Studies on the classic shell colour and banding polymorphism of the land snail Cepaea played a crucial role in establishing the importance of natural selection in maintaining morphological variation. Cepaea is also a pre-eminent model for ecological genetics because the outward colour and banding phenotype is entirely genetically determined, primarily by a ‘supergene’ of at least five loci. Unfortunately, progress in understanding the evolution and maintenance of the Cepaea polymorphism stalled, partly because of a lack of genetic markers. With a view to re-establish Cepaea as a prominent model of molecular ecology, we made six laboratory crosses of Cepaea nemoralis, five of which segregated for shell ground colour (C) and the presence or absence of bands (B). First, scoring of colour and banding in 323 individuals found no recombination between the C and B loci of the supergene. Second, using restriction site–associated DNA sequencing (RAD-Seq) of two parents and 22 offspring, we identified 44 anonymous markers putatively linked to the colour (C) and banding (B) loci. The genotype of eleven of the most promising RAD-Seq markers was independently validated in the same 22 offspring, then up to a further 146 offspring were genotyped. The closest RAD-Seq markers scored are within ~0.6 centimorgan (cM) of the C-B supergene linkage group, with the combined loci together forming a 35.8 cM linkage map of markers that flank both sides of the Cepaea C-B supergene.
Linkage map marker sequence assemblies
RAD-tag sequences and paired-end contig assemblies for 11 RAD loci, which were used to develop a linkage map of genetic markers flanking the shell colour (C) and banding presence (B) loci of the Cepaea nemoralis supergene. The sequences were generated by Illumina sequencing of three RAD-Seq libraries comprising both parents and a total 24 F1 offspring from a single cross (C100 x C101) that segregated for shell colour (C) and presence or absence of banding (B). De novo assembly of RAD loci was performed using RADtools 1.2.4 (http://www.RAD-Seq.info). A combination of Unix and Excel commands were then used to identify candidate RAD loci that cosegregate with colour and banding. Paired-end reads were assembled into contigs using VelvetOptimiser version 2.1.7, separately for each of the three RAD libraries. For the RAD markers shown, the corresponding contigs were concatenated and then manually aligned into longer, high-quality contigs. Each sequence is labelled: RAD marker ID – allelic phase with the supergene loci – sequence type – length – coverage. Contigs that could not be aligned are labelled as such.
Cepaea nemoralis RAD markers RAD-tag and paired-end sequence assemblies.fas
Linkage mapping genotypes dataset
Genotypes used to produce a linkage map of 11 RAD-Seq derived markers flanking the shell colour (C) and banding presence (B) loci of the Cepaea nemoralis supergene. Data for two crosses are shown: C100 x C101 was a cross between a double homozygous recessive yellow-banded mother and a heterozygous father, resulting in either yellow-unbanded or pink-banded F1 offspring (n = 102). C118 x C119 was a cross between a double homozygous recessive yellow-banded parent and a pink-banded parent heterozygous for colour, but not banding. Full details of the crosses and the genotype assays are provided in the paper. Marker genotypes are provided in CRIMAP formatting.
Cepaea linkage map genotypes dataset.xlsx
Shell colour and banding phenotype data from 6 Cepaea nemoralis crosses
Phenotype data for the shell ground colour (C)and banding presence (B) supergene loci, scored in 398 individuals from 6 crosses of Cepaea nemoralis. As C. nemoralis is a simultaneous hermaphrodite, offspring from both parents were used, except for crosses C100 x C101 and C108 x C109, where a non-virgin snail (C100 and C108 respectively) was removed after mating. Full details of the crosses, rearing of the snails and analysis of the data are provided in the paper.
Cepaea crosses colour and banding phenotype data.xlsx
