scholarly journals Molecular and morphological approaches for species delimitation and hybridization investigations of two Cichla species

2017 ◽  
Vol 107 (0) ◽  
Author(s):  
Andrea A. F. Mourão ◽  
Diogo Freitas-Souza ◽  
Diogo T. Hashimoto ◽  
Daniela C. Ferreira ◽  
Fernanda D. do Prado ◽  
...  
Keyword(s):  
Species Delimitation ◽  
Parental Species ◽  
Future Research ◽  
Correct Identification ◽  
Pcr Multiplex ◽  
Pcr Rflp ◽  
Genetic Pattern ◽  
Morphometric Methods ◽  
Species Specific ◽  
Specific Nucleotide

ABSTRACT The hybridization is a widely-discussed issue in several studies with fish species. For some authors, hybridization may be related with diversification and speciation of several groups, or also with the extinction of populations or species. Difficulties to differentiate species and hybrids may be a problem to correctly apply a management of wild species, because hybrid lineages, especially the advanced ones, may resemble the parental species. The genus Cichla Bloch & Schneider, 1801 constitutes an interesting experimental model, considering that hybridization and taxonomic uncertainties hinder a correct identification. Considering these problems, in this study, we developed genetic methodologies and applied meristic and morphometric approaches in wild samples in order to identify species and for test a possible hybridization between Cichla kelberi Kullander & Ferreira, 2006 and Cichla piquiti Kullander & Ferreira, 2006. For this, C. kelberi, C. piquiti and potential hybrid ( carijó) individuals were collected in Paraná and Tietê rivers (SP, Brazil). For meristic and morphometric methods, the individuals were analyzed using the statistical software Pcord 5:31, while for molecular methods, primers for PCR-multiplex were designed and enzyme for PCR-RFLP were selected, under the species-specific nucleotide. All results indicated that the carijó is not an interspecific hybrid, because it presented identical genetic pattern and morphology closed to C. piquiti. Thus, we propose that carijó is a C. piquiti morphotype. In addition, this study promotes a new molecular tool that could be used in future research, monitoring and management programs of the genus Cichla.

scholarly journals Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections

2020 ◽  
Vol 6 (4) ◽  
pp. 308
Author(s):  
Joana Carvalho-Pereira ◽  
Filipa Fernandes ◽  
Ricardo Araújo ◽  
Jan Springer ◽  
Juergen Loeffler ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Correct Identification ◽  
Colony Pcr ◽  
Pcr Multiplex ◽  
Pathogen Dna ◽  
Species Specific

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

Molecular identification of some Hoplolaimus species from the USA based on duplex PCR, multiplex PCR and PCR-RFLP analysis

Nematology ◽  
2009 ◽  
Vol 11 (3) ◽  
pp. 471-480 ◽  
Author(s):  
Robert Robbins ◽  
Allen Szalanski ◽  
Chang-Hwan Bae
Keyword(s):  
Multiplex Pcr ◽  
Dna Sequences ◽  
Rflp Analysis ◽  
Pcr Primers ◽  
Interspecific Variation ◽  
Molecular Techniques ◽  
Specific Pcr ◽  
Pcr Multiplex ◽  
Pcr Rflp ◽  
Species Specific

AbstractTwo different molecular approaches, a multiplex PCR and PCR-RFLP of ITS-rDNA, were developed for the identification of Hoplolaimus species. DNA sequences of H. columbus, H. galeatus, H. concaudajuvencus, H. magnistylus, H. seinhorsti and three undescribed Hoplolaimus species were used to design species-specific primers. Three reverse species-specific PCR primers for H. columbus, H. galeatus and H. magnistylus were developed using the ITS1 region exhibiting interspecific variation. Three species-specific PCR primers in combination with the forward primer, Hoc-1f, produced distinct amplicons of 580 bp for H. columbus, 120 bp for H. galeatus and 340 bp for H. magnistylus. We successfully identified each of three species by multiplex PCR when all three were mixed in a single PCR reaction. Restriction enzyme digests of the PCR amplicon using HaeIII and RsaI permitted discrimination of H. columbus, H. galeatus, H. magnistylus, H. concaudajuvencus, H. sp. 1, H. sp. 2 and H. sp. 3 from each other. These results suggest that these molecular techniques allow for rapid, easy and reliable identification of Hoplolaimus species.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera

2018 ◽  
Vol 57 (5) ◽  
pp. 643-648
Author(s):  
Milena Kordalewska ◽  
Joanna Kalita ◽  
Zofia Bakuła ◽  
Anna Brillowska-Dąbrowska ◽  
Tomasz Jagielski
Keyword(s):  
Specific Identification ◽  
Pcr Rflp ◽  
Identification Of Fungi ◽  
Species Specific

scholarly journals Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass

Plant Disease ◽  
2019 ◽  
Vol 103 (8) ◽  
pp. 2010-2014 ◽  
Author(s):  
J. Francisco Iturralde Martinez ◽  
Francisco J. Flores ◽  
Alma R. Koch ◽  
Carla D. Garzón ◽  
Nathan R. Walker
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Temperature Cycling ◽  
Molecular Technique ◽  
Correct Identification ◽  
Internal Transcribed Spacer Region ◽  
Spring Dead Spot ◽  
End Point ◽  
Specificity And Sensitivity ◽  
Species Specific

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5′ terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

scholarly journals Leafhoppers and cixiids in phytoplasma-infected carrot fields: Species composition and potential phytoplasma vectors

2010 ◽  
Vol 25 (4) ◽  
pp. 311-318 ◽  
Author(s):  
Tanja Drobnjakovic ◽  
Pantelija Peric ◽  
Dejan Marcic ◽  
Luca Picciau ◽  
Alberto Alma ◽  
...  
Keyword(s):  
Control Strategies ◽  
Species Level ◽  
Effective Control ◽  
Correct Identification ◽  
Vegetable Crops ◽  
Vegetative Period ◽  
Aster Yellows ◽  
Scaphoideus Titanus ◽  
The Family ◽  
Pcr Rflp

The first molecular analysis of samples collected in southern Backa (Serbia) confirmed the presence of aster yellows (16SrI) and stolbur phytoplasmas (16SrXII) in insects belonging to the family Cicadellidae, as well as in carrot plants where the insects were collected. A correct identification of the phytoplasmas and their vectors is essential to arrange effective control strategies to prevent diseases associated with phytoplasmas from spreading to carrots and other vegetable crops. In order to enhance knowledge about insect vectors of aster yellows and stolbur phytoplasmas in Serbia, Cicadellidae and Cixiidae (Homoptera Auchenorrhyncha), the most common vectors of these phytoplasmas, were monitored in southern Backa during 2008. Adults leaf- and planthoppers were collected and identified at species level using standard entomological methods, and tested for phytoplasma presence by means of PCR/RFLP. A total of 13 insect species of Cicadellidae were identified, as follows: a) three species of the subfamily Agallinae: Anaceratagallia ribauti (Ossiannilsson), Anaceratagallia venosa (Fourcroy), and Anaceratagallia laevis (Ribaut); b) seven species of the subfamily Deltocephalinae: Psammotettix confinis (Dahlbom), Psammotettix striatus (Linnaues) Psammottettix alienus (Dahlbom), Macrosteles sexnotatus (Fall?n), Ophiola decumana (Kontkanen), Errastunus ocellaris Fall?n, and Scaphoideus titanus Ball; c) three species of the subfamily Typhlocibinae: Eupteryx atropunctata (Goeze), Eupteryx mellissae Curtis, Zyginidia pullula (Boheman). Female specimens of the genus Euscelis (Deltocephalinae) were also collected, as well as one species of Reptalus quinquecostatus (Dufour) of the family Cixiidae. Stolbur phytoplasmas were detected in A. laevis, A. ribauti, A. venosa, P. striatus, P. confinis and P. alienus. The species: A. laevis, O. decumana, and P. confinis were AY-infected (subgroup 16SrI-A), while subgroup 16SrI-C was found only in one specimen of P. confinis. Since some aster yellows- and stolbur-infected species of the genera Psammotettix and Anaceratagallia (especially P. confinis and A. laevis) were regularly and commonly found in the infected carrot fields during the whole vegetative period, they could play a significant role in transmitting and spreading these pathogens in natural environment.

Systematics of the Australian golden trapdoor spiders of the Euoplos variabilis-group (Mygalomorphae : Idiopidae : Euoplini): parapatry and sympatry between closely related species in subtropical Queensland

2021 ◽  
Author(s):  
Jeremy D. Wilson ◽  
Michael G. Rix
Keyword(s):  
Species Delimitation ◽  
Systematic Approach ◽  
Sequence Data ◽  
Future Research ◽  
Populated Area ◽  
Closely Related Species ◽  
Taxonomic Description ◽  
Eastern Australia ◽  
First Time ◽  
Adequate Data

The Australian golden trapdoor spiders of the tribe Euoplini (family Idiopidae) are among the most abundant and diverse of mygalomorph lineages in subtropical eastern Australia. Throughout this highly populated area, species in the monophyletic Euoplos variabilis-group are largely ubiquitous; however, species delimitation has long proven difficult in the group because species are morphologically very similar and have parapatric or even sympatric distributions. We address these challenges in the variabilis-group, and explore the phylogeny and taxonomy of species using an integrative systematic approach. In doing so, we apply a conservative, pragmatic methodology, naming only species for which adequate data are available (namely sequence data and unequivocally linked male specimens), and explicitly stating and mapping material that could not be linked to a species, to aid future research on the group. We describe five new species from south-eastern Queensland –E. booloumba sp. nov., E. jayneae sp. nov., E. raveni sp. nov., E. regalis sp. nov. and E. schmidti sp. nov.; we redescribe two previously named species – E. similaris (Rainbow & Pulleine, 1918) and E. variabilis (Rainbow & Pulleine, 1918); and we reillustrate the recently described E. grandis Wilson & Rix, 2019. The nominate species, E. variabilis, is shown to have a far smaller distribution than previously thought, and E. similaris is given a modern taxonomic description for the first time. A key to adult male specimens is also provided. This study further reveals a case of sympatry between two species within the variabilis-group; both E. raveni sp. nov. and E. schmidti sp. nov. occur in the Brisbane Valley, south of the Brisbane River – a notable result given that closely related mygalomorph species usually occur allopatrically. This work updates what is currently known of the phylogeny and diversity of one of the dominant mygalomorph lineages of subtropical eastern Australia, resolving a complex and highly endemic fauna. http://zoobank.org/urn:lsid:zoobank.org:pub:A4FB92F6-EFFF-4468-B1D8-000D69923996

scholarly journals PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

2010 ◽  
Vol 43 (1) ◽  
pp. 100-101 ◽  
Author(s):  
Aline Weber Medeiros ◽  
Pedro d'Azevedo ◽  
Rebeca Inhoque Pereira ◽  
Ana Paula Cassenego ◽  
Sueli Van Der Sand ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Food Samples ◽  
Correct Identification ◽  
Accurate Identification ◽  
16S Ribosomal Dna ◽  
Pcr Rflp ◽  
Enterococcus Casseliflavus ◽  
Dna Pattern ◽  
Enterococcus Gallinarum ◽  
Rdna Analysis

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

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 Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae

2014 ◽  
Vol 23 (4) ◽  
pp. 495-500 ◽  
Author(s):  
Michelle Cardoso dos Santos ◽  
Mônica Regina Vendrame Amarante ◽  
Maria Regina Lucas da Silva ◽  
Alessandro Francisco Talamini do Amarante
Keyword(s):  
Haemonchus Contortus ◽  
Tail Length ◽  
Pcr Analysis ◽  
Correct Identification ◽  
Specific Primer ◽  
Male Adult ◽  
Domestic Ruminants ◽  
Third Stage ◽  
Species Specific ◽  
Haemonchus Placei

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

PCR-based molecular discrimination betweenMaritrema eroliaeandProbolocoryphe uca(Digenea: Microphallidae) in Kuwait Bay

2013 ◽  
Vol 88 (2) ◽  
pp. 177-182
Author(s):  
W.Y. Al-Kandari ◽  
S.A. Al-Bustan ◽  
M. Alnaqeeb ◽  
A.M. Isaac
Keyword(s):  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Direct Pcr ◽  
Specific Primers ◽  
Kuwait Bay ◽  
Future Studies ◽  
Larval Stages ◽  
Marine Snails ◽  
Pcr Rflp ◽  
Species Specific

AbstractMicrophallid trematodes are common parasites in marine snails and crustacean hosts at Kuwait Bay. The larval stages of two microphallids,Maritrema eroliaeandProbolocoryphe uca, are difficult to differentiate morphologically. In this study, two PCR-based techniques were established for quick and accurate discrimination between the larval stages of the two microphallid species, employing restriction fragment length polymorphism (PCR-RFLP) and species-specific primers. Both techniques utilized nucleotide differences in the second internal transcribed region (ITS2) of the ribosomal DNA (rDNA) in the two species. For the PCR-RFLP technique, restriction enzymeAvaII was selected and it generated different restriction profiles among the two microphallids. In addition, species-specific primers were prepared for each microphallid species that amplified distinctive fragments. Both techniques showed that the larval stages of the two microphallid species can be identified accurately. However, direct PCR amplification using species-specific primers was more advantageous than the PCR-RFLP technique since it allowed rapid and specific discrimination between the two species. This technique provides a useful tool that can be used in future studies for the study of the distribution of microphallid species and their definitive hosts at different localities of Kuwait Bay.

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