Fine Mapping of the Pond Snail Left-Right Asymmetry (Chirality) Locus Using RAD-Seq and Fibre-FISH

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.

A twisting story: how a single gene twists a snail? Mechanogenetics

“…This is a strong indication that dextrality is determined by a single gene or by genes at closely linked loci. This was recently confirmed by DNA mapping using RAD-Seq and Fibre-FISH techniques (Liu et al 2013). Furthermore, we could show that the gene dictates the cytoskeletal dynamics at the third cleavage.…”

Chromosome engineering in zygotes with CRISPR/Cas9

“…These were hybridized with probes using a standard FISH protocol (Liu et al ., 2013). The following BAC (Bacterial artificial chromosome) clones were use: RP23‐63G7 (87,292,751–87,445,311) labeled with Aminoallyl‐dUTP‐Texas Red (Jena‐Bioscience), RP24‐102H24 (87,884,864–88,075,187) labeled with Aminoallyl‐dUTP‐XX‐Atto‐488 (Jena‐Bioscience) within the genome edited region.…”

“…For the deletion, B lymphocytes isolated from spleens were grown in RPMI 1 10% FBS, stimulated using lipopolysaccharide (LPS, Sigma-Aldrich) and after 44 hours blocked in metaphase with 0.1 lg/mL KaryoMax Colcemid (Gibco Life Technologies) for 2 hours prior to harvesting chromosomes. These were hybridized with probes using a standard FISH protocol (Liu et al, 2013). The following BAC (Bacterial artificial chromosome) clones were use: RP23-63G7 (87,292,445,311) labeled with Aminoallyl-dUTP-Texas Red (Jena-Bioscience), RP24-102H24 (87,884,864-88,075,187) labeled with Aminoallyl-dUTP-XX-Atto-488 (Jena-Bioscience) within the genome edited region.…”

The report of my death was an exaggeration: A review for researchers using microsatellites in the 21st century

“…These methods enable discovery of thousands of markers, even in nonmodel organisms, and characterization of different levels of genetic variation across the genome (Hohenlohe et al, 2010; Rowe et al, 2011; Liu et al, 2013; Lu et al, 2013). The main differences between RAD-Seq and GBS are methodological, relating to which REs are used to digest DNA, how sequencing adapters and multiplexing barcodes are added to samples, and the use of a size selection step (Elshire et al, 2011; Cronn et al, 2012).…”