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Abstract:
Phenotypic plasticity describes the property of a genotype to respond to environmental variation by producing distinct phenotypes. In Pristionchus pacificus, the mouth form is developmentally plastic, resulting in two alternative mouth forms: the eurystomatous (Eu) predatory form has two teeth, whereas the alternative bacteriovorus stenostomatous (St) form has a single tooth. The switch between the two forms is environmentally sensitive, and a previous forward genetic approach showed a key switch function for the sulfatase-coding gene eud-1, mutations in which result in all-St worms. In this study, we used P. pacificus natural isolates with different Eu/St ratios to generate Recombinant Inbred Lines (RIL) and performed Quantitative Trait Locus (QTL) analysis to dissect the genetic architecture underlying mouth dimorphism. Our result showed the involvement of one major locus on the X chromosome, spanning a recently described multi‐gene locus containing eud-1, its paralog, and two more genes encoding α-N-acetylglucosaminidases (nag), all of which were shown to be involved in mouth form regulation. RNA-seq analysis of parental strains revealed 40% higher expression of eud-1 in the high Eu parental strain, and CRISPR-Cas9 mutants of the two sulfatases paralogous in the high Eu parental strain showed a complete switch to the St form. With the absence of non-synonymous substitutions in eud-1 between the parental lines, we used CRISPR-Cas9 technology to perform variant swapping in the eud-1 regulatory region to define potential causative SNPs. Our experimental analysis identified variations in different cis-regulatory components of eud-1. Copy number differences in a potential Forkhead transcription factor binding site within the promoter/enhancer region, besides a SNP in the eud-1 first intron between the parental lines, caused differences in mouth-form ratios phenotype. Mutant lines showed an additive effect of these cis-regulatory elements, with a systematic change in the mouth-form phenotype and downregulation of eud-1 expression. Currently, we are using CRISPR-Cas9 technology to examine the potential involvement of various Forkhead genes in controlling eud-1 expression, while also expanding our analysis to test variations in the causative region within 30 more P. pacificus natural isolates.
