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

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. Zebrafish (Danio rerio) is the vertebrate animal model most rapidly adopted in biomedical research. The zebrafish genome is 70% similar to the human, and 80% percent of the genes responsible for human diseases have an orthologue in zebrafish 1. It is relatively easy to generate mutant animals, and the cost of zebrafish maintenance is low. Applications of this animal model include, among the many, toxicology, developmental biology, genetics and degenerative diseases, psychiatric conditions, cancer, and metabolic disorders. Researchers can choose from a variety of wild-type and mutant zebrafish lines for their studies. All available from the Zebrafish International Resource Center (ZIRC), wild-type zebrafish lines include Tübingen, AB, NHGRI-1, Sanger AB Tübingen (SAT), WIK, Tüpfel long fin (TL), Nadia, and Cooch Behar. Also available, are more than forty thousand mutant lines. Despite previous reports of the derivation of the IM inbred zebrafish line in 2011, homozygous zebrafish lines for all loci are lacking 2,3. Some initiatives are in progress that may fulfill the need for fish lines that have an identical genetic identity. We are currently working on a strategy to generate homozygous diploid fish using the method developed by Streisinger and colleagues in 1981, followed by somatic cell nuclear transfer (SCNT) 4-9. Once a gynogenetic embryo is developed, we isolate cells from them, and subsequently, after testing for normal k...

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses

“…In addition, the primers may serve as a foundation for the development of genetic identification systems of related equids, such as the donkey and zebra. We also note for those who wish to use some of the methods reported here for other species, that each species seems to have its own unique challenges for tetraSTR development [e.g., horse has a limited number of good tetraSTRs, zebrafish appears to have many null alleles, and the honeybee may require a longer LUS for good variability ( 46 ), this work, and Venta, unpublished results].…”

“…Some experimental work is currently being done in horse cell lines which may be used in investigations of pathology ( 45 ). In human cell line work, a significant number of cell lines have been misidentified, resulting in irreproducibility in the scientific literature ( 46 ). The markers reported here can be used to verify that the correct horse cell lines have been used, possibly using the new universal primers for labeling, mentioned in the methods section, and a subset of the full 17-plex panel in order to speed the process and limit the cost.…”

Quality of DNA extracted from freshwater fish scales and mucus and its application in genetic diversity studies of Perca fluviatilis and Rutilus rutilus

“…DNA extraction from the mucus such as skin swabbing has been used successfully in many fish species including Neolamprologus pulcher [ 36 ], Gadus morhua [ 37 ], Lepomis macrochirus [ 38 ], Oreochromis niloticus [ 39 ], or zebrafish [ 17 ]. So far, there has been a lack of information on whether extracting DNA from the mucus is suitable for studies into a genetic structure of a population due to a higher risk of DNA contamination [ 18 ] as compared to DNA obtained from the scales. In this study, we extracted DNA from the scale and mucus of two freshwater fish species perch and roach using the Aljanabi and Martinez [ 24 ] method.…”

“…Swabbing appears to be less invasive than DNA extracted from the muscle or fin; since it requires no anesthetic or the need to remove a tissue, it should be considered as the most efficient non-invasive method. So far, the DNA extraction method from the mucus has been still questionable due to DNA yield, polymerase chain reaction (PCR) amplification, and the potential for cross-contamination [ 17 , 18 ].…”

Skin swabbing protocol to collect DNA samples from small-bodied fish species

“…Other research groups have reported DNA extraction by the hot-shot method following skin swabbing: e.g. Venta et al (2020).…”