The Laboratory Domestication of Zebrafish: From Diverse Populations to Inbred Substrains

Abstract

We know from human genetic studies that practically all aspects of biology are strongly influenced by the genetic background, as reflected in the advent of "personalized medicine." Yet, with few exceptions, this is not taken into account when using laboratory populations as animal model systems for research in these fields. Laboratory strains of zebrafish (Danio rerio) are widely used for research in vertebrate developmental biology, behavior, and physiology, for modeling diseases, and for testing pharmaceutic compounds in vivo. However, all of these strains are derived from artificial bottleneck events and therefore are likely to represent only a fraction of the genetic diversity present within the species. Here, we use restriction site-associated DNA sequencing to genetically characterize wild populations of zebrafish from India, Nepal, and Bangladesh, and to compare them to previously published data on four common laboratory strains. We measured nucleotide diversity, heterozygosity, and allele frequency spectra, and find that wild zebrafish are much more diverse than laboratory strains. Further, in wild zebrafish, there is a clear signal of GC-biased gene conversion that is missing in laboratory strains. We also find that zebrafish populations in Nepal and Bangladesh are most distinct from all other strains studied, making them an attractive subject for future studies of zebrafish population genetics and molecular ecology. Finally, isolates of the same strains kept in different laboratories show a pattern of ongoing differentiation into genetically distinct substrains. Together, our findings broaden the basis for future genetic, physiological, pharmaceutic, and evolutionary studies in Danio rerio.

Keywords: RAD-seq; genetic differentiation; genetic diversity; inbreeding; laboratory strains; wild populations; zebrafish.

FIg. 1.

Zebrafish samples used in the study. Sample descriptions were obtained from publications first describing the fish (Whiteley et al. 2011; Wilson et al. 2014), when applicable. n, number of individuals sampled. The map of sampling locations was obtained from Google Earth v7.3.2 (September 23, 2019). Data SIO, NOAA, U.S. Navy, NGA, GEBCO. Image Landsat/Copernicus.

FIg. 2.

Population structure of the zebrafish samples. (A) Admixture plot generated by the R package LEA. Each column corresponds to one fish. Colors indicate the proportion of variation shared between individuals. Thirteen distinct subpopulations are identified, three of them within CB. (B) Unrooted Maximum Likelihood tree, generated with 1,000 bootstrap replicates.

FIg. 3.

Population differentiation in wild and laboratory zebrafish. (Blue) F_ST, relative genetic differentiation, shows the amount of genetic variation that can be explained by differences between (sub)populations. (Green) D_xy, absolute genetic differentiation, average amount of pairwise differences between two chromosomes taken from different (sub)populations.

FIg. 4.

Within-strain variability of zebrafish. (A) Three different estimators of the scaled mutation rate, calculated independently for each population, show wild fish (CB, UT, KHA, CHT) to be genetically much more diverse than any of the laboratory strains. θ_π, average number of pairwise differences, divided by total length of the sequence; θ_w, proportion of polymorphic sites, normalized with sample size; θ₁, observed number of singleton mutations, divided by total length of the sequence; Fis, coefficient of inbreeding. (B) The genomes of laboratory strains contain long stretches of reduced heterozygosity that can vary even between isolates of the supposedly same strain. In CB, observed heterozygosity is usually slightly higher than expected under Hardy–Weinberg equilibrium. Almost no polymorphic sites were retrieved for the long arm of chromosome 4 (the natural sex chromosome; Anderson et al. 2012) in any of the populations. Individual data points are not indicated; lines represent loess-smoothed averages calculated from the heterozygosity of polymorphic sites. The positions of identified polymorphic sites themselves are shown for each population as a separate track at the bottom.

FIg. 5.

Derived allele frequency in zebrafish. (A) The frequency spectra in wild population closely follow the expectations (black line). In contrast, laboratory strains have a lack of low-frequency alleles, with the most inbred strain (TU_2018) demonstrating a nearly flat spectrum. These spectra look similar for G/C->A/T and A/T->G/C substitution types. In the wild fish and the “newest” laboratory strain Nadia, biases can be seen for the substitutions that change GC-content, with A/T to G/C substitutions being more common among high-frequency variants and G/C to A/T among low-frequency variants. In UT and CHT, the observed spectra are generally close to what would be expected under neutrality. The irregular shape of spectra for these two populations is caused by the uneven distribution of genomes among the bins, resulting from relatively small sample sizes. In the KHA population from Nepal, numerous alleles that are rare in other populations are present at high frequencies. Populations with significant differences between the A/T -> G/C and G/C -> A/T spectra are marked with an asterisk (*). n, number of available genomes.