A rapid <scp>qPCR</scp> method for genetic sex identification of <i>Salmo salar</i> and <i>Salmo trutta</i> including simultaneous elucidation of interspecies hybrid paternity by high‐resolution melt analysis

d’Auriac, Marc Anglès; Urke, Henning Andre; Kristensen, Torstein

doi:10.1111/jfb.12401

Cited by 12 publications

(17 citation statements)

References 15 publications

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“…Microsatellite loci str543INRA [24], str60INRA [25], and T3-13 [26] were used for the identification of the parental and gynogenetic genotype. The genetic sex of the gynogenetic offspring was evaluated using the Y-chromosome-related DNA markers (sdY-Fw) [27]. PCR reactions were conducted using the Eppendorf Mastercycler Personal thermocycler and the reaction mixture 2xPCR Master Mix (A&A Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

High Rate of Deformed Larvae among Gynogenetic Brown Trout (Salmo trutta m. fario) Doubled Haploids

Jagiełło

Zalewski

Dobosz

et al. 2017

BioMed Research International

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Mitotic gynogenesis results in the production of fully homozygous individuals in a single generation. Since inbred fish were found to exhibit an increased frequency of body deformations that may affect their survival, the main focus of this research was to evaluate the ratio of individuals with spinal deformities among gynogenetic doubled haploids (DHs) brown trout as compared to nonmanipulated heterozygous individuals. Gynogenetic development was induced by the activation of brown trout eggs by UV-irradiated homologous and heterologous (rainbow trout) spermatozoa. The subsequent exposure of the activated eggs to the high hydrostatic pressure disturbed the first cleavage in gynogenetic zygotes and enabled duplication of the maternal haploid set of chromosomes. The survival rate was significantly higher among gynogenetic brown trout hatched from eggs activated with the homologous UV-irradiated spermatozoa when compared to DHs hatched from eggs activated by the heterologous spermatozoa. More than 35% of the gynogenetic larvae exhibited body deformities, mostly lordosis and scoliosis. The percentage of malformed brown trout from the control group did not exceed 15%. The increased number of deformed larvae among DHs brown trout suggested rather a genetic background of the disease related to the fish spine deformities; however, both genetic and environmental factors were discussed as a cause of such conditions in fish.

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Section: Methodsmentioning

confidence: 99%

High Rate of Deformed Larvae among Gynogenetic Brown Trout (Salmo trutta m. fario) Doubled Haploids

Jagiełło

Zalewski

Dobosz

et al. 2017

BioMed Research International

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“…DNA quality of the O . tshawytscha individuals had been previously successfully tested [45] and gave negative results when used with the S . salar and S .…”

Section: Resultsmentioning

confidence: 99%

COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

d’Auriac

2016

PLoS ONE

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Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered.

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“…erinaceum with six Single Nucleotide Polymorphisms (SNPs). Sequence variation among PCR products of same length may be easily differentiated, without sequencing, by using HRM for various diagnostic purposes such as for example fish 35,36 , mollusc 37,38 and microalgae 39 species and genotypic sex identification. In this study HRM was used on the 350 bp psb A amplicons to identify three maerl species, Lithothamnion erinaceum , Lithothamnion cf.…”

Section: Discussionmentioning

confidence: 99%

Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

d’Auriac

Gall

Peña

et al. 2019

Sci Rep

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Coralline algae form extensive maerl and rhodolith habitats that support a rich biodiversity. Calcium carbonate harvesting as well as trawling activities threatens this ecosystem. Eleven species were recorded so far as maerl-forming in NE Atlantic, but identification based on morphological characters is unreliable. As for most red algae, we now use molecular characters to resolve identification of these taxa. However, obtaining DNA sequences requires time and resource demanding methods. The purpose of our study was to improve methods for achieving simple DNA extraction, amplification, sequencing and sequence analysis to allow robust identification of maerl species and other coralline algae. Our novel and easy DNA preparation method for coralline algae was of sufficient quality for qPCR amplification and sequencing of all 47 tested samples. The new psbA qPCR assay successfully amplified a 350 bp fragment identifying six species and uncovering two new Operational Taxonomic Units. Molecular results were corroborated with anatomical examination using i.e. scanning electron microscopy. Finally, the qPCR assay was coupled with High Resolution Melt analysis that successfully differentiated the closely related species Lithothamnion erinaceum and L. cf. glaciale. This DNA preparation and qPCR technique should vitalize coralline research by reducing time and cost associated with molecular systematics.

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A rapid qPCR method for genetic sex identification of Salmo salar and Salmo trutta including simultaneous elucidation of interspecies hybrid paternity by high‐resolution melt analysis

Cited by 12 publications

References 15 publications

High Rate of Deformed Larvae among Gynogenetic Brown Trout (Salmo trutta m. fario) Doubled Haploids

High Rate of Deformed Larvae among Gynogenetic Brown Trout (Salmo trutta m. fario) Doubled Haploids

COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

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