Rapid Genetic Identification of the Blue Crab Callinectes sapidus and Other Callinectes spp. Using Restriction Enzyme Digestion and High Resolution Melt (HRM) Assays

Abstract

The blue crab Callinectes sapidus is one of the most widely studied marine crustaceans due to its high economic value and ecological significance. Despite extensive research on the blue crab in North America, many questions remain about the distribution and abundance of the species in the subtropics and tropics. In many places, C. sapidus is sympatric with morphologically similar Callinectes spp., which has implications for seafood mislabeling. To enable rapid identification of the species, we designed and tested two PCR-based assays targeting the 12S rRNA mitochondrial gene. The first assay discriminates C. sapidus from other Callinectes spp. via post-PCR restriction digestion (PCR-RFLP) and the second assay discriminates among multiple Callinectes spp. through High Resolution Melting (HRM) analysis and supervised machine learning analyses. A total of 58 DNA samples from five Callinectes spp. (validated via 12S gene sequencing) were used for assay testing. The PCR RFLP assay was 100% accurate identifying C. sapidus from other Callinectes spp. HRM analysis of amplicons showed good discrimination among species, with distinct clusters formed between species with higher sequence homology. Linear discriminant analysis (LDA) classification of HRM curves was quite successful given the small dataset available, producing ∼90-91% mean accuracy in classification over all species with 100-fold cross validation. Much of the error came from misclassifications between C. similis and C. danae, which are ∼99% similar in sequence for the amplicon; collapsing them into a single class increased overall classification success to 94%. Error also arose from C. bocourti classifications, which had a reference set containing only three samples. Classification accuracy of C. sapidus alone via HRM was 97.5%. Overall, these assays show great promise as rapid and inexpensive methods to identify Callinectes spp. and have application for both ecological research and seafood identification or labeling.

Chromosome-level genome assembly of the blue crab,Callinectes sapidus

“…The lack of recombination-based linkage data or other cytogenetic information such as karyotypes for blue crab means that there is little outside reference data to guide the assembly. However, the chromosome level-genome assembly, together with that of P. trituberculatus , will permit genomic work assessing genetic diversity and species identification across the range ( Place and Plough 2017 ; Plough 2017 ; Windsor et al 2019 ; Lee et al 2020 ). Future work to advance blue crab aquaculture through selective breeding could make use of the genome through a genomic selection approach ( Meuwissen et al 2001 ; Heffner et al 2009 ), which has recently been applied to aquaculture species like shrimp and mollusks ( Hollenbeck and Johnston 2018 ; Zenger et al 2019 ; Houston et al 2020 ).…”

Determination of genetic diversity between natural and cultured populations of Common Dentex (Dentex dentex) fish in the East Aegean Sea

“…In addition, genetic identification of the blue crab Callinectes sapidus and other Callinectes spp. was also performed using as few samples as 1 for a particular location (Lee et al 2020). Although more than 25 species would need to be collected in order to achieve maximum accuracy, Goodall-Copestake et al ( 2012) note that a sample size of five individuals per species population is adequate to differentiate between extremes in assessing the haplotype and nucleotide COI variation across wide-ranging animal taxa.…”