Data_Sheet_1_Rapid Genetic Identification of the Blue Crab Callinectes sapidus and Other Callinectes spp. Using Restriction Enzyme Digestion and High Resolution Melt (HRM) Assays.fasta

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.

蓝蟹（Callinectes sapidus）是当前研究最为广泛的海洋甲壳类动物之一，兼具极高经济价值与生态重要性。尽管北美地区针对蓝蟹已开展了大量研究，但该物种在亚热带与热带海域的分布及种群丰度仍有诸多未解之谜。在诸多海域，C. sapidus会与形态相似的蓝蟹属（Callinectes）其他物种同域共存，这一情况与海鲜产品的标签误标问题密切相关。为实现该物种的快速鉴定，本研究设计并验证了两种靶向线粒体12S rRNA基因的聚合酶链式反应（PCR）检测方法。第一种检测方法通过PCR后限制性酶切（PCR-RFLP）实现C. sapidus与其他蓝蟹属物种的区分；第二种检测方法则借助高分辨率熔解（HRM）分析与监督机器学习分析，实现多种蓝蟹属物种的分类鉴别。本研究共使用58份来自5种蓝蟹属物种的DNA样本（经12S基因测序验证）开展检测方法验证。PCR-RFLP检测方法可100%准确区分C. sapidus与其他蓝蟹属物种。对扩增产物的HRM分析可实现物种间的良好区分，序列同源性较高的物种间可形成清晰的聚类簇。鉴于可用数据集规模较小，对HRM曲线开展线性判别分析（LDA）分类的效果已较为理想：经100折交叉验证后，所有物种的平均分类准确率约为90%~91%。多数分类误差出现在C. similis与C. danae之间，二者的扩增子序列同源性高达约99%；若将二者合并为单一类别，则整体分类准确率可提升至94%。分类误差还出现在C. bocourti的鉴定中，该物种的参考样本集仅包含3份样本。仅针对C. sapidus的HRM分类准确率可达97.5%。总体而言，这两种检测方法作为快速且低成本的蓝蟹属物种鉴定手段展现出巨大应用潜力，可同时应用于生态学研究与海鲜产品的身份鉴定及标签规范工作。