An Efficient Duplex PCR Method for Sex Identification of the European Grapevine Moth Lobesia botrana (Lepidoptera: Tortricidae) at Any Developmental Stage

2020 ◽  
Vol 113 (5) ◽  
pp. 2505-2510
Author(s):  
Carlos Aguirre ◽  
Natalia Olivares ◽  
Patricio Hinrichsen
Keyword(s):  
Developmental Stage ◽  
Lobesia Botrana ◽  
Sex Identification ◽  
Morphological Identification ◽  
Sequence Information ◽  
Duplex Pcr ◽  
W Chromosome ◽  
Starting Point ◽  
Pcr Method ◽  
Specific Sequences

Abstract Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult. The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.

scholarly journals Morphological and molecular identification of potato cyst nematode populations in Serbia

2010 ◽  
Vol 62 (3) ◽  
pp. 747-754 ◽  
Author(s):  
Violeta Oro ◽  
Z. Ivanovic ◽  
B. Nikolic ◽  
L. Barsi ◽  
M. Radivojevic ◽  
...  
Keyword(s):  
Sibling Species ◽  
Dna Isolation ◽  
Cyst Nematode ◽  
Morphological Identification ◽  
Potato Cyst Nematodes ◽  
Duplex Pcr ◽  
Cyst Nematodes ◽  
Quarantine Species ◽  
Pcr Method ◽  
Morphological Differences

Quarantine species such as potato cyst nematodes Globodera rostochiensis and G. pallida are present in Serbia since 1999 and 2005, respectively. These nematodes are sibling species and their morphological identification is complex due to their morphometric overlap. The cysts from the localities of Kladnica, Sanac, Gojna Gora and Milatovici were grown on susceptible potato varieties and their morphological differences have been discussed. To avoid ambiguities in species morphological designation a duplex PCR method was chosen for a rapid and accurate species identification. The whole procedure, from DNA extraction to DNA isolation, can be performed in a single day. .

scholarly journals Gonoactivity of Culex (Culex) (Diptera: Culicidae) Mosquitoes During Winter in Temperate Argentina

2021 ◽  
Author(s):  
María Florencia Branda ◽  
Magdalena Laurito ◽  
Andrés Mario Visintin ◽  
Walter Ricardo Almirón
Keyword(s):  
Developmental Stage ◽  
Culex Quinquefasciatus ◽  
Culex Pipiens ◽  
Life Cycles ◽  
Morphological Identification ◽  
Dry Ice ◽  
Culex Pipiens Pipiens ◽  
Host Seeking ◽  
Baited Traps ◽  
The City

Abstract The subgenus Culex L. includes species involved in summer–autumn arbovirus transmission but studies during winter are scarce in temperate Argentina. Female specimens were collected host-seeking at dry-ice-baited traps during autumn–winter–spring at two sites in Córdoba City during 2016 and 2017. The specimens were morphologically identified and dissected to determine the follicular developmental stage (gonotrophic activity). Females with advanced follicular stages (≥III) were subjected to molecular procedures to confirm or re-identify previous morphological identification. Five species (Culex apicinus Philippi (Diptera: Culicidae), Culex dolosus (Lynch-Arribálzaga) (Diptera: Culicidae), Culex maxi Dyar (Diptera: Culicidae), Culex pipiens pipiens L. (Diptera: Culicidae), and Culex quinquefasciatus Say (Diptera: Culicidae)) were collected and found gonoactive during winter; showing that a high proportion of Culex (Culex) females remain reproductively active during the unfavorable season for mosquito populations. Among them, it is worth noting the collection of Cx. quinquefasciatus, vector of the St. Louis encephalitis virus (endemic in the city), a specimen of Cx. p. pipiens, and a hybrid of Cx. p. pipiens/Cx. quinquefasciatus (during autumn). The study of this community during winter should continue because a high gonoactive female proportion with advanced follicular stages was found: 29.12 and 13.07% in 2016 and 2017, respectively. Local studies such as this one provide evidence about ornithophilic Culex species with active year-round life cycles, species that could favor arbovirus overwintering.

Sex identification of pigeons using polymerase chain reaction analysis with simple DNA extraction

2019 ◽  
Vol 12 (2) ◽  
pp. 45-48
Author(s):  
Shao-jie Liang ◽  
Ming-xia Chen ◽  
Chun-qi Gao ◽  
Hui-chao Yan ◽  
Guo-long Zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Polymerase Chain Reaction Analysis ◽  
Sex Identification ◽  
Z Chromosome ◽  
W Chromosome ◽  
Chain Reaction ◽  
Assay Time ◽  
Polymerase Chain ◽  
The Cost

Sex identification plays an important role in avian production. Hitherto, it is difficult to distinguish the sexes of monomorphic birds based on their external features. The chromo-helicase-DNA-binding genes contain CHD-W gene and CHD-Z gene, which are located on the W chromosome and Z chromosome, respectively. Since CHD-W gene is unique to females, the polymerase chain reaction can be used for sex identification. However, extracting DNA procedures for verifying the sex is tedious and expensive. To address these disadvantages, the objective of this study was to develop a simple DNA extraction assay to efficiently process blood, liver, and feather samples. The results showed that 2% dimethylsulfoxide was suitable for processing blood, and phosphate-buffered saline was suitable for processing liver and feather samples. The specific primers were designed, and the length of the targets is 474 bp on Z chromosome and 319 bp on W chromosome. The pigeons were identified as females based on the presence of two bands on the gel, and as males based on the presence of one band. Taken together, our results suggested that feather samples were more appropriate than blood or liver for sex identification of pigeons. Compared to the traditional DNA extraction, this method shortened the assay time and reduced the cost.

369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR

2007 ◽  
Vol 19 (1) ◽  
pp. 300
Author(s):  
M. Zhang ◽  
Q. Fu ◽  
W. S. Qin ◽  
H. Y. Zheng ◽  
Y. Q. Lu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Pcr Amplification ◽  
Single Copy ◽  
Bubalus Bubalis ◽  
Sex Identification ◽  
Sry Gene ◽  
Embryo Sex ◽  
Pcr Method ◽  
Male Specific

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos. This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).

scholarly journals Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1967
Author(s):  
J. Dorado ◽  
M. Bottrel ◽  
I. Ortiz ◽  
M. Díaz-Jiménez ◽  
B. Pereira ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Embryo Transfer ◽  
Recovery Rate ◽  
Embryo Quality ◽  
Donor Age ◽  
Successive Cycle ◽  
Assisted Reproductive Technique ◽  
Factors Affecting ◽  
Embryo Recovery ◽  
Starting Point

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds.

The concept of temperament in personality research

1987 ◽  
Vol 1 (2) ◽  
pp. 107-117 ◽  
Author(s):  
Jan Strelau
Keyword(s):  
Developmental Stage ◽  
Personality Research ◽  
Starting Point

The place of temperament in personality research has been broadly discussed taking into account different understandings of both concepts ‐ temperament and personality. Temperament may be regarded as (a) one of the elements of personality, (b) as a synonym of personality, and (c) as a phenomenon with its own specificity, not belonging to the structure of personality. Taking the latter position as a starting point, the author discusses five aspects in which temperament and personality differ: (a) the determinants of development, (6) the developmental stage in which temperament and personality are thought to be shaped, (c) the populations to which they refer, (d) the degree to which they are saturated with contents of behaviour, and (e) the role both personality and temperament play in integrating behaviour. It is concluded that the phenomena in which both concepts differ are not as strongly opposed as they are complementary.

scholarly journals Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Kebo Xie ◽  
Xiaoxiang Dou ◽  
Ridao Chen ◽  
Dawei Chen ◽  
Cheng Fang ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Molecular Level ◽  
Sequence Information ◽  
Content Type ◽  
C Terminus ◽  
Starting Point ◽  
Absidia Coerulea ◽  
Fungal Kingdom

ABSTRACT In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level. IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

scholarly journals COMPARISON OF METHODS FOR DETECTION OF MICROSPORIDIA SPECIES OF THE GENUS NOSEMA IN HONEY BEES (APIS MELLIFERA)

2013 ◽  
Vol 6 (1) ◽  
pp. 19-27
Author(s):  
Uroš Glavinić ◽  
Aleksandar Stanković ◽  
Jevrosima Stevanović ◽  
Predrag Simeunović ◽  
Nevenka Aleksić ◽  
...  
Keyword(s):  
Honey Bees ◽  
Pcr Analysis ◽  
Duplex Pcr ◽  
Infection Level ◽  
Nosema Species ◽  
Pcr Method ◽  
Biological Methods ◽  
Pcr Techniques ◽  
Microsporidia Species

Two microsporidia species of the Nosema genus cause nosemosis in the adult honeybee: N. apis and N. ceranae. For diagnostic purposes and the determination of infection level various microscopic and molecular biological methods are used. Th e aim of this research was to compare the reliability of the traditional microscopic assessment and two PCR techniques: simplex- and duplex-PCR. Honey bee samples were taken from 150 colonies. Microscopic examination, performed according to the recommendations of the OIE, revealed Nosema spores in 68.67% samples analysed, whilst with the simplex-PCR method all samples (100.0%) proved positive. On the other hand, duplex-PCR method used for the identifi cation of Nosema species resulted in 84.0% positive samples, all of which were N. ceranae. Our recommendation of the simplex-PCR method for the monitoring of honey bees in fi eld conditions is based on its higher reliability than the microscopic assessment in the detection of low-level infections, as well as its potential for the detection of vegetative Nosema sp. stages; thus the early detection and timely prevention of Nosema infection would be possible. Nosema species identifi cation is simplest and most cost-eff ective if performed with the duplex-PCR analysis. However, the simplex-PCR is more reliable, thus, it is suggested that samples that were negative when assessed with microscopy and duplex-PCR analysis undergo simplex-PCR.

Identification and authentication of commercial mi-iuy croaker (Miichthys miiuy) products by two PCR-based methods

2020 ◽  
Author(s):  
tae sun kang
Keyword(s):  
Economic Value ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Duplex Pcr ◽  
Consensus Sequences ◽  
Pcr Product ◽  
Otolithes Ruber ◽  
Pcr Method ◽  
First Time ◽  
Miichthys Miiuy

Mi-iuy croaker (Miichthys miiuy) is one of the most important ingredients of Korean cuisine and thus has the highest economic value. However, the similar morphological traits among croaker fish belonging to family Sciaenidae are often exploited for seafood fraud. In this study, M. miiuy-specific primer set was designed and further improved by the development of a rapid and cost-effective duplex PCR method. The specificity of M. miiuy-specific duplex PCR was tested using 22 seafood species, and no cross-reactivity was observed. The sensitivity of the PCR assay was found to be 0.1 ng/µL. For the first time, labeling compliance of 43 commercial m-iuy croaker products was verified using both full DNA barcoding and M. miiuy-specific duplex PCR methods. For species identification, BOLDSYSTEMS and GenBank database were screened with the consensus sequences of each PCR product as a query. This identification result was further confirmed using the M. miiuy-specific duplex PCR method. The findings of this study revealed that principal species substituted were law croaker (Pseudotolithus senegallus, n=4), bigeye croaker (Micropogonias megalops, n=3), whitemouth croaker (Micropogonias furnieri, n=1), and tigertoothed croaker (Otolithes ruber, n=1). A significant percentage (21%) of mislabeling was present in commercial mi-iuy products sold on the South Korea market.

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