Fine mapping of the Cepaea nemoralis shell colour and mid-banded loci using a high-density linkage map

Abstract

Molluscs are a highly speciose phylum that exhibits an astonishing array of colours and patterns, yet relatively little progress has been made in identifying the underlying genes that determine phenotypic variation. One prominent example is the land snail Cepaea nemoralis for which classical genetic studies have shown that around nine loci, several physically linked and inherited together as a 'supergene', control the shell colour and banding polymorphism. As a first step towards identifying the genes involved, we used whole-genome resequencing of individuals from a laboratory cross to construct a high-density linkage map, and then trait mapping to identify 95% confidence intervals for the chromosomal region that contains the supergene, specifically the colour locus (C), and the unlinked mid-banded locus (U). The linkage map is made up of 215,593 markers, ordered into 22 linkage groups, with one large group making up ~27% of the genome. The C locus was mapped to a ~1.3 cM region on linkage group 11, and the U locus was mapped to a ~0.7 cM region on linkage group 15. The linkage map will serve as an important resource for further evolutionary and population genomic studies of C. nemoralis and related species, as well as the identification of candidate genes within the supergene and for the mid-banding phenotype.

Fig. 1. Linkage map showing the density of markers across all chromosomes and approximate position of mapped C and U loci.

Black lines indicate marker positions, and colours show increasing marker density, reflecting the number of markers at each map location, from least dense (blue) to most dense (red). Also shown are exemplar C. nemoralis shells, from left: yellow unbanded, pink unbanded, yellow mid-banded, pink mid-banded, yellow five-banded, and pink five-banded.

Fig. 2. Results of the genome scans showing LOD scores across all linkage groups.

a Results for the yellow phenotype and b results for the mid-banded phenotype. The red line indicates the 5% genome-wide significance level derived from the permutation test.

Fig. 3. Effect plots of genotypes for each phenotype investigated with associated LOD scores.

a Effects of the different genotypes on the presence of yellow shell colour phenotype on chromosome 11. b Effects of the different genotypes on the presence of the mid-banded phenotype on chromosome 15.

Fig. 4. Raw genotypes for all individuals at each putative locus associated with yellow shell ground colour within the interval Y2 (1 = yellow, 0 = not yellow).

a Raw genotypes at position 31.385, b raw genotypes at position 32.056, c raw genotypes at position 32.727, d raw genotypes at position 33.398. All positions except one, 33.398 d, show the same segregation pattern between alleles, where allele B is associated with yellow shell ground colour and A is associated with a non-yellow.

Fig. 5. Raw genotypes for all individuals at each putative locus associated with the mid-band phenotype within interval M2 (1 = mid-banded, 0 = not mid-banded).

a Raw genotypes at position 39.511 and b showing raw genotypes at position 40.182. The two positions shown here are the only two positions within this that fit the expected pattern, such that all BB genotypes correspond to mid-banded phenotype, and all AA genotypes correspond to lack of a mid-banded (usually five-banded). The other six locations within this interval did not fit the pattern and are shown in Supplementary Fig. S5.