A simple molecular method for rapid identification of commercially used Amblyseius and Neoseiulus species (Acari: Phytoseiidae)

Predatory mites from the Amblyseius (Neoseiulus) genus (Family Phytoseiidae, Order Parasitiformes) are widely used for protecting plants against pests, especially thrips. The differentiation of Amblyseius and Neoseiulus species by their morphological features is problematic despite the fact that they are taxonomically different genera. The Phytoseiidae family includes a lot of species that are extremely difficult to distinguish from each other. The discovery of new molecular genetic markers might considerably facilitate the express identification of commercial mite species. Despite their high morphological similarity, the three common commercial Amblyseius and Neoseiulus mite species (Neoseiulus cucumeris, Amblyseius swirskii, and Neoseiulus barkeri) differed significantly in the nucleotide sequences of DNA fragments containing the ITS1 and ITS2 internal transcribed spacers. We found that when PCR products of the amplified DNA fragments from A. swirskii, N. cucumeris, and N. barkeri were treated with a combination of AccB1I, AspLEI, and SspI endonucleases and the resulting products were separated by electrophoresis in agarose gel, the obtained picture was sufficiently specific to provide accurate identification of the analyzed mite species. The lengths of the digestion products were different enough to allow their resolution in agarose gels. The PCR-RFLP method developed by us allows the rapid and accurate identification of commercially used Amblyseius and Neoseiulus species without DNA sequencing. The combination of the three endonucleases (AccB1I, AspLEI, and SspI) and FaeI can be used for the differentiation of all other but less frequently commercially used Phytoseiidae mites.

Download Full-text

Phylogenetic Relationships of Local Durian Species based on Morphological Characteristics and PCR-RFLP Analysis of the Ribosomal Internal Transcribed Spacer (ITS) DNA

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v8i3.6748 ◽

2016 ◽

Vol 8 (3) ◽

pp. 362

Author(s):

Fidia Fibriana ◽

Lutfia Nur Hadiyanti

Keyword(s):

Phylogenetic Relationships ◽

Internal Transcribed Spacer ◽

Morphological Characteristics ◽

Biology Education ◽

Rflp Analysis ◽

Restriction Pattern ◽

Internal Transcribed Spacers ◽

Central Java ◽

Pcr Rflp ◽

Pcr Products

In this study, twenty local durian accessions obtained from Central Java in situ collection were characterized using the morphological characteristics and the restriction patterns which generated from the region spanning the internal transcribed spacers ITS LEU and ITS 4. Morphological characteristics of durian leaf, stem, tree, and fruit showed variations for the different accessions, whereas polymerase chain reaction (PCR) products of ribosomal DNA region showed a low length of variation. The size of the PCR products and the restriction analyses with the restriction endonucleases Bsp1431yielded a restriction pattern for each accessions. The results of this study can be utilized by local durian farmers as a preliminary reference for durian propagation. The data obtained need to be supported by further research using the other molecular markers to obtain more accurate data. The clear identity of durian species can help the management of propagation systems by farmers to get superior local durian.How to CiteFibriana, F., & Hadiyanti, L. N. (2016). Phylogenetic Relationships of Local Durian Species based on Morphological Characteristics and PCR-RFLP Analysis of the Ribosomal Internal Transcribed Spacer (ITS) DNA. Biosaintifika: Journal of Biology & Biology Education, 8(3), 362-370.

Download Full-text

Molecular Identification and Characterisation of Aspergillus Flavus Isolates Originating from Serbian Wheat Grains

Acta Alimentaria ◽

10.1556/066.2020.49.4.3 ◽

2020 ◽

Vol 49 (4) ◽

pp. 382-389

Author(s):

J. Krulj ◽

N. Ćurčıć ◽

A. Bočarov Stančıć ◽

J. Kojıć ◽

L. Pezo ◽

...

Keyword(s):

Aspergillus Flavus ◽

Molecular Genetic ◽

Pcr Amplification ◽

Its Region ◽

Holistic Approach ◽

Accurate Identification ◽

Essential Point ◽

Climate Conditions ◽

Wheat Grains ◽

Pcr Rflp

During previous years, regarding the shifts in climate conditions in temperate region, such as occurrence of high temperatures and prolonged drought, increased occurrence frequencies of Aspergillus flavus and aﬂatoxins in cereal grains were recorded. A reliable and accurate identiﬁcation of the fungi is of great importance for evaluating the microbiological risks of contamination. The essential point of the present investigation was molecular characterisation and identiﬁcation of A. flavus isolates originating from common wheat and spelt grains collected after harvest during the period of three years (2015–2017) in Northern Serbia. A holistic approach that included PCR ampliﬁcation of two DNA genomic regions and PCR-RFLP assay followed by fragment length analysis, provided complete and comprehensive characterisation of A. flavus isolated from wheat grains. The presented results indicate that there was no diﬀerence among the tested Aspergillus isolates on the molecular–genetic level. All 38 strains were identiﬁed as A. flavus by sequencing of combined ITS region and β-tubulin gene fragments (acc. no.: MH582473 to MH582510). PCR-RFLP method in combination with a Lab-on-a-chip (LoaC) electrophoresis can be successfully used to rapidly identify A. flavus isolates.

Download Full-text

Identification and phylogeny of some species of the genera Sporidiobolus and Rhodotorula using analysis of the 5.8s rDNA gene and two ribosomal internal transcribed spacers

Archives of Biological Sciences ◽

10.2298/abs1101079m ◽

2011 ◽

Vol 63 (1) ◽

pp. 79-88 ◽

Cited By ~ 1

Author(s):

M. Mokhtari ◽

H.R. Etebarian ◽

S.H. Mirhendi ◽

M. Razavi

Keyword(s):

Yeast Species ◽

Fragment Size ◽

Internal Transcribed Spacers ◽

Rhodotorula Mucilaginosa ◽

Yeast Identification ◽

5.8S Rdna ◽

Electrophoretic Patterns ◽

Yeast Strains ◽

Pcr Products ◽

Its1 And Its2

Due to the problems encountered in routine morphological and physiological procedures that are used in yeast identification, DNA-based methods have recently been developed. In the present study, l66 yeast strains were isolated from several apple and citrus cultivars. After analysis by basic morphological methods, the ITS1 and ITS2 regions of the isolates were amplified separately, and the isolates were grouped based on fragment size polymorphism (FSP) of the amplicons. By comparing the electrophoretic patterns of the PCR products with Rhodotorula mucilaginosa, species were identified as Rhodotorula. For precise and final identification, the ITS-PCR products were subjected to sequencing followed by Blast analysis. As a result, eight isolates were identified as belonging to the Rhodotorula genus, of which five were identified as R. mucilaginosa and three as R. glutinis, and one as a Sporidiobolus. We conclude that the method PCR-FSP, in combination with other approaches, is useful for the identification of yeast species.

Download Full-text

Early Detection and Identification of the Main Fungal Pathogens for Resistance Evaluation of New Genotypes of Forest Trees

Forests ◽

10.3390/f9120732 ◽

2018 ◽

Vol 9 (12) ◽

pp. 732 ◽

Cited By ~ 2

Author(s):

Konstantin Shestibratov ◽

Oleg Baranov ◽

Natalya Subbotina ◽

Vadim Lebedev ◽

Stanislav Panteleev ◽

...

Keyword(s):

Early Detection ◽

Fungal Pathogens ◽

Molecular Genetic ◽

Internal Transcribed Spacers ◽

Silver Birch ◽

Forest Trees ◽

In Planta ◽

Genetic Method ◽

Detection And Identification ◽

Its1 And Its2

The growing importance of forest plantations increases the demand for phytopathogen resistant forest trees. This study describes an effective method for early detection and identification of the main fungal phytopathogens in planting material of silver birch (Betula pendula) and downy birch (B. pubescens), based on the estimation of the size of the internal transcribed spacers (ITS1 and ITS2) in the 18S-5.8S-28S rDNA gene cluster, which are species-specific for most micromycetes. The electrophoretic assay of the ITS1 and ITS2 loci has allowed us to identify predominant phytopathogenic fungal species in downy and silver birch in planta. This new molecular genetic method can be used to screen birch and other forest trees for different fungal pathogens to evaluate disease resistance. This information can be useful in breeding new genotypes of forest trees, including transgenic clones with modified wood composition.

Download Full-text

SELECTION AND ASSESSMENT OF ISSR-MARKERS FOR ANALYSIS OF SEPARATE CATTLE POPULATIONS

Animal Breeding and Genetics ◽

10.31073/abg.61.15 ◽

2021 ◽

Vol 61 ◽

pp. 137-145

Author(s):

О. M. Мaherovska

Keyword(s):

Genetic Diversity ◽

Genetic Structure ◽

Dairy Cattle ◽

Dna Sequences ◽

Biological Material ◽

Dna Polymorphism ◽

Molecular Genetic ◽

Issr Markers ◽

Dna Fragments ◽

Pcr Products

The article covers molecular genetic analysis of intermicrosatellite DNA sequences of dairy cattle productivity. Molecular markers based on DNA polymorphism were used for this monitoring. Such markers make it possible to assess quickly the genetic polymorphism of taprin in the herd. Іnsofar as a large number of intermicrosatellite repeats is in the genome of cattle, that increases the probability of detecting polymorphic loci. The ISSR markers selected for the study are based on multiclocus synthesis in polymerase chain reaction (PCR) and allow an objective study of the breed and interbreed diversity. And it makes possible to assess quickly and accurately genetic diversity for the presence of genes associated with economically useful traits. The purpose of this work is the selection and evaluation of ISSR-markers for the analysis and study of genetic diversity of Ukrainian and imported breeds of dairy cattle. Samples of biological material from representatives of three herds of cattle (Ukrainian Red-and-White spotted dairy, Montbeliard breed and their crossbreeds) were selected for the study by the method of groups-analogues. For the analysis of this material the generally accepted zootechnical methods of studying of a selection material and methods of an estimation of animals on molecular – genetic markers are included. According to standard methods, DNA was isolated from peripheral blood lymphocytes using a set of reagents "DNA Sorb B". Amplification of total DNA with ISSR primers was performed on a thermal cycler "Tertsyk" ("DNA technology" of the Russian Federation). Electrophoretic separation of DNA fragments was performed in an agarose gel according to conventional methods. The size of the obtained PCR products was detected using a molecular weight marker SM1343. As a result of the study of the biological material of these animals, the obtained ISSR-PCR products were quite heterogeneous. The vast majority of polylocus spectra had clear discrete bands, but there were amplicons without clear discrete bands. Analyzing the results of the study of the genetic structure of animals of the Ukrainian Red-and-White dairy breed, using primers ISSR-1, ISSR-2, ISSR-3 and ISSR-4, the range of obtained PCR products ranges from 250 bp. up to 3000 bp. The range of amplification products in Montbeliard animals has a smaller range and ranges from 250 bp, respectively. up to 1500 bp.The obtained amplicons for the use of primers ISSR-1 and ISSR-2, ISSR-3 and ISSR-4 in the turf of Ukrainian Red-and-White dairy and Montbeliard breeds have sizes from 350 bp to 2000 bp. Having analyzed the information you can determine the distribution of the number and length of DNA fragments using 4 ISSR-markers. The total number of amplified DNA fragments varies depending on the primer from 21 to 106, and their size ranges from 250 BP up to 3000 bp. Based on the analysis of DNA plymorphism, it is possible to assess the heterogeneity of selected populations of cattle. Thus, as a result of studying the genetic structure of animals of two breeds of dairy cattle and their crossbreeds by intermicrosatellite DNA loci, their individual polymorphism was revealed. The amplification products have significant variations depending on the primer used. Primers ISSR-1 and ISSR-2 were the most informative for the analysis of cattle DNA polymorphism.

Download Full-text

Optimization of PCR Purification Using Silica-Coated Magnetic Beads

Eurasian Journal of Applied Biotechnology ◽

10.11134/btp.1.2020.8 ◽

2020 ◽

Author(s):

K.T. Berdimuratova ◽

A.O Amirgazin ◽

М.А. Kuibagarov ◽

V.B. Lutsay ◽

K.K. Mukanov ◽

...

Keyword(s):

Nucleic Acids ◽

Magnetic Beads ◽

Molecular Genetic ◽

Genetic Research ◽

Buffer System ◽

Dna Fragments ◽

Dna Molecules ◽

Sequencing Technologies ◽

Pcr Products ◽

The Cost

Purification of nucleic acids is still an important step in molecular genetic research. The development of whole genome sequencing technologies has increased the requirements for the purity of the nucleic acids used, and also required the selection of DNA fragments by size. Buffer systems that contain PEG/NaCl solutions and silica-coated magnetic beads allow to purify nucleic acids and selectively sorb certain sizes of DNA. In this article, we present a simple protocol for the purification of PCR products with the ability to absorb the required DNA molecules. It was determined that the use of an optimized PEG / NaCl buffer system with magnetic silica gel in a ratio of 1.5: 1 with a PCR product allows to get rid of DNA fragments 100 and less base pairs (bp), as well as other contaminants, while maintaining this is more than 90% of the DNA in solution. The ratio of 0.35: 1 allows for high-affinity sorption of DNA molecules larger than 400 bp. The practical use of the obtained data allows us to improve the quality of sequencing without increasing the cost of research.

Download Full-text

Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653864 ◽

1995 ◽

Vol 73 (05) ◽

pp. 756-762 ◽

Cited By ~ 25

Author(s):

Yoshiaki Tomiyama ◽

Hirokazu Kashiwagi ◽

Satoru Kosugi ◽

Masamichi Shiraga ◽

Yoshio Kanayama ◽

...

Keyword(s):

Dna Analysis ◽

Molecular Genetic ◽

Genetic Defect ◽

Nucleotide Sequence Analysis ◽

Type I ◽

Normal Amount ◽

Immunoblot Assay ◽

Glanzmann’S Thrombasthenia ◽

Glanzmann's Thrombasthenia ◽

Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

Download Full-text

Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽

10.3390/genes12020146 ◽

2021 ◽

Vol 12 (2) ◽

pp. 146

Author(s):

Catarina Xavier ◽

Mayra Eduardoff ◽

Barbara Bertoglio ◽

Christina Amory ◽

Cordula Berger ◽

...

Keyword(s):

Dna Extraction ◽

Ancient Dna ◽

Extraction Method ◽

Fragment Size ◽

Molecular Genetic ◽

Extraction Methods ◽

Degraded Dna ◽

Size Analysis ◽

Dna Fragments ◽

Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Download Full-text

In Silico Study Suggesting the Bias of Primers Choice in the Molecular Identification of Fungal Aerosols

Journal of Fungi ◽

10.3390/jof7020099 ◽

2021 ◽

Vol 7 (2) ◽

pp. 99

Author(s):

Hamza Mbareche ◽

Marc Veillette ◽

Guillaume J. Bilodeau

Keyword(s):

In Silico ◽

Fungal Diversity ◽

High Throughput Sequencing ◽

In Silico Analysis ◽

Its Region ◽

Internal Transcribed Spacers ◽

Sequence Length ◽

Universal Method ◽

Continuous Work ◽

Its1 And Its2

This paper presents an in silico analysis to assess the current state of the fungal UNITE database in terms of the two eukaryote nuclear ribosomal regions, Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2), used in describing fungal diversity. Microbial diversity is often evaluated with amplicon-based high-throughput sequencing approaches, which is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of the primers used for amplification. The goal of this study is to validate if the mismatches of the primers on the binding sites of the targeted taxa could explain the differences observed when using either ITS1 or ITS2 in describing airborne fungal diversity. Hence, the choice of the pairs of primers for each barcode concur with a study comparing the performance of ITS1 and ITS2 in three occupational environments. The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2. However, the database contains an equal number of unidentified taxa from ITS1 and ITS2 regions in the six taxonomic levels employed (phylum, class, order, family, genus, species). The chosen ITS primers showed differences in their ability to amplify fungal sequences from the UNITE database. Eleven taxa consisting of Trichocomaceae, Dothioraceae, Botryosphaeriaceae, Mucorales, Saccharomycetes, Pucciniomycetes, Ophiocordyceps, Microsporidia, Archaeorhizomycetes, Mycenaceae, and Tulasnellaceae showed large variations between the two regions. Note that members of the latter taxa are not all typical fungi found in the air. As no universal method is currently available to cover all the fungal kingdom, continuous work in designing primers, and particularly combining multiple primers targeting the ITS region is the best way to compensate for the biases of each one to get a larger view of the fungal diversity.

Download Full-text

Patterns of intraspecific morphological variability in soil mites reflect their dispersal ability

Experimental and Applied Acarology ◽

10.1007/s10493-020-00587-y ◽

2021 ◽

Vol 83 (2) ◽

pp. 241-255

Author(s):

Julia Baumann

Keyword(s):

Molecular Genetic ◽

Morphological Variability ◽

Mite Species ◽

Molecular Genetic Analysis ◽

Dispersal Ability ◽

Geometric Morphometric ◽

Geographic Population ◽

Mobile Species ◽

Population Structures ◽

Subdivided Populations

AbstractThe ability to disperse is one of the most important factors influencing the biogeography of species and speciation processes. Highly mobile species have been shown to lack geographic population structures, whereas less mobile species show genetically strongly subdivided populations which are expected to also display at least subtle phenotypic differences. Geometric morphometric methods (GMM) were now used to analyze morphological differences between European populations of a presumed non-phoretic, little mobile mite species in comparison to a highly mobile, phoretic species. The non-phoretic species Scutacarus carinthiacus showed a phenotypic population structure, whereas the phoretic species S. acarorum displayed homogeneity. These different patterns most probably can be explained by different levels of gene flow due to different dispersal abilities of the two species. GMM proved to be a sensitive tool that is especially recommendable for the analysis of (old) museum material and/or specimens in microscopic slides, which are not suitable for molecular genetic analysis.

Download Full-text