Molecular markers for detection and diagnosis of the giant grouper (Epinephelus lanceolatus)

Abstract

The giant grouper (Epinephelus lanceolatus) is a high-value fish in Taiwan. This study used random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and the cytochrome oxidase subunit I gene (CoxI) of mitochondrial DNA to analyze genetic variation in E. lanceolatus. Specific primers derived from these methods were developed. Wild and cultivated giant grouper were collected in the Penghu, Pingtung, and Kaohsiung regions of Taiwan and confirmed to species levels. DNA extracted from each sample was analyzed followed by analysis using 95 primers of RAPD and 59 primers of ISSR to investigate genetic variation among samples. Results showed that RAPD analysis using 21 primers yielded 279 bands and 86 polymorphic bands (31% polymorphism). Both RAPD115 and RAPD73 primers were capable of discriminating wild and cultivated giant groupers. Seventeen of the 59 ISSR primers (29.3% of total primers) produced 166 bands and 58 polymorphic bands (34.9%). In CoxI analysis, the sequences could differentiate wild and cultivated populations of E. lanceolatus. For the development of sequence characterized amplified region (SCAR) markers, a 747 bp amplicon was generated by the ISSR method and sequenced. Primer pairs targeting the SCAR marker sequence were designed, which enabled species-specific detection and avoided cross-amplification of other species of Serranidae fish. In our study, the specific primers (SCAR1/SCAR2) amplified a product in cultured giant grouper but not in the wild population. The specific DNA fragment produced from SCARF/SCAR2 primers was amplified in all giant grouper samples but not in other Serranidae fish. Specific ISSR-SCAR markers were developed in this study to distinguish wild and cultivated populations of giant grouper and also to distinguish them from other Serranidae and giant grouper fish. We developed DNA molecular marker techniques and CoxI sequences which could be used to generate information for species identification, trace genetic variation between different individuals in aquaculture, and authenticate fish and fishery products.