Mauremys is a globally traded species group that was newly identified in 2012 in the South Korean wild (National Institute of Environmental Research 2012) and was designated as an invasive alien species in South Korea in 2020. The National Institute of Ecology (2018) confirmed the sympatric distribution of M. sinensis and M. reevesii, and hybrids as well were identified, indicating a serious threat to the conservation of domestically protected species. Therefore, this study aimed to develop genotypic markers for application in high-resolution melting (HRM) analysis for the rapid and reliable identification of M. sinensis, M. reevesii, and their hybrids.
HRM analysis enables genotyping by determining DNA variations, such as single nucleotide polymorphisms (SNPs), based on the melt transition (Tm) shape of real-time PCR products (Reed et al. 2007; Wittwer 2009; Wittwer et al. 2003). It has been applied extensively in hybridization studies and species identification (Buglione et al. 2020; Ganopoulos et al. 2013; Minett et al. 2021; Ouso et al. 2020; Park et al. 2023). Therefore, this study aimed to develop HRM markers that can distinguish M. sinensis, M. reevesii, and their hybrids for future monitoring.
Thirteen blood and tissue samples were used to assess HRM marker development (Online Resource 1). The samples belonged to three, six, and four individual M. sinensis, M. reevesii, and hybrids, respectively. DNA was extracted using a Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For molecular identification, the R35 region of nuclear DNA intron 1 was amplified using MAU-R35-1f (5′-CAAAAGTCATTCTCTGGCTTC-3′), MAU-R35-2f (5′-GTCAGACTTCTTTGCATATTTGTAA-3′), and MAU-R35-1r (5′-CAACTATGTGCTGGACAG-3′), which were designed in the present study. The sequences of the R35 fragments were edited and aligned using Geneious 5.3.6 (BIOMATTERS, Auckland, New Zealand) and Multiple-sequence alignments were performed using CLUSTAL X (Thompson et al. 1997).
We analyzed the DNA sequence data of 10 Mauremys species from the NCBI GenBank database to design a primer combination for PCR amplification of the HRM molecular marker in the intron 1 region of the nuclear R35 gene belonging to M. sinensis and M. reevesii. These sequences were aligned with the newly produced sequence data using ClustalW in BioEdit 7.2.5 (Özvegy et al. 2015), and a DNA sequence matrix was created. Based on these results, the ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) was used to design new PCR primer combinations containing SNPs that could distinguish between M. sinensis, M. reevesii, and their hybrids. The parameters included an 18–20-mer length of the oligonucleotide sequence, 40–55% GC content, and a Tm of 50–60 °C. The novel PCR primers for HRM analysis were MAU-R36-0658f (5′-TCAGCTTCTCAGCTTCTTTC-3′) and MAU-R36-0713r (5′-CAGTGCCAGGCAGGATT-3′) (Fig 1). PCR amplification was carried out using the QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific) with MeltDoctor HRM Master Mix (Thermo Fisher Scientific) to perform HRM analysis. The holding stage consisted of an enzyme-activation step at 95 °C for 10 min, whereas the cycling stage consisted of a 40-cycle denaturation step at 95 °C for 15 s and an annealing/elongation step at 60 °C for 1 min. The melt curve/dissociation stage included denaturation at 95 °C for 10 s, annealing at 60 °C for 1 min, HRM at 95 °C for 15 s, and annealing at 60 °C for 15 s.
SNPs that could distinguish between M. sinensis, M. reevesii, and their hybrids were examined in intron 1 of R35. Novel molecular markers, including PCR primer combinations, were subsequently established. The forward and reverse primers were highly conserved, with no genetic variation between M. sinensis and M. reevesii. In addition, the SNPs showed a 1-bp genetic variation between the two species (Fig 1). Thirteen specimens were subjected to HRM analysis and divided into three genotypes: M. sinensis (A), M. reevesii (G), and M. sinensis × M. reevesii (R).
HRM analysis using the genotyping markers revealed that three specimens produced a single peak with a Tm of 74.9 °C that corresponded to M. sinensis (A). Moreover, six specimens with a Tm of 75.4–75.7 °C corresponded to M. reevesii (G), whereas four specimens with a Tm of 75.2 °C corresponded to M. sinensis × M. reevesii (G) (Online Resource 1). Further analysis using the melting curves produced visibly different plot shapes (Fig 2). These results were consistent with those observed for R35 (Online Resource 1).
In the present study, we successfully developed HRM markers to distinguish the hybrids from M. sinensis and M. reevesii. The HRM markers developed in this study will be useful for the rapid identification of M. sinensis and interspecific hybrids. However, they had limitations in the identification of other turtles in the genus Mauremys. Therefore, further studies should be conducted to develop markers that can compensate for these limitations.