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Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis

Identifying and understanding mechanisms that generate phenotypic diversity is a fundamental goal of evolutionary biology. With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail Cepaea nemoralis (Linnaeus, 1758) has been a model for evol...

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Autores principales: Affenzeller, Susanne, Cerveau, Nicolas, Jackson, Daniel John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114279/
https://www.ncbi.nlm.nih.gov/pubmed/30157182
http://dx.doi.org/10.1371/journal.pone.0201396
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author Affenzeller, Susanne
Cerveau, Nicolas
Jackson, Daniel John
author_facet Affenzeller, Susanne
Cerveau, Nicolas
Jackson, Daniel John
author_sort Affenzeller, Susanne
collection PubMed
description Identifying and understanding mechanisms that generate phenotypic diversity is a fundamental goal of evolutionary biology. With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail Cepaea nemoralis (Linnaeus, 1758) has been a model for evolutionary biologists and population geneticists for decades. However, the genetic mechanisms by which C. nemoralis generates this pigmented shell diversity remain unknown. An important first step in investigating this pigmentation pattern is to establish a set of validated reference genes for differential gene expression assays. Here we have evaluated eleven candidate genes for reverse transcription quantitative polymerase chain reaction (qPCR) in C. nemoralis. Five of these were housekeeping genes traditionally employed as qPCR reference genes in other species, while six alternative genes were selected de novo from C. nemoralis transcriptome data based on the stability of their expression levels. We tested all eleven candidates for expression stability in four sub-adult tissues of C. nemoralis: pigmented mantle, unpigmented mantle, head and foot. We find that two commonly employed housekeeping genes (alpha tubulin, glyceraldehyde 3-phosphate dehydrogenase) are unsuitable for use as qPCR reference genes in C. nemoralis. The traditional housekeeping gene UBIquitin on the other hand performed very well. Additionally, an RNA-directed DNA polymerase (RNAP), a Potassium Channel Protein (KCHP) and a Prenylated Rab acceptor protein 1 (PRAP), identified de novo from transcriptomic data, were the most stably expressed genes in different tissue combinations. We also tested expression stability over two seasons and found that, although other genes are more stable within a single season, beta actin (BACT) and elongation factor 1 alpha (EF1α) were the most reliable reference genes across seasons.
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spelling pubmed-61142792018-09-17 Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis Affenzeller, Susanne Cerveau, Nicolas Jackson, Daniel John PLoS One Research Article Identifying and understanding mechanisms that generate phenotypic diversity is a fundamental goal of evolutionary biology. With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail Cepaea nemoralis (Linnaeus, 1758) has been a model for evolutionary biologists and population geneticists for decades. However, the genetic mechanisms by which C. nemoralis generates this pigmented shell diversity remain unknown. An important first step in investigating this pigmentation pattern is to establish a set of validated reference genes for differential gene expression assays. Here we have evaluated eleven candidate genes for reverse transcription quantitative polymerase chain reaction (qPCR) in C. nemoralis. Five of these were housekeeping genes traditionally employed as qPCR reference genes in other species, while six alternative genes were selected de novo from C. nemoralis transcriptome data based on the stability of their expression levels. We tested all eleven candidates for expression stability in four sub-adult tissues of C. nemoralis: pigmented mantle, unpigmented mantle, head and foot. We find that two commonly employed housekeeping genes (alpha tubulin, glyceraldehyde 3-phosphate dehydrogenase) are unsuitable for use as qPCR reference genes in C. nemoralis. The traditional housekeeping gene UBIquitin on the other hand performed very well. Additionally, an RNA-directed DNA polymerase (RNAP), a Potassium Channel Protein (KCHP) and a Prenylated Rab acceptor protein 1 (PRAP), identified de novo from transcriptomic data, were the most stably expressed genes in different tissue combinations. We also tested expression stability over two seasons and found that, although other genes are more stable within a single season, beta actin (BACT) and elongation factor 1 alpha (EF1α) were the most reliable reference genes across seasons. Public Library of Science 2018-08-29 /pmc/articles/PMC6114279/ /pubmed/30157182 http://dx.doi.org/10.1371/journal.pone.0201396 Text en © 2018 Affenzeller et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Affenzeller, Susanne
Cerveau, Nicolas
Jackson, Daniel John
Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title_full Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title_fullStr Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title_full_unstemmed Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title_short Identification and validation of reference genes for qPCR in the terrestrial gastropod Cepaea nemoralis
title_sort identification and validation of reference genes for qpcr in the terrestrial gastropod cepaea nemoralis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114279/
https://www.ncbi.nlm.nih.gov/pubmed/30157182
http://dx.doi.org/10.1371/journal.pone.0201396
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