Genetic insight into a polygenic trait using a novel Genome Wide Association approach in a wild amphibian population

Body size variation is central in the evolution of life history traits in amphibians, but the underlying genetic architecture of this complex trait is still largely unknown. Herein, we studied the genetic basis of body size and fecundity of the alternative morphotypes in a wild population of the Greek smooth newt (Lissotriton graecus). By combining a Genome-wide association approach with linkage disequilibrium network analysis, we were able to identify clusters of highly correlated loci thus maximizing sequence data for downstream analysis. The putatively associated variants explained 12.8% to 44.5% of the total phenotypic variation in body size and were mapped to genes with functional roles in the regulation of gene expression and cell cycle processes. Our study is the first to provide insights into the genetic basis of complex traits in newts and provides a useful tool to identify loci potentially involved in fitness related traits in small data sets from natural populations in non-model species.

Methods
Phenotypic measurments were collected from female Greek smooth newts, including snout to vent length, body weight and reproductive components.

Genomic DNA was extracted from tissue samples. ddRAD libraries were produced using an IGATech custom protocol (IGA Technology Services, Udine, Italy), with minor modifications with respect to Peterson’s double digest restriction-site associated DNA preparation (Peterson et al., 2012), using SphI (5’GCATG 3’) and BamHI (5’GGATCC 3’) endonucleases. Library pools were selected on a BluePippin (Sage Science Inc. Beverly, MA, USA) setting the range to 380–500 bp. The resulting libraries were checked with both a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and by Bioanalyzer DNA Assay (Agilent Technologies, Santa Clara, CA). Libraries were sequenced with 150 cycles in paired end mode on a NovaSeq 6000 Sequencing System following the manufacturer’s instructions (Illumina, San Diego, CA).

Raw reads were demultiplexed and trimmed to remove adaptors using the process_radtags utility included in Stacks v.2.0 (Catchen et al., 2013). Short reads were de novo assembled, catalogued and matched using the ustacks cstacks, sstacks and tsv2bam (for paired-end reads) utilities in Stacks. SNPs were called using gstacks which assembles and genotypes contigs. SNP filtering was done under the populations component included in Stacks.

Here we provide the vcf files used in the study along with the imputed genotyping file and the genetic relationship matrix (GRM) that were generated.  We also provide files containing individual phenotypic measurments used for the association analysis in Tassel and LDna.