ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Nematology ◽  
2009 ◽  
Vol 11 (5) ◽  
pp. 649-668 ◽  
Author(s):  
Wolfgang Burgermeister ◽  
Helen Braasch ◽  
Kai Metge ◽  
Jianfeng Gu ◽  
Thomas Schröder ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Restriction Analysis ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Its Sequence ◽  
Additional Species ◽  
New Information ◽  
Specific Fragment ◽  
Species Specific ◽  
Different Origins

Abstract Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

PCR-based genetic identification of marlin and other billfish

1998 ◽  
Vol 49 (5) ◽  
pp. 383 ◽  
Author(s):  
B. H. Innes ◽  
P. M. Grewe ◽  
R. D. Ward
Keyword(s):  
Mitochondrial Dna ◽  
Genetic Test ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Genetic Identification ◽  
Dna Molecule ◽  
Pcr Rflp ◽  
D Loop ◽  
Specific Variation ◽  
Species Specific

A genetic test was developed for the identification of the six species of billfish found in Australian waters (black marlin, Indo–Pacific blue marlin, striped marlin, Indo–Pacific sailfish, shortbill spearfish and broadbill swordfish). The test was based on the PCR–RFLP analysis of a 1400 bp region of the mitochondrial DNA molecule, the d-loop, using four restriction enzymes (Hinf I, Rsa I and Sau3A I andTaq I). A total of 33 composite haplotypes were observed among 160 fish; all were species-specific. Three of the species—black marlin, striped marlin and broadbill swordfish—showed sufficient intra-specific variation to be useful in population structure analyses.

scholarly journals Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

2020 ◽  
Vol 59 (1) ◽  
pp. 55-61
Author(s):  
Ghobad BABAEI ◽  
Seyyed Alireza ESMAEILZADEH-HOSSEINI ◽  
Mahbobeh ZANDIAN ◽  
Vahid NIKBAKHT
Keyword(s):  
Restriction Analysis ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Reference Pattern ◽  
Consensus Sequences ◽  
Rdna Sequences ◽  
Rflp Profile ◽  
Ziziphus Jujube ◽  
East Of Iran ◽  
Virtual Rflp

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

scholarly journals Plasmid profiles, restriction fragment length polymorphisms and O-serotypes amongVibrio anguillarumisolates

1996 ◽  
Vol 117 (3) ◽  
pp. 471-478 ◽  
Author(s):  
K. Pedersen ◽  
T. Tiainen ◽  
J. L. Larsen
Keyword(s):  
Restriction Fragment ◽  
Fragment Length ◽  
Restriction Analysis ◽  
Vibrio Anguillarum ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Plasmid Profile ◽  
Length Polymorphisms ◽  
Environmental Bacteria ◽  
Restriction Fragment Length

SummaryA total of 279Vibrio anguillarumstrains were serotyped and examined for plasmid content. Plasmids were subjected to digestion with restriction enzymes. Most strains belonged to serogroup O1 (39%) and O2 (16%). In total 164 strains (53%) carried plasmids. Of the O1 and O2 isolates, 92% and 30%, respectively, carried one or more plasmids. Restriction fragment length polymorphism (RFLP) analysis of plasmid DNA indicated that plasmids belonged to several groups. Each group seemed to be restricted to a single O-serovar. The largest group was the pJM1-like plasmids among most serovar O1 strains. Most of these plasmids were about 67 kb like the pJM1 plasmid, but various derivatives ranged from 26–77 kb. RFLP studies of the 67 kb plasmids revealed 17 different restriction patterns. Some patterns were dominant among European strains whereas others were dominant among North American strains. The results confirmed the applicability of O-serotyping together with plasmid profile and restriction analysis of plasmids for typing ofV. anguillarum. They also indicated that plasmids among strains which belonged to the traditional fish pathogenic serogroups, O1 and O2, showed more homology than did strains from most other serogroups, that were usually non-pathogenic, environmental bacteria.

Rapid identification of Amaranthus caudatus and Amaranthus hypochondriacus by sequencing and PCR–RFLP analysis of two starch synthase genes

Genome ◽  
2012 ◽  
Vol 55 (8) ◽  
pp. 623-628 ◽  
Author(s):  
Young-Jun Park ◽  
Tomotaro Nishikawa
Keyword(s):  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Rapid Identification ◽  
Starch Synthase ◽  
Amaranthus Caudatus ◽  
Grain Amaranth ◽  
Amaranthus Hypochondriacus ◽  
Pcr Rflp ◽  
Cultivated Species ◽  
Species Specific

The objective of this study was to develop a PCR–RFLP method to identity the cultivated species of grain amaranth based on variations in the sequences of their starch synthase genes. We sequenced the SSSI and GBSSI loci in 126 accessions of cultivated grain amaranth collected from diverse locations around the world. We aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR–RFLP analysis. Our analyses indicated that EcoRI would recognize the sequence 5′-GAATT/C-3′ in the SSSI gene from Amaranthus caudatus L., and TaqI would recognize the sequence 5′-T/CGA-3′ in the GBSSI gene from Amaranthus hypochondriacus L. The PCR products obtained using gene-specific primers were 423 bp (SSSI) and 627 or 635 bp (GBSSI) in length. These products were cut with different restriction enzymes resulting in species-specific RFLP patterns that could be used to distinguish among the cultivated grain amaranths. The results clearly showed that A. caudatus and A. hypochondriacus were easily differentiated at the species level using this method. Therefore, the PCR–RFLP method targeting amaranth starch synthase genes is simple and rapid, and it will be a useful tool for the identification of cultivated species of grain amaranth.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia

Parasitology ◽  
1998 ◽  
Vol 117 (1) ◽  
pp. 1-13 ◽  
Author(s):  
K. VICTOIR ◽  
A. L. BAÑULS ◽  
J. AREVALO ◽  
A. LLANOS-CUENTAS ◽  
R. HAMERS ◽  
...  
Keyword(s):  
Gene Locus ◽  
Genetic Characterization ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Natural Isolates ◽  
Phenotype Diversity

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

First report of Bursaphelenchus xylophilus in Portugal and in Europe

Nematology ◽  
1999 ◽  
Vol 1 (7) ◽  
pp. 727-734 ◽  
Author(s):  
Manuel M. Mota ◽  
Helen Braasch ◽  
Maria Antonia Bravo ◽  
Ana Catarina Penas ◽  
Wolfgang Burgermeister ◽  
...  
Keyword(s):  
Pinus Pinaster ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Bursaphelenchus Xylophilus ◽  
Female Tail ◽  
Dna Fragment ◽  
Species Specific ◽  
First Time ◽  
Very High ◽  
Pin Maritime

AbstractA survey of aphelenchid nematodes (Nematoda: Aphelenchida) associated with maritime pine, Pinus pinaster, was conducted in Portugal in 1996 and 1999. A Bursaphelenchus species has been identified for the first time in the Iberian Peninsula. B. xylophilus is reported for the first time in Europe. It was found in very high numbers - up to 38 000 per 10 g of pine wood - inside a few declining trees infested with curculionid, cerambycid and scolytid beetles. Morphological observations, including shape of spicules, bursa, vulva, female tail end and stylet as well as morphometrics, were in accordance with the species description. Species-specific DNA fragment patterns were obtained using ITS-RFLP analysis, with five different restriction enzymes. The importance and implications of this finding are discussed. Premiere signalisation de Bursaphelenchus xylophilus au Portugal, at en Europe - Une enquete sur les nematodes Aphelenchides (Nematoda: Aphelenchida) associes au pin maritime (Pinus pinaster) a ete realisee au Portugal de 1996 a 1999. Une espece de Bursaphelenchus a ete identifiee pour la premiere fois dans la Peninsule Iberique. B. xylophilus est signale pour la premiere fois en Europe. Il a ete trouve en tres grand nombre - jusqu'a 38 000 individus pour 10 g de bois de pin - dans des arbres deperissants infestes par des Coleopteres Curculionides, Cerambycides et Scolytides. Les observations concernant la morphologie - en particulier la forme des spicules, la bourse, la vulve, l'extremite de la queue de la femelle et le stylet - de meme que les donnees morphometriques correspondent a la description de l'espece. Des sequences de fragments d'ADN specifique de l'espece ont ete obtenus par analyse ITS-RFLP a l'aide de cinq enzymes de restriction. L'importance et les implications de cette decouverte sont discutees.

Mitochondrial DNA and isozyme electrophoretic analyses of the endangered Acadian whitefish, Coregonus huntsmani Scott, 1987

1991 ◽  
Vol 69 (2) ◽  
pp. 311-316 ◽  
Author(s):  
L. Bernatchez ◽  
T. A. Edge ◽  
J. J. Dodson ◽  
S. U. Qadri
Keyword(s):  
Mitochondrial Dna ◽  
Genetic Model ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Electrophoretic Analysis ◽  
The Other ◽  
Lake Whitefish ◽  
Mitochondrial Genotype ◽  
Species Specific ◽  
Mtdna Restriction Analysis

Electrophoretic analysis of isozymes and mitochondrial DNA (mtDNA) restriction analysis were used to study the genetic divergence between the Acadian whitefish, Coregonus huntsmani, and members of the subgenera Coregonus (lake whitefish, C. clupeaformis) and Leucichthys (Arctic cisco, C. autumnalis, and lake cisco, C. artedii). Results obtained from both studies demonstrated that the Acadian whitefish is genetically highly distinct from the other coregonines examined. mtDNA restriction analysis revealed that the Acadian whitefish possesses a unique mitochondrial genotype which is divergent from that of the two cisco species or lake whitefish. Twelve of 13 restriction enzymes used were informative in distinguishing the Acadian whitefish from the other species, and species-specific fragment patterns were observed for 10 enzymes. In isozyme analysis of five loci, the Acadian whitefish was monomorphic at two loci for alleles not found in lake whitefish. Acadian whitefish also possessed an additional isozyme at another locus that was not found in lake whitefish and Arctic cisco specimens. This isozyme is unknown from the genetic model for lake whitefish at this locus. These results provided useful genetic markers to identify the Acadian whitefish. They emphasize that the extinction of the species would represent a major loss of both genetic diversity and potential information concerning the contentious phylogeny of coregonine fishes.

scholarly journals Identification and differentiation among chicken’s, duck’s, quail’s, rabbit’s and turkey's meat using PCR-RFLP technique

2015 ◽  
Vol 31 (1) ◽  
pp. 101-108 ◽  
Author(s):  
S.M. Abdel-Rahman ◽  
A.M. Elmaghraby ◽  
A.S. Haggag
Keyword(s):  
Mitochondrial Dna ◽  
Cytochrome B ◽  
Fragment Length ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Species Specific ◽  
Rflp Assay

PCR-RFLP technique was developed for identification and differentiation among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat. DNA from small amount of muscles (0.05 g) was extracted and a region of mitochondrial DNA (cytochrome-b gene) in chicken, duck, quail, rabbit and turkey was amplified by PCR. Fragment length of the PCR product was 371 bp in chicken, 374 bp in duck and rabbit and 377 bp in both quail and turkey. Six nucleotides different makes it difficult to differentiate among these five species-specific meat. For differentiation, three different restriction enzymes (DdeI, MspI and TaqI) were used to digest the PCR products. Restriction analysis showed difference among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat. Where, DdeI yielded two fragments (291 and 83 bp) only in rabbit?s meat. MspI yielded three fragments (221, 85 and 65 bp) in chicken?s meat and two fragments (290 and 87 bp) in both quail?s and turkey's meat. TaqI yielded three fragments (146, 134 and 94 bp) in duck?s meat and two fragments (226 and 151 bp) in quail?s meat. The use of Cytb- PCR-RFLP assay allowed a direct and fast authentication and differentiation among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat.

scholarly journals SHV-1 β-Lactamase Is Mainly a Chromosomally Encoded Species-Specific Enzyme in Klebsiella pneumoniae

2001 ◽  
Vol 45 (10) ◽  
pp. 2856-2861 ◽  
Author(s):  
José Chaves ◽  
Margarita G. Ladona ◽  
Concepción Segura ◽  
Amparo Coira ◽  
Roser Reig ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Chromosomal Dna ◽  
Promoter Regions ◽  
Specific Pcr ◽  
Positive Hybridization ◽  
Species Specific

ABSTRACT The nature of the SHV-1 β-lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. β-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of β-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples withK. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 β-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 β-lactamase originated from the K. pneumoniaechromosome.

Satellite DNA and heterochromatin of the flour beetle Tribolium confusum

Genome ◽  
1993 ◽  
Vol 36 (3) ◽  
pp. 467-475 ◽  
Author(s):  
Miroslav Plohl ◽  
Vlatka Lucijanić-Justić ◽  
Durdica Ugarković ◽  
Eduard Petitpierre ◽  
Carlos Juan
Keyword(s):  
Satellite Dna ◽  
Tandem Repeats ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Tribolium Confusum ◽  
Inverted Repeats ◽  
Metaphase Chromosomes ◽  
Bent Dna ◽  
Distamycin A

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40–45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 × 105 per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.Key words: tandem repeats, DNA sequence, bent DNA, inverted repeats, Coleoptera.

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