Abstract
The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, “D”. First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.
Highlights
Consistent left-right asymmetry is a decisive feature of embryonic development, yet the breaking of bilateral symmetry is still poorly understood [1]
A lesser number of snails were generated by backcrossing Dd heterozygotes to dd snails, with the Dd snail ‘‘father’’ being removed after mating and the dd used as the ‘‘mother’’
The genotype of 389 offspring was inferred, of which 221 were genetically dextral (DD or Dd) and 168 sinistral. This ratio differed from the 1:1 expected (X2 = 7.221; P,0.007). As it has previously been shown [13,14] that a high proportion of embryos fail to develop from a mother that is genetically sinistral, dd, we speculate that one explanation is differential mortality of dd individuals compared with Dd individuals from a common dd mother
Summary
Consistent left-right asymmetry is a decisive feature of embryonic development, yet the breaking of bilateral symmetry is still poorly understood [1]. In the hypothetical view of Brown and Wolpert [2], the general solution to the problem of symmetry breaking is provided by a pre-existing asymmetric molecular reference, or ‘‘F-molecule’’, which aligns with anteriorposterior and dorsal-ventral axes and creates an asymmetric signal, perhaps transporting an effector molecule towards the left or right. Pond snails of the genus Lymnaea have been used to study asymmetry for nearly 120 years. It has been known since 1894 that both the coil of the shell and the entire body asymmetry of a snail are entirely predicated by the spiral twist that takes place as the 4cell embryo divides [3]. In dominant dextral-coiling L. stagnalis (genotype DD or Dd) the movement of cells in the early embryo, and the twist of the adult animal’s shell is clockwise, whereas in genetically sinistral (recessive, dd) snails it is anticlockwise
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