of large COI sequence differences between H. armigera and H. assulta suggested that it could be used to distinguish the two morphologically alike species and be employed for quick species identification during pest control.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Onset of cryptic vicariance in the Japanese dormouse Glirulus japonicus (Mammalia, Rodentia) in the Late Tertiary, inferred from mitochondrial and nuclear DNA analysis

The sequences of the mitochondrial cytochrome b gene and restriction site variation in the spacer region of the nuclear ribosomal RNA gene [rDNA-restriction fragment length polymorphism (RFLP)] were analysed to determine the phylogeographic structure of the Japanese dormouse ( Glirulus japonicus ), which is threatened by deforestation and has been designated an endangered species in Japan. The phylogenetic tree of cytochrome b grouped G. japonicus into six geographical populations: north-eastern Honshu (I), central Honshu (II), west-central Honshu/Kii Peninsula (III), western Honshu (IV), Shikoku (V), and westernmost Honshu/Kyushu (VI); the genetic distances among these groups suggest divergence in the Late Tertiary. The lineage of group VI was located at the basal position in the phylogenetic tree, followed by the radiation of the other lineages. An rDNA-RFLP analysis of 15 restriction sites roughly supported such genetic isolation; groups I, II, III, IV, V and VI have five, two, one, one, one and four unique restriction sites, respectively, revealing four geographic groups as cryptic species: I, II, III + IV + V and VI. Our results reveal the ancient divergences of the local population, which has a complicated evolutionary history, and should be useful in developing a framework for the conservation of this species.     

Screening for restriction endonucleases in methane-oxidizing bacteria.

51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.     

Identification of enterohepatic Helicobacter species by restriction fragment-length polymorphism analysis of the 16S rRNA gene

Background. Restriction fragment-length polymorphism (RFLP) analysis of a 1,200-bp polymerase chain reaction–amplified DNA fragment of gene coding for 16S rRNA was used to generate restriction profiles of 11 enterohepatic Helicobacter spp. isolated from various animals and humans.
Methods. The amplicon from each Helicobacter sp. was digested with four restriction endonucleases: Alu I, Hinf I, Hha I, and Dde I. Alu I digestion produced five patterns that were useful for initial differentiation.
Results. Most Helicobacter spp. isolated from rodents had the same RFLP profiles by Alu I digestion (except H. rodentium and H. cholecystus ), but they had different RFLP profiles by Hha I digestion. Only H. bilis and " H. rappini" mouse isolates could not be readily distinguished by the polymerase chain reaction-RFLP method. However, these two species can be distinguished using H. bilis specific primers. Some of the Helicobacter spp. have an intervening sequence in their 16S rRNA gene, which changes the RFLP patterns; in these cases, sequencing is the preferred method to make an appropriate diagnosis.
Conclusions. The RFLP method used in this study was straightforward and rapid and should prove useful as an adjunct for identification and classification of multiple enterohepatic Helicobacter spp.     

Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed.     

A Brave New World for an Old World Pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil

The highly polyphagous Old World cotton bollworm Helicoverpa armigera is a quarantine agricultural pest for the American continents. Historically H. armigera is thought to have colonised the American continents around 1.5 to 2 million years ago, leading to the current H. zea populations on the American continents. The relatively recent species divergence history is evident in mating compatibility between H. zea and H. armigera under laboratory conditions. Despite periodic interceptions of H. armigera into North America, this pest species is not believed to have successfully established significant populations on either continent. In this study, we provide molecular evidence via mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) partial gene sequences for the successful recent incursion of H. armigera into the New World, with individuals being detected at two sites (Primavera do Leste, Pedra Preta) within the State of Mato Grosso in Brazil. The mtDNA COI and Cyt b haplotypes detected in the Brazilian H. armigera individuals are common throughout the Old World, thus precluding identification of the founder populations. Combining the two partial mtDNA gene sequences showed that at least two matrilines are present in Brazil, while the inclusion of three nuclear DNA Exon-Primed Intron-Crossing (EPIC) markers identified a further two possible matrilines in our samples. The economic, biosecurity, resistance management, ecological and evolutionary implications of this incursion are discussed in relation to the current agricultural practices in the Americas.     

Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA

A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)-based method for species identification was applied to seven Japanese Lymantria species, including four Asian gypsy moth (AGM) species. We sequenced the partial end of the cytochrome c oxidase I (COI) gene, tRNA leucine, COII gene, and partial end of the tRNA lysine in mitochondrial DNA (mtDNA) for one individual of each of the seven species. We analyzed the recognition sites of three restriction endonucleases and constructed a scheme for Lymantria species identification using PCR–RFLP. We then applied the scheme to 291 individuals from 45 populations of seven species. We found that all seven species were correctly identified using PCR–RFLP. These results suggest that PCR–RFLP is useful for identifying Japanese Lymantria species, which may be detected at Japanese ports.     

Molecular phylogeny of the Canary Island lacertids (Gallotia): mitochondrial DNA restriction fragment divergence in relation to sequence divergence and geological time

Restriction fragment length polymorphisms of 6 base pair recognising endonucleases are used to reconstruct the phylogeny of the endemic Canary Island lacertid, Gallotia. The division into conventional species is upheld by this molecular analysis and the western Canary Island lizard (G. galloti) and eastern Canary Island lizard (G. atlantica) are hypothesized to be sister species. A more comprehensive study of the intraspecific relationships of G. galloti, based on nineteen restriction enzymes, indicates that there are distinct southern and northern lineages within this species. The phylogenetic analysis does not uphold the conventional subspecies, but suggests an alternative arrangement with one northern (La Palma, Tenerife) and one southern (Gomera, Hierro) subspecies. The inferred timing of molecular divergence of populations of G. galloti, based on RFLP analysis, is compatible with the geological timing for island origin and fossil data. Mantel tests show that mitochondrial RFLP divergence is correlated with mitrochondrial 12s rRNA and cytochrome oxidase I sequence divergence and highly correlated with mitochondrial cytochrome b sequence divergence.     

Mitochondrial DNA variation in European populations of Silurus glanis

Mitochondrial DNA diversity of 13 wild Silurus glanis populations (covering the entire range of the species) and eight hatchery populations was investigated. PCR-RFLP analysis of four regions of mitochondrial DNA (cytochrome b, control region, ND-5/6) was used. Nineteen haplotypes were found. Thirteen of them were private. The proportion of total genetic diversity attributable to population differentiation was almost 80%. Despite the existence of significant differentiation between populations for mtDNA variation, no consistent pattern of geographic structuring was revealed and nucleotide divergence among S. glanis populations was low. These phenomena are discussed with regard to the impact of glaciation events. The domesticated stocks show less genetic diversity than natural ones, possibly due to their mode of management. Analysis of three European catfish species S. glanis, S. aristotelis and Silurus triostegus (sampled in the Euphrates river) revealed several endonucleases which produced restriction phenotypes diagnostic for the three species.     

Multiple sequences related to a constant-region kappa light chain gene in the rabbit genome

Four allelic genes control kappa light chain allotypes in the rabbit. Amino acid sequence studies have revealed an extensive divergence (22%--33%) in the alternative forms of the kappa constant region (b4, b5, b6 and b9). Furthermore, independent studies have shown that a rabbit could express a wrong allotype. To assess the hypothesis that b allotypes are encoded by duplicated genes with a polymorphic control mechanism, we have analyzed the DNAs of four different homozygous rabbits using the Southern blot hybridization technique, with a cloned b4 C kappa probe. DNAs of individual b4, b5, b6 and b9 rabbits were cleaved with Eco RI, Kpn I and Pst I restriction endonucleases. Comparative analysis of restriction patterns shows that all four rabbit DNAs contain multiple DNA fragments hybridizing with the probe under low- and high-stringency washing conditions. In addition, each restriction pattern is distinct, suggesting that the C kappa genes are organized differently in animals expressing different allotypes.     

四种白蚁的线粒体DNA限制性片段长度多态性研究(等翅目:鼻白蚁科,木白蚁科)

用６种限制性内切酶分析了４种白蚁的线粒体 Ｄ Ｎ Ａ 限制性片段长度多态性,根据限制性片段差异计算了４种白蚁之间的遗传距离,利用 Ｕ Ｐ Ｇ Ｍ Ａ 聚类分析法构建了分子聚类图。结果表明：尖唇异白蚁与散白蚁属关系很近,聚类分析结果显示应将其归于散白蚊属     

Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases

300 clones of microorganisms isolated at different stations and from different depths in the Black Sea were screened for restriction endonucleases production. The production of restriction endonucleases was found in 17 clones screened. Three of them were identified to be Alteromonas haloplanktis B1. Restriction endonuclease AhaB1 is an isoshizomer of Sau961. An identified Alteromonas haloplanktis clone B8 produces AhaB8I restriction endonuclease the prototype to which is KpnI. Of the clones isolated three are Moraxella species B4 producing MapB4I restriction endonuclease analogous to BanI, three are Bacillus species producing BspB2I and one is Micrococcus lylae 113 producing Mly1131 analogue of NarI, six Moraxella species B6 produce MspB6I. The isolated producer strains may be used for isolation of above mentioned restriction endonucleases.     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.     

Identification of Trichinella isolates by polymerase chain reaction--restriction fragment length polymorphism of the mitochondrial cytochrome c-oxidase subunit I gene.

We developed a polymerase chain reaction based approach using restriction fragment length polymorphisms of the mitochondrial cytochrome c-oxidase subunit I to identify nine genotypes (Trichinella spiralis, Trichinella britovi-European strains, Trichinella britovi-Japanese strains, Trichinella nativa, Trichinella nelsoni, Trichinella T5, Trichinella T6, Trichinella T8 and Trichinella pseudospiralis) in the genus Trichinella. Partial mitochondrial cytochrome c-oxidase subunit I genes of nine genotypes were amplified by polymerase chain reaction, sequenced, and digested with three restriction endonucleases (Mse I, Alu I and Bsp1248 I). This polymerase chain reaction based restriction fragment length polymorphism method allowed the identification of Trichinella genotypes. Trichinella spiralis, Trichinella britovi-Japanese strains, Trichinella nelsoni, T5 and Trichinella pseudospiralis were distinguishable by digestion with Mse I. Trichinella britovi-European strains and Trichinella T8 were distinguishable by digestion using Alu I, and Trichinella nativa and Trichinella T6 were distinguishable by double-digestion with Mse I and Bsp1286 I. The results obtained with this polymerase chain reaction based restriction fragment length polymorphism assay confirmed those previously reported by others and support the separation of the Japanese isolates as a new genotype, namely Trichinella T9.     

中国常见仓储书虱PCR-RFLP快速识别

应用PCR-RFLP技术,对我国4种常见仓储书虱即嗜虫书虱Liposcelis entomophila(Enderlein)、嗜卷书虱L.bostrychophila(Badonnel)、无色书虱L.decolor(Pearman)和小眼书虱L.paeta(Pearman)开展快速识别方法的研究。采用CTAB法从上述4种书虱体内提取基因组DNA,选用1对引物扩增16S rDNA基因区域,分别用5种限制性内切酶对PCR产物进行酶切。结果表明,PCR扩增片段大小约为500bp,用限制性内切酶DraI酶切得到的片段可将供试书虱区分开。该方法不受4种供试书虱地理种群、虫态(成虫、若虫)和性别的影响,可用于4种书虱的快速识别。     

Assessment of concordance among genealogical reconstructions from various mtDNA segments in three species of Pacific salmon (genus Oncorhynchus)

Seven segments of mitochondrial DNA (mtDNA), comprising 97% of the mitochondrial genome, were amplified by polymerase chain reaction (PCR) and examined for restriction site variation using 13 restriction endonucleases in three species of Pacific salmon: pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon. The distribution of variability across the seven mtDNA segments differed substantially among species. Little similarity in the distribution of variable restriction sites was found even between the mitochondrial genomes of the even- and odd-year broodlines of pink salmon. Significantly different levels of nucleotide diversity were detected among three groups of genes: six NADH-dehydrogenase genes had the highest; two rRNA genes had the lowest; and a group that included genes for ATPase and cytochrome oxidase subunits, the cytochrome b gene, and the control region had intermediate levels of nucleotide diversity. Genealogies of mtDNA haplotypes were reconstructed for each species, based on the variation in all mtDNA segments. The contributions of variation within different segments to resolution of the genealogical trees were compared within each species. With the exception of sockeye salmon, restriction site data from different genome segments tended to produce rather different trees (and hence rather different genealogies). In the majority of cases, genealogical information in different segments of mitochondrial genome was additive rather than congruent. This finding has a relevance to phylogeographic studies of other organisms and emphasizes the importance of not relying on a limited segment of the mtDNA genome to derive a phylogeographic structure.     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.