Identication of Four Mud Crab Species (Genus Scylla) based on High-resolution Melting (HRM) Analysis

In this study, we described the successful identication of four mud crab species (genus Scylla) by high-resolution melting curve analysis (HRM) of single nucleotide polymorphisms (SNPs). The complete mitochondrion genome sequences of Scylla serrata, Scylla paramamosain, Scylla olivacea and Scylla tranquebarica were aligned and screened for distinct fragments. A single pair of primers were designed to amplify the 203-bp consensus amplicon. As a result, a total of 96 samples from the four mud crab species were clearly separated. The melting proles from HRM analysis were found to be distinct across the species tested, and a number of 28 SNP sites were conrmed by sequencing. This new identication method will be a useful tool for discrimination of the four Scylla species and contribute to the effective breeding management of these species.


Introduction
The mud crabs, Scylla spp. are a group of 4 commercially important portunid species. They are important components of the mangrove ecosystem and distributed widely throughout the Indo-West-Paci c region (Macnae 1968; Keenan et al., 1998). However, over shing and the damage of natural habitat of mud crabs has been observed at varying levels in different countries as both national and international markets have developed (Lebata et al. 2009). In recent years, recovery efforts such as arti cial breeding and release are in progress for the stock enhancement purpose (Le Vay et al. 2008; Lebata et al. 2009).
Considering the distinct larval rearing procedure and population habitat of different Scylla species in arti cial breeding and release program (Waiho et al. 2018), the accurate taxonomic assessment of genus Scylla is necessary for the conservation and recovery of local Scylla germplasm resources.
Because of their high morphological plasticity and the absence of distinct morphological diagnostic characters, it is di cult to identify Scylla species visually. In the previous studies, molecular identi cation methods based on the variation in product fragment length of mitochondrial DNA and nuclear ribosomal

PCR ampli cation and HRM analysis
The complete mitochondrion genome of the four species were downloaded from NCBI database

Results And Discussion
The accuracy of species identi cation is crucial for breeding, husbandry, and consumers. The overlapping of distribution areas and similarity of morphology make it di cult to identify Scylla species. HRM analysis is sensitive and can distinguish single-site mutations in a sequence, it has been widely used in . Compared with existing gel-based species discrimination methods of Scylla species, HRM analysis is a very promising technique, which provides a rapid, sensitive and high-throughput alternative for species identi cation (Jin et al. 2015). In this study, a 203-bp fragment of mitochondrion sequence of the four mud crab species was ampli ed using the CSN9 primers 5'-AATTAACTAATGACATCATCTCATGG-3'(forward) and 5'-CGAGTTACATCTCGTCAT CATTG − 3' (reverse).
The melting pro les from HRM analysis were successfully obtained and found to be distinct across the four mud crab species (Fig. 1). T m calling analysis showed that the melting points of S. serrata and S. paramamosain were 79.55 and 80.12°C, while the S. tranquebarica and S. olivacea exhibited two melting peaks at 78.12 and 82.04, 78.42 and 84.40°C respectively. These results proved that HRM is a sensitive technique for distinguishing these four species.
Sequencing analysis was performed in order to con rm the species-speci c mutation which resulted in the distinct HRM pro les. A number of 9 haplotypes were identi ed across all the sequenced samples, including 2 for S. serrata, 3 for S. olivacea, 2 for S. paramamosain, and 2 for S. tranquebarica respectively. The alignment of haplotype fragments was presented in Fig. 2. The results demonstrated that the distinct HRM curves of each Scylla species were due to the interspeci c differences in a number of SNPs. In total, 28 SNP sites were detected from the 203-bp amplicon of the target region. Transitions were observed at twenty-seven sites and transversions at one site. These sequencing results proved the robustness of HRM analysis to distinguish the four Scylla species with only one pair of primers.
In conclusion, the results obtained in this study indicated that the consensus 203-bp fragment of mitochondrial DNA sequence contained SNPs was appropriate for HRM analysis, the distinct melting pro les of the four mud crab species provide a rapid and high-throughput method for the discrimination of Scylla species, which will be a useful tool for the genetic research and establishment of effective breeding management of these species. Figure 1 Discriminations of single-nucleotide polymorphisms (SNPs) among four genus Scylla species using highresolution melting analysis A Normalized and temp-shifted difference plot. B Normalized melting curves.  Alignment of haplotype fragments from the mitochondrial DNA sequence of the four species using the pair of primers. Nucleotides identical to the sequence on the top are shown with dots