Validation of SSR-tetranucleotide multiplex panel for kinship evaluation in tilapia breeding programs

Suzane Fonseca Freitas, Heden Luiz Marques Moreira, Daiane Machado Souza, Carla Giovane Ávila Moreira, Rafael Aldrighi Tavares, Nelson José Laurino Dionello


This study aimed to develop PCR assays, reaction combinations, and validation of tetranucleotide SSR loci for tilapia, to minimize the impact of erroneous allele inferences on genotype determination of this marker. Microsatellites containing tetranucleotide repeats were obtained from tilapia genome, version 2.1, avoiding loci in the same linking group. Primers were designed for different fragment sizes, and fluorescence added to each locus. A total of 10 loci were amplified, separately and in combination, and loaded into a single capillary sequencer panel. Alleles were amplified without stutters and easily interpreted. PCR amplifications of DNA repeatedly extracted from samples, and genotyping at different PCR rounds were performed to infer allele signaling errors. The panel obtained in this study is currently used in kinship analyses and pedigree corrections in a tilapia breeding program. 


Genetic variability; Genotyping; Microsatellite markers

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AMOUSSOU, T. O. et al. An insight into advances in fisheries biology, genetics and genomics of African tilapia species of interest in aquaculture. Aquaculture Reports, v.14, 2019.

BAGSHAW, A. T. M. Functional mechanisms of microsatellite DNA in eukaryotic genomes. Genome Biology and Evolution, v. 9, n. 9, p. 2428-2443, 2017.

BOTSTEIN, D. et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, v. 32, n. 3, p. 314, 1980.

DIAS, M. A. D. et al. Evaluation of the genetic diversity of microsatellite markers among four strains of Oreochromis niloticus. Animal Genetics, v. 47, n. 3, p. 345-353, 2016.

GUICHOUX, E. et al. Current trends in microsatellite genotyping. Molecular Ecology Resources, v. 11, n. 4, p. 591-611, 2011.

HAMOY, I. G. et al. Isolation and characterization of tri and tetranucleotide microsatellite markers for the tambaqui (Colossoma macropomum, Serrasalmidae, Characiformes). Conservation Genetic Resources, v. 3, n. 1, p. 33-36, 2011.

HUYNH, O. A. et al. Low-cost methods for DNA extraction and quantification. In: JANKONWICZ-CIESLACK, J. et al. Biotechnologies for Plant Mutation Breeding. Switzerland: Springer International Publishing, 2017. cap. 14, p. 227-239.

JONES, A. G. et al. A practical guide to methods of parentage analysis. Molecular Ecology Resources, v. 10, n. 1, p. 6-30, 2010.

JOSHI, D.; RAM, R. N.; LOHANI, P. Microsatellite markers and their application in fisheries. International Journal of Advances in Agricultural Science and Technology, v. 4, n. 10, p. 67-104, 2017.

KORESSAAR, T.; REMM, M. Enhancements and modifications of primer design program Primer3. Bioinformatics Applications Note, v. 23, n. 10, p. 1289-1291, 2007.

LEE-MONTERO, I. et al. Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.). Animal Genetics, v. 44, n. 1, p. 533-546, 2013.

MILLER, W. L. et al. Identification and evaluation of a core microsatellite panel for use in white-tailed deer (Odocoileus virginianus). BMC Genetics, v. 20, n. 49, p. 1-20, 2019.

OLAFSSON, K. et al. Novel set of multiplex assays (SalPrint15) for efficient analysis of 15 microsatellite loci of contemporary samples of the Atlantic salmon (Salmo salar). Molecular Ecology Resources, v. 10, n. 1, p. 533-537, 2010.

PIMENTEL, J. S. M. et al. High-throughput sequencing strategy for microsatellite genotyping using neotropical fish as a model. Frontiers in Genetics, v. 9, n. 1, 2018.

POMPANON, F. et al. Genotyping erros: causes, consequences and solutions. Nature Reviews: Genetics, v. 6, p. 847-859, 2005.

PUCKETT, E. Variability in total project and per sample genotyping costs under varying study designs including with microsatellites or SNPs to answer conservation genetic questions. Conservation Genetic Resources, v. 9, n. 2, p. 289-304, 2017.

PUTMAN, A. I.; CARBONE, I. Challenges in analysis and interpretation of microsatellite data for population genetic studies. Ecology and Evolution, v. 14, n. 4, p. 4399-4428, 2014.

PUTNAM, A. S.; IVY, J. A. Kinship-based management strategies for captive breeding programs when pedigrees are unknown or uncertain. Journal of Heredity, v. 105, n. 3, p. 303-311, 2014.

RUTTEN, M. J. M. et al. Genetic characterization of four strains of Nile tilápia (Oreochromis niloticus L.) using microsatellite markers. Animal Genetics, v. 35, p. 93-97, 2004.

SULLIVAN, K. M. et al. Automated DNA profiling by fluorescent labeling of PCR products. Genome Research, v. 2, p. 34-40, 1992.

TIBIHIKA, P. D. et al. Application of microsatellite genotyping by sequencing (SSR-GBS) to measure genetic diversity of the East African Oreochromis niloticus. Conservation Genetics, v. 20, n. 1, p. 57-372, 2019.

TURCHETTO-ZOLAT, A. C. et al. Marcadores moleculares na era genômica: metodologias e aplicações. Ribeirão Preto: Sociedade Brasileira de Genética, 2017. 181 p.

UNTERGASSER, A. et al. Primer3: new capabilities and interfaces. Nucleic Acids Research, v. 40, n. 15, p. e115, 2012.

YANG, M. et al. Parentage determination in golden mandarin fish (Siniperca scherzeri) based on microsatellite DNA markers. Aquaculture International, v. 23, n. 2, p. 499-507, 2014.

ZHAN, L. et al. MEGASAT: automated inference of microsatellite genotypes from sequence data. Molecular Ecology Resources, v. 17, p. 247-256, 2017.

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