Abstract
Astyanax mexicanus, a teleost fish that exists in a river-dwelling surface form and multiple cave-dwelling forms, is an excellent system for studying the genetic basis of evolution. Cavefish populations, which independently evolved from surface fish ancestors multiple times, have evolved a number of morphological and behavioral traits. Quantitative trait loci (QTL) analyses have been performed to identify the genetic basis of many of these traits. These studies, combined with recent sequencing of the genome, provide a unique opportunity to identify candidate genes for these cave-specific traits. However, tools to test the requirement of these genes must be established to evaluate the role of candidate genes in generating cave-specific traits. To address this need, we designed transcription activator-like effector nucleases (TALENs) to target two genes that contain coding changes in cavefish relative to surface fish and map to the same location as QTL for pigmentation, oculocutaneous albinism 2 (oca2) and melanocortin 1 receptor (mc1r). We found that surface fish genes can be mutated using this method. TALEN-induced mutations in oca2 result in mosaic loss of melanin pigmentation visible as albino patches in F0 founder fish, suggesting biallelic gene mutations in F0s and allowing us to evaluate the role of this gene in pigmentation. The pigment cells in the albino patches can produce melanin upon treatment with L-DOPA, behaving similarly to pigment cells in albino cavefish and providing additional evidence that oca2 is the gene within the QTL responsible for albinism in cavefish. This technology has the potential to introduce a powerful tool for studying the role of candidate genes responsible for the evolution of cavefish traits.
Highlights
Identifying and characterizing causative mutations is crucial to understanding the evolution of traits
As both of these mutations are thought to result in loss of function of mc1r, a transcription activatorlike effector nucleases (TALENs) was designed targeting the region around the missense mutation
Our results established that TALENs in Astyanax mexicanus can be highly efficient, with one TALEN pair generating up to 50% mutant alleles in individual embryos
Summary
Identifying and characterizing causative mutations is crucial to understanding the evolution of traits. A substantial amount of work has been done to identify loci responsible for natural variation using genetic mapping techniques (for example [1,2]). A few of these studies have identified the genes likely to be responsible for this phenotypic variation (for example [3,4]). A significant challenge that remains is testing for the functional consequences. TALENs in the Cavefish Astyanax mexicanus founding member of Recombinetics, Inc and holds shares in this company. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials
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