A 13-plex of tetra- and penta-STRs to identify zebrafish

Patrick J. Venta,William G. Poulos,Marie-Claude Senut,Sukumal Prukudom,Jose B. Cibelli,Anthony K. Nguyen

doi:10.1038/s41598-020-60842-5

Patrick J. Venta, William G. Poulos + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-60842-5

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Abstract

The zebrafish species Danio rerio has become one of the major vertebrate model organisms used in biomedical research. However, there are aspects of the model that need to be improved. One of these is the ability to identify individual fish and fish lines by DNA profiling. Although many dinucleotide short tandem repeat (diSTR) markers are available for this and similar purposes, they have certain disadvantages such as an excessive polymerase slippage (“stutter”) that causes difficulties in automated genotyping and cross-laboratory comparisons. Here we report on the development of a 13-plex of tetranucleotide and pentanucleotide STRs (tetraSTRs and pentaSTRs, respectively) that have low stutter. The system uses an inexpensive universal primer labelling system, which can easily be converted to a direct labeling system if desired. This 13-plex was examined in three zebrafish lines (NHGRI-1, kca33Tg, and kca66Tg, originally obtained from ZIRC). The average observed heterozygosity (Ho) and expected heterozygosity (He) in these highly inbred lines were 0.291 and 0.359, respectively, which is very similar to what has been found with diSTRs. The probability of identity (PI) for all fish tested was 2.1 × 10−5 and the PI for siblings (PIsib) was 6.4 × 10−3, as calculated by the Genalex package. Ninety percent of the fish tested were correctly identified with their respective strains. It is also demonstrated that this panel can be used to confirm doubled-haploid cell lines. This multiplex should find multiple uses for improving the accuracy and reproducibility of studies using the zebrafish model.

Highlights

MethodsTen fish (five of each sex) were used from each of the wild-type line NHGRI-1, the kca33Tg, and the kca66Tg transgenic reporter lines (these lines were all originally obtained from the Zebrafish International Resource Center [ZIRC] and any family relationships for the individual fish are unknown)
We report a 13-plex of seven tetra- and six pentaSTRs that should prove useful for a variety of genetic studies in zebrafish, including verification of the correct background line that has been used to produce knock-out and knock-in alleles and, further, we show here that these markers can be used to monitor the production of doubled haploid zebrafish cell lines
A zebrafish 13-plex was developed that consists of seven tetraSTRs and six pentaS

Summary

Methods

Ten fish (five of each sex) were used from each of the wild-type line NHGRI-1, the kca33Tg, and the kca66Tg transgenic reporter lines (these lines were all originally obtained from the Zebrafish International Resource Center [ZIRC] and any family relationships for the individual fish are unknown). DNA samples were taken at our animal facility at MSU. For NHGRI-1 lines we used F0 animals i.e. adult-founders from ZIRC. For Kca33Tg and Kca66Tg lines we used the F2 progeny of founders obtained from ZIRC. DNA was obtained by “scale swabs” from fish present in the laboratory using a modified protocol of previously established methods[19]. Dried swabs were stored at room temperature (~20 °C for up to two weeks) if they were not processed immediately, as described below under DNA preparation. Control tailfin DNA was used from previous isolations taken in the lab

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Discussion

Conclusion