Genotyping-by-sequencing as an economical method of SNP discovery in Pinus contorta and Picea glauca

K. Nurkowski, K. Hodgins, S. Yeaman, L. Rieseberg, S.N. Aitken

Genotyping-by-Sequencing is an economical way to reduce the complexity of a genome before sequencing to make the best use of expensive sequencing space. Here we present a modified protocol to produce GBS libraries that reduces hands-on sample preparation time and increases the total amount of library produced. We use the restriction enzyme Pst1 to cleave the genomic DNA sample,and then we ligate double stranded adapters onto each end of the cleaved fragments. We then amplify the fragments that have the adapters correctly ligated using a high-fidelity polymerase, and perform a final size selection for fragments 400 – 600 bp using magnetic beads. We have sequenced a preliminary plate of 96 lodgepole pine samples using this technique. We aligned the GBS sequences to a draft the draft lobolly genome (PineRefSeq Project) using bwa. After filtering we identified 168,326 SNPs, but only 34,025 SNPs representing 9437 genes, were shared across 50 or more individuals. We plan to fine-tune our modified protocol for interior spruce and lodgepole pine and use the SNPs we discover to complement our sequence capture data to assess the genetic basis of adaptation to climate in those species. This will complement a large sequence capture data set that will capture SNPs across much of the exome. The GBS will allow us to expand our search to other areas of the genome, such regulatory regions or other genes not included in our re-sequencing project. It will also allow us to identify more putatively neutral loci that will allow us to assess the effects of demographic history on the genome.