Many turtle and tortoise populations in Asia have sharply decreased due to international trade, habitat destruction, and degradation due to urbanization and pollution (IUCN 2020; Suzuki et al. 2011). The Reeves turtle, Mauremys reevesii (Reptilia: Geoemydidae), is native to Korea and China, and has recently been introduced in Japan and Taiwan (Lee et al. 2011; Lovich et al. 2011; NIBR 2019). This species is designated as an endangered species and is legally protected in the Republic of Korea (ROK). For the successful conservation and restoration of endangered species, it is necessary to develop a rapid and accurate identification method for indigenous populations in the ROK. However, international trade for medicine, food, religious release, and pets among neighboring countries of M. reevesii (Cheng and Dudgeon 2006; Kaiser et al. 2010; Suzuki et al. 2011) have made it difficult to identify its indigenous population in the ROK because of the lack of morphological traits that can easily distinguish it from those of neighboring countries.
High-resolution melting (HRM) technology can easily and rapidly detect nucleotide sequence variations in target species (Reed and Wittwer 2004; Vossen et al. 2009; Er and Chang 2012). Itis easier to use and cheaper than standard sequencing methods (Radvánský et al. 2011; Ramón-Laca et al. 2014; Chen et al. 2020). Furthermore, a 2-hour running time in a closed-tube performance enables high-throughput genotyping of target organisms while preventing cross-contamination (Vossen et al. 2009). In this study, we developed a genotyping marker for HMR analysis to distinguish and identify M. reevesii populations.
A total of 27 blood samples of M. reevesii were collected from freshwater reservoirs, rivers, and pet trade markets in the ROK (Table 1). Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s protocol. To sequence the complete mitochondrial cytochrome b (COB) region, PCR amplification was carried out using AccuPower® PCR PreMix (Bioneer, Daejeon, Republic of Korea) and the primer set TES-14180f (5'-GATTYGAAAAACCACCGTTG-3') and TES-15384r (5'-GGTTTACAAGACCAATGCTT-3') newly designed in this study using the ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were as follows: initial denaturation at 95°C, followed by 35 cycles at 95°C for 20 s, 55°C for 20 s, 72°C for 1 min, and a final elongation step at 72°C for 5 min. After purification using theAccuPrep® PCR Purification Kit (Bioneer), the PCR products were sequenced according to the manufacturer's protocol of the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) using a DNA Analyzer 3730xl (Thermo Fisher Scientific). After manual corrections, the nucleotide sequences were aligned using Clustal W in BioEdit 7.2.5 (Özvegy et al. 2015) to construct the nucleotide sequence matrix.
Based on the matrix, we designed a new PCR primer set, Mre-COB-0526f (5'-CTAACCCGATTCTTCACCTT-3') and Mre-COB-0604r (5'-TTGTTTGATCCGGTTTCGTG-3'), for HRM analysis of M. reevesii populations. 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, and the cycling stage consisted of a 40-cyle denaturation step at 95°C for 15 s and an annealing/elongation step at 60°C for 1 min. The meltcurve/dissociation stage consisted of 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.
Sequence analyses and comparisons of the complete mitochondrial COB region of 27 blood samples of M. reevesii from freshwater bodies and pet trade markets in the ROK revealed that there were three single nucleotide polymorphism (SNP) sites that were significant enough to distinguish its populations. They were found at downstream positions 530, 536, and 545 bp from the start codon of the COB gene (Table 1). According to their positions and combinations of nucleotide substitutions, three genotypes were identified and designated as the Korean Type (TTT), Chinese Type (TCT), and Minor Type (CCC), which correspond to Hap03, Hap01, and Hap04, respectively of Oh et al.(2017). Hap03 was dominant in Korea (51.1%) and Japan (76.9%); Hap01 was dominant in China (85.0%); Hap04 was much less abundant in Korea (4.2%), China (2.5%), and Taiwan (20.0%) (Oh et al. 2017).
The HRM analysis using the genotyping markers revealed that 12 specimens produced a single peak with Tm (melting temperature) at 77.6-77.7°C that corresponds to the Korean Type (TTT), 13 specimens at 77.9–78.1°C to the Chinese Type (TCT), and 2 specimens at 79.0°C to the Minor Type (CCC) (Fig. 1A). Further analysis using difference plot melt curves also produced visibly different shapes of plots (Fig. 1B). These results were consistent with those of the COB sequence analysis (Table 1).
In this study, we developed and applied a genotyping marker to M. reevesii populations for HRM analysis for the first time. The marker was applied to 27 blood specimens collected from the ROK and successfully distinguished the dominant population in the ROK. It will be useful for the rapid and accurate identification of the native population of M. reevesii in the ROK. However, mitochondrial genes represent maternal inheritance (Slowinski and Keogh 2000; Vun et al. 2011) and have an apparent disadvantage in distinguishing the F1 produced by hybridization within species or populations. Therefore, it is necessary to study the combination of nuclear short tandem repeat (STR) markers (Miller et al. 2011; Ruíz-Rivero et al. 2021) with our HRM markers in future studies.