Inbred zebrafish lines: A genetic repository for zebrafish researchers

Abstract

Genetically uniform strains/inbred strains of animals are used in experiments that demand minimal genetic background variability and highly reproducible results. Modern genomic research is heavily dependent on inbred strains and they are widely used for identification of quantitative loci that affect evolutionarily and biomedically important traits, dissection of gene-gene and gene environmental interaction patterns, toxicity studies, immunology, reverse genetics, cancer research, etc. In mice, over 450 inbred strains have been described and in medaka over 13 inbred strains have been established. Owing to its numerous attributes zebrafish, Danio rerio, is one of the widely studied vertebrate model organisms, but comparable zebrafish inbred strains are not available to the scientific community. This is because of early onset of inbreeding depression causing high mortality of embryos and larvae and biased sex ratio that have been reported from earlier attempts to produce inbred lines. At CZeBraG-Centre for Zebrafish Breeding and Genetics Research facility of ICARCIFE, the inbred lines of zebrafish are being developed. The strategy followed is intense inbreeding coupled with strong positive selection for selected traits. The base population consisted of three wild stocks of zebrafish collected from rivers of North East India. Using nested mating design 84 families were produced in F1 generation and 46 inbred families were produced in F2 generation. Genetic parameters for growth traits like body weight and standard length were estimated. The overall heritability for body weight and standard length was estimated using animal model and found to be 0.43 ± 0.05 and 0.36 ± 0.04 respectively. Fecundity was also recorded but showed no significant variation among stocks. Inbreeding depression was studied by comparing the population means for body weight and standard length of F1 and F2 generation. The analysis suggested there was no inbreeding depression. Genetic monitoring of zebrafish is conducted using microsatellite markers. New PCR primers were designed for 20 selected microsatellite loci with high stringency. High Resolution Melting Curve analysis was used for genotyping individual zebrafish. In addition to developing inbred lines the centre is also maintaining the wild zebrafish stocks and has commenced supply of pedigreed zebrafish for researchers. In future zebrafish of known inbred levels (12.5 to 50% and above) can be supplied. Currently, the CZeBraG can house about 10000 adults, with separate facility for holding individual fish.

Genetic Basis for Morphological Variation in the Zebrafish Danio rerio: Insights from a Low-Heterozygosity Line

“…Examples of inbred fish strains and lines include the clean livebearer Poeciliopsis lucida [76][77][78], species of Xiphophorus-platyfish and swordtails [79,80], and the Japanese medaka Oryzias latipes, in which strains have been inbred for more than 100 generations [81]. Inbreeding promotes stronger long-term effects, primarily in reproduction (e.g., reduction in fecundity) or sex-ratio skewing, with growth performance being less affected [25,82,83]. However, variation in growth performance occurs in inbred zebrafish [19].…”

“…The use of homozygous strains/lines is generally employed in experimentation which requires low variability in their genetic background to facilitate the reproducibility of results because of their lower heterozygosity and long-term stability [24,25]. Despite the existing evaluation of different traits between homozygous and heterozygous strains/lines (see references above), most information is related to genetic studies including single-nucleotide polymorphism (SNP) variation, assessing copy number variants between zebrafish lines, the role of xenosensors in nuclear receptor function, building genetic repositories, and comparing genetic backgrounds from wild and inbred populations, [14,15,[25][26][27][28], with little study in this context of morphology, physiology, or behavior.…”