A simple PCR-based method for the rapid and accurate identification of spider mites (Tetranychidae) on cassava

Abstract The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism–polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

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Discrimination of Diploid Crucifer Species Using PCR-RFLP of Chloroplast DNA

HortScience ◽

10.21273/hortsci.39.7.1575 ◽

2004 ◽

Vol 39 (7) ◽

pp. 1575-1577 ◽

Cited By ~ 6

Author(s):

Claudia Cunha ◽

Muhammet Tonguç ◽

Phillip D. Griffiths

Keyword(s):

Chloroplast Dna ◽

Dna Sequences ◽

Raphanus Sativus ◽

Fragment Length Polymorphism ◽

Diploid Species ◽

Restriction Enzymes ◽

Chain Reaction ◽

Species Identity ◽

Pcr Rflp ◽

Polymerase Chain

Chloroplast DNA (cpDNA) was used to identify polymorphisms between crucifer species using the polymerase chain reaction-random fragment-length polymorphism (PCR-RFLP) technique. Ten primer pairs based on cpDNA gene sequences were used to amplify cpDNA fragments in Brassica oleracea L., B. rapa L., B. nigra (L.) Koch, B. napus L., B. carinata Braun, B. juncea (L.) Czern, and Raphanus sativus L. accessions. Amplified DNA sequences were then digested using 11 restriction enzymes to identify polymorphisms between the 7 species. Of the 110 combinations, 38 generated polymorphisms that discriminated one or more of the species. Genotyping of these polymorphisms in 10 accessions of each of the diploid species (B. oleracea, B. nigra, B. rapa and R. sativus) did not reveal segregating polymorphisms among accessions within species, indicating that they can be used to help determine species identity. Ten accessions of each of the amphidiploids B. napus, B. carinata and B. juncea were genotyped to infer their maternal ancestry. The diploid source of cpDNA in B. carinata was B. nigra in all accessions tested and B. rapa for nine of ten B. juncea accessions tested. Two B. napus accessions amplified polymorphisms shared with B. rapa, and eight accessions produced unique polymorphisms from neither B. rapa, B. oleracea or B. nigra. The polymorphisms identified in this study can be used to help confirm identity of the diploid crucifer species for taxonomic and conservation studies.

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Molecular identification studies on Oestrus ovis L. (Diptera:Oestridae) larvae infested sheep in jazan region , Saudi Arabia

Indian Journal of Animal Research ◽

10.18805/ijar.v0iof.8487 ◽

2017 ◽

Cited By ~ 2

Author(s):

Bosly . A. Hanan

Keyword(s):

Saudi Arabia ◽

Molecular Identification ◽

Gene Sequence ◽

Morphological Identification ◽

Oestrus Ovis ◽

Chain Reaction ◽

Polymerase Chain ◽

Partial Fragment ◽

Mitochondrial Cytochrome Oxidase ◽

Mtcoi Gene

Oestrus ovis L. (O. ovis) (Diptera: Oestridae) (Sheep Bot Fly) is ubiquitous in distribution. Myiasis causing larvae collected from Abu-Arish area (Eastern Jazan), Saudi Arabia were confirmed as O. ovis based on morphological traits. Polymerase chain reaction (PCR) studies targeting amplification of partial fragment (606 bp) of mitochondrial cytochrome oxidase subunitI (mtCOI) gene further confirmed the species. Phylogenetic analysis based on the partial (mtCOI) gene sequence revealed that the accession KU921431 showed 97% similarity based on nucleotide pairs of O.ovis accessions, retrieved from the GenBank. Thus, a method of molecular identification of O.ovis larvae was established as a credible substitution to morphological identification in Jazan region, Saudi Arabia.

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Profile of follicle-stimulating hormone and polymorphism of follicle-stimulating hormone receptor in Madrasin cattle with ovarian hypofunction

Veterinary World ◽

10.14202/vetworld.2020.879-883 ◽

2020 ◽

Vol 13 (5) ◽

pp. 879-883

Author(s):

Budi Utomo ◽

Emmanuel Djoko Putranto ◽

Amaq Fadholly

Keyword(s):

Fragment Length Polymorphism ◽

Follicle Stimulating Hormone ◽

Restriction Enzymes ◽

Chain Reaction ◽

Blood Samples ◽

Fshr Gene ◽

Polymerase Chain ◽

Genetic Makeup ◽

Restriction Fragment Length ◽

Stimulating Hormone

Background and Aim: The follicle-stimulating hormone (FSH) gene is an essential regulator of fertility in livestock. This study aims to provide information on the genetic makeup of Madrasin cattle experiencing hypofunction by the FSH profile and FSH receptors (FSHR) polymorphism. Materials and Methods: Blood samples were collected from the Bangkalan regency in Indonesia. DNA was isolated and purified following the extraction protocol of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Results: Our results showed that the FSH gene had a band length of 310 bp and produce two alleles (A and B) with restriction enzymes at 250 bp, 230 bp, and 145 bp. Furthermore, the FSHR gene had a band length of 303 bp and produced two homozygous genotypes: GG at bp 239 and CC at bp 188. Conclusion: Based on these differences, there was no change in allele frequency and genotype between Madura and Madrasin cattle due to crossbreeding with Limousin cattle. Thus, further detailed investigations of Madrasin cattle are required to elucidate the profile of the LH and LHR genes.

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Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

Phytopathology ◽

10.1094/phyto.2000.90.4.345 ◽

2000 ◽

Vol 90 (4) ◽

pp. 345-353 ◽

Cited By ~ 33

Author(s):

Frank N. Martin

Keyword(s):

Polymerase Chain Reaction ◽

Fragment Length Polymorphism ◽

Coastal Region ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Molecular Techniques ◽

Chain Reaction ◽

Polymerase Chain ◽

Collection Sites ◽

Restriction Fragment Length

Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.

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Molecular and morphological discrimination betweenTylospora fibrillosaandTylospora asterophoramycorrhizae

Canadian Journal of Botany ◽

10.1139/b98-182 ◽

1999 ◽

Vol 77 (1) ◽

pp. 11-21 ◽

Cited By ~ 11

Author(s):

Ursula Eberhardt ◽

Lutz Walter ◽

Ingrid Kottke

Keyword(s):

Fragment Length Polymorphism ◽

Pcr Primers ◽

Morphological Identification ◽

Chain Reaction ◽

Specific Primers ◽

Specific Pcr ◽

Pcr Rflp ◽

Polymerase Chain ◽

Species Specific ◽

First Time

Among the mycorrhizal types of spruce, Tylospora-type mycorrhizae are the most constant and abundant. Two species of the genus Tylospora occur in Europe, Tylospora fibrillosa and Tylospora asterophora. Mycorrhizae of T. asterophora are described in detail for the first time. Sequences of the internal transcribed spacer (ITS) of the ribosomal genes were obtained from T. fibrillosa and T. asterophora mycorrhizae, sporocarps, and cultured mycelium. Discrimination and identification of the two species by ITS polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) are discussed in the light of inter- and intra-specific variability. Species-specific PCR primers were designed to distinguish both species. Molecular screening of Tylospora-type mycorrhizae from field material led to unambiguous results, whereas morphological identification is likely to fail because of great similarity even at the microscopic level.Key words: Tylospora asterophora, Tylospora fibrillosa, ectomycorrhizae, taxon specific primers (TSOPs), ITS sequences.

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Identitas dan Keragaman Genetik Begomovirus yang Berasosiasi dengan Penyakit Keriting pada Tomat Berdasarkan Teknik Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP)

Jurnal AgroBiogen ◽

10.21082/jbio.v4n1.2008.p9-17 ◽

2016 ◽

Vol 4 (1) ◽

pp. 9 ◽

Cited By ~ 1

Author(s):

Tri Joko Santoso ◽

Sri H. Hidayat ◽

M. Herman ◽

H. Aswidinnoor ◽

Sudarsono Sudarsono

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Restriction Enzymes ◽

Chain Reaction ◽

Polymerase Chain ◽

Leaf Curl Virus ◽

Restriction Fragment Length

Begomoviruses, members of the Geminivirus, are considered as emerging plant viruses. This was due to the increasing incidences and severities of the diseases in a number of economically important crops, including tomato. Genetic diversities of the Begomovirus isolates infecting tomato (Lycopersicon esculentum) of several areas in Indonesia were analyzed by using Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) technique. A 1500 base pairs of PCR fragments amplified by using degenerate primers for Begomovirus was digested using four restriction enzymes, i.e., DraI, EcoRI, RsaI, and PstI. The pattern of RE digested fragments of 8 Begomovirus isolates and the predicted RFLP fragments of the Begomovirus isolates in the GeneBank database were used to determine the genetic identities and diversities among the isolates. Positive results of the PCR amplifications proved that diseased tomato plant samples collected from 8 locations in Java and Sumatra were infected with at least one Begomovirus isolate. The PCR amplification products, which were digested using the four restriction enzymes indicated the presence of polimorfisms among the DNA fragments of the Begomovirus isolates. Identifications of the Begomovirus indicated that the Brastagi, Bogor, Sragen, Ketep, and Boyolali isolates were Tomato Leaf Curl Virus (ToLCV); the isolates from Malang and Blitar isolates were Ageratum Yellow Vein Virus (AYVV), while one isolate from Kaliurang was Tomato Yellow Leaf Curl Virus (TYLCV). Results of the phylogenetic analysis of the 8 Begomovirus isolates based on Begomoviruses from the DNA database indicated that they belonged to three different groups.

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Cloning, characterization and identification of polymorphism in TCR Zeta Gene in deoni cattle

Indian Journal of Animal Research ◽

10.18805/ijar.b-3389 ◽

2018 ◽

Author(s):

K. Swathi ◽

M. Gnana Prakash ◽

D. Sakaram ◽

T. Raghunandan ◽

A. Sarat Chandra ◽

...

Keyword(s):

T Cell Receptor ◽

Fragment Length Polymorphism ◽

Bos Indicus ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Cell Receptor ◽

Chain Reaction ◽

Reading Frame ◽

Pcr Rflp ◽

Polymerase Chain

The cDNA encoding, T-cell receptor zeta (TCR z; CD247) molecule of Deoni cattle (Bos indicus), was isolated, cloned and sequenced in the present study. The CD247 cDNA comprised 1078 nucleotides including a 30 nucleotide 5¹-untranslated region (UTR), 495 nucleotide single open reading frame (ORF) and 553 nucleotide 3¹-UTR. Deduced amino acid of cattle CD247 sequence was two residues shorter than the corresponding sheep sequences. However, ruminant-specific insertions and substitutions in transmembrane (TM) and intra-cytoplasmic (IC) domain were present in cattle. Immunoreceptor tyrosine-based activation motifs (ITAMs), the important motifs for TCR signalling, were totally conserved among ruminants including cattle. The 3¹ - UTR region of the cattle CD247 was highly homologous to the corresponding region in the buffalo sequence and showed lack of polymorphism after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using Hae III and Mse I restriction enzymes in cattle population. Phylogenetically, cattle sequence was closer to buffalo sequence under the ruminant’s lineage. The conserved nature of this gene ensures TCR integrity which is vital for induction of optimal and efficient immune response.

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rDNA-RFLP identification ofCandidaspecies in immunocompromised and seriously diseased patients

Canadian Journal of Microbiology ◽

10.1139/w04-025 ◽

2004 ◽

Vol 50 (7) ◽

pp. 514-520 ◽

Cited By ~ 7

Author(s):

Patrícia M Pinto ◽

Maria A Resende ◽

Cristiane Y Koga-Ito ◽

José A.G Ferreira ◽

Miriam Tendler

Keyword(s):

Fragment Length Polymorphism ◽

Restriction Enzymes ◽

Clinical Analysis ◽

Chain Reaction ◽

Candida Spp ◽

Pcr Products ◽

Analysis Laboratory ◽

Polymerase Chain ◽

Belo Horizonte ◽

Its1 And Its2

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.Key words: polymerase chain reaction, restriction fragment length polymorphism, candidosis, Candida spp.

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Identification of Phytophthora Isolates to Species Level Using Restriction Fragment Length Polymorphism Analysis of a Polymerase Chain Reaction-Amplified Region of Mitochondrial DNA

Phytopathology ◽

10.1094/phyto.2004.94.9.983 ◽

2004 ◽

Vol 94 (9) ◽

pp. 983-991 ◽

Cited By ~ 24

Author(s):

Frank N. Martin ◽

Paul W. Tooley

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Restriction Enzymes ◽

Species Level ◽

Chain Reaction ◽

Polymerase Chain ◽

Restriction Fragment Length

Polymerase chain reaction primers spanning the mitochondrially encoded coxI and II genes have been identified that were capable of amplifying target DNA from all 152 isolates of 31 species in the genus Phytophthora that were tested. Digestion of the amplicons with restriction enzymes generated species-specific restriction fragment length polymorphism banding profiles that were effective for isolate classification to a species level. Of the 24 species in which multiple isolates were examined, intraspecific polymorphisms were not observed for 16 species, while 5 species exhibited limited intraspecific polymorphism that could be explained by the addition/loss of a single restriction site. Intraspecific polymorphisms were observed for P. megakarya, P. megasperma, and P. syringae; however, these differences may be a reflection of the variation that exists in these species as reported in the literature. Although digestion with AluI alone could differentiate most species tested, single digests with a total of four restriction enzymes were used in this investigation to enhance the accuracy of the technique and minimize the effect of intraspecific variability on correct isolate identification. The use of the computer program BioNumerics simplified data analysis and identification of isolates. Successful template amplification was obtained with DNA recovered from hyphae using a boiling miniprep procedure, thereby reducing the time and materials needed for conducting this analysis.

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