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 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.