Iwagaki oyster, Crassostrea nippona, widely distributes along the seashore of Eastern Asia, and has been considered to be a potential breeding species due to its delicious taste and high commercial value. In order to study its genetic background and population structure, we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals. The number of alleles ranged from 3 to 22, while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949, respectively. Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C. nippona. We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring. In actual parentage analysis, 80% of the offspring were correctly assigned to their parental pairs using three multiplex PCRs. Furthermore, the success rate of parentage assignment reached 96% when the other two multiplex PCRs were added. These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction, resource conservation and selective breeding program of C. nippona.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Blacket, M. J., Robin, C., Good, R. T., Lee, S. F., and Miller, A. D., 2012. Universal primers for fluorescent labelling of PCR fragments–An efficient and cost-effective approach to genotyping by fluorescence. Molecular Ecology Resources, 12: 456–463.
Botstein, D., White, R. L., Skolnick, M., and Davis, R. W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32: 314–331.
Boudry, P., Collet, B., Cornette, F., Hervouet, V., and Bonhomme, F., 2002. High variance in reproductive success of the Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses. Aquaculture, 204: 283–296.
Itoh, N., Tun, K. L., Komiyama, H., Ueki, N., and Ogawa, K., 2004. An ovarian infection in the Iwagaki oyster, Crassostrea nippona, with the protozoan parasite Marteilioides chungmuensis. Journal of Fish Diseases, 27: 311–314.
Jones, O. R., and Wang, J., 2010. COLONY: A program for parentage and sibship inference from multilocus genotype data. Molecular Ecology Resources, 10: 551–555.
Kalinowski, S. T., Taper, M. L., and Marshall, T. C., 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology, 16: 1099–1106.
Li, N., Li, Q., Kong, L. F., and Yu, H., 2016. Development of three multiplex PCR primer sets for ark shell (Scapharca broughtonii) and their validation in parentage assignment. Journal of Ocean University of China, 15: 311–317.
Li, Q., Liu, S. K., and Kong, L. F., 2009. Microsatellites within genes and ESTs of the Pacific oyster Crassostrea gigas and their transferability in five other Crassostrea species. Electronic Journal of Biotechnology, 12: 15–16.
Li, Q., Park, C., and Kijima, A., 2003. Allelic transmission of microsatellites and application to kinship analysis in newly hatched Pacific abalone larvae. Fisheries Science, 69: 883–889.
Li, Q., Yu, H., and Yu, R. H., 2006. Genetic variability assessed by microsatellites in cultured populations of the Pacific oyster (Crassostrea gigas) in China. Aquaculture, 259: 95–102.
Li, R. H., Li, Q., Cornette, F., Dégremont, L., and Lapègue, S., 2010. Development of four EST-SSR multiplex PCRs in the Pacific oyster (Crassostrea gigas) and their validation in parentage assignment. Aquaculture, 310: 234–239.
Liu, T., Li, Q., Song, J. L., and Yu, H., 2017. Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster (Crassostrea gigas). Journal of Ocean University of China, 16: 151–160.
Liu, Z. J., and Cordes, J. F., 2004. DNA marker technologies and their applications in aquaculture genetics. Aquaculture, 238: 1–37.
Li, W. J., 2007. Biology and cultivation of oyster Crassostrea nippona. Fisheries Science, 26: 689–690 (in Chinese with English abstract).
Marshall, T. C., Slate, J., Kruuk, L. E. B., and Pemberton, J. M., 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology, 7: 639–655.
Nie, H. T., Li, Q., and Kong, L. F., 2012. Development of four multiplex PCRs in the Zhikong scallop (Chlamys farreri) and their validation in parentage assignment. Biochemical Systematics and Ecology, 44: 96–101.
Ostrander, E. A., Jong, P. M., Rine, J., and Duyk, G., 1992. Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences. Proceedings of the National Academy of Sciences, 89: 3419–3423.
Porta, J., Porta, J. M., Martínez-Rodríguez, G., and del Carmen Alvarez, M., 2006. Development of a microsatellite multiplex PCR for Senegalese sole (Solea senegalensis) and its application to broodstock management. Aquaculture, 256: 159–166.
Rousset, F., 2008. GENEPOP’007: S complete re-implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources, 8: 103–106.
Sekino, M., Hamaguchi, M., Aranishi, F., and Okoshi, K., 2003. Development of novel microsatellite DNA markers from the Pacific oyster Crassostrea gigas. Marine Biotechnology, 5: 227–233.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., and Rozen, S. G., 2012. Primer3–New capabilities and interfaces. Nucleic Acids Research, 40: e115–e115.
Wang, J., 2004. Sibship reconstruction from genetic data with typing errors. Genetics, 166: 1963–1979.
Yoon, H. S., Jung, H. T., and Choi, S. D., 2008. Suminoe oyster (Crassostrea ariakensis) culture in Korea. Journal of Shellfish Research, 27: 505–508.
Yuan, M. Y., Mao, L. J., and Zhou, W., 2008. Chromosome karyotypic analysis of oyster Crassostrea nippona. Journal of Dalian Fisheries University, 23: 318–320 (in Chinese with English abstract).
This study was supported by the National Natural Science Foundation of China (No. 31772843), the Natural Science Foundation of Guangxi Province (No. AA17204080-4), and the Fundamental Research Funds for the Central Universities (No. 201762014).
About this article
Cite this article
Liu, K., Li, Q. & Li, Q. Multiplex PCR Sets of Novel Microsatellite Loci for Iwagaki Oyster Crassostrea nippona and Their Application in Parentage Assignment. J. Ocean Univ. China 19, 191–198 (2020) doi:10.1007/s11802-020-4212-z
- Crassostrea nippona
- multiplex PCR
- parentage assignment