Utility of pooled sequencing for association mapping in nonmodel organisms

Abstract

High-density genome-wide sequencing increases the likelihood of discovering genes of major effect and genomic structural variation in organisms. While there is an increasing availability of reference genomes across broad taxa, the greatest limitation to whole-genome sequencing of multiple individuals continues to be the costs associated with sequencing. To alleviate excessive costs, pooling multiple individuals with similar phenotypes and sequencing the homogenized DNA (Pool-Seq) can achieve high genome coverage, but at the loss of individual genotypes. Although Pool-Seq has been an effective method for association mapping in model organisms, it has not been frequently utilized in natural populations. To extend bioinformatic tools for rapid implementation of Pool-Seq data in nonmodel organisms, we developed a pipeline called PoolParty and illustrate its effectiveness in genetic association mapping. Alignment expectations based on five pooled Chinook salmon (Oncorhynchus tshawytscha) libraries showed that approximately 48% genome coverage per library could be achieved with reasonable sequencing effort. We additionally examined male and female O. tshawytscha libraries to illustrate how Pool-Seq techniques can successfully map known genes associated with functional differences among sexes such as growth hormone 2. Finally, we compared pools of individuals of different spawning ages for each sex to discover novel genes involved with age at maturity in O. tshawytscha such as opsin4 and transmembrane protein19. While not appropriate for every system, Pool-Seq data processed by the PoolParty pipeline is a practical method for identifying genes of major effect in nonmodel organisms when high genome coverage is necessary and cost is a limiting factor.

A mobile sex-determining region, male-specific haplotypes, and rearing environment influence age at maturity in Chinook salmon

“…This haplotype was also associated with the largest fish in Alaska and the Wenatchee River, suggesting a conserved genetic basis for older age at maturity among these haplotypes. Two previous studies of Chinook salmon failed to find a signal of age at maturity on the Ots17 (Micheletti and Narum 2018, Waters et al 2018). It is possible that these populations had little or no male-specific haplotype variation to detect; however, it is also possible that pooling samples by age class (e.g.…”

“…It is possible that these populations had little or no male-specific haplotype variation to detect; however, it is also possible that pooling samples by age class (e.g. Micheletti and Narum 2018) could have masked signals of male-specific haplotypes. If multiple haplotypes are present, but each at low frequency, there may not be enough individuals with haplotype-specific alleles to reach significance in a GWAS.…”

“…In an aquacultural strain of Atlantic salmon, multiple genomic regions, including the VGLL3 gene, explained a total of 78% of the variation in age at maturity (Sinclair-Waters et al 2020). In Chinook salmon, the specific genes underlying variation in age at maturity are unknown but GWAS has identified SNPs associated with age at maturity on several autosomes (Micheletti and Narum 2018, Waters et al 2018) and male-specific sex chromosome haplotypes are associated with variation in size and age at maturity in male Chinook salmon from Alaska (McKinney et al 2019b). Despite the lack of specific knowledge of genes governing age at maturity in most salmon species, studies have consistently shown high heritability for this trait (Gall et al 1988, Heath et al 2002, Reed et al 2018) and QTL/GWAS studies have identified genomic regions associated with age at maturity in multiple species (Moghadam et al 2007, Haidle et al 2008, Ayllon et al 2015).…”

“…This includes sex-linked heritability (Hankin et al 1993), heritability of male reproductive strategies (Heath et al 2002), and male-specific haplotypes associated with size and age at maturity in Chinook salmon from Alaska (McKinney et al 2019b). While the sex chromosome (Ots17) has been strongly implicated in sex-specific age at maturity, genes on other chromosomes have also shown associations (Micheletti and Narum 2018, Waters et al 2018).…”

Genomic variation underlying complex life-history traits revealed by genome sequencing in Chinook salmon

“…For each Pool-seq library, raw 150 bp paired-end reads (2 Â 150 bp) were processed using the PoolParty pipeline (Micheletti & Narum [21]) that integrates several existing resources into a single pipeline. Briefly, this included multiple steps that started with trimming reads (to a minimum of 50 bp) with a quality score less than 20 using the trim-fastq.pl script part of Popoolation2 [22].…”