Development of the Genotyping Marker for Reeves’ Turtle (Mauremys Reevesii) Using High-resolution Melting (HRM) Analysis

DOI: https://doi.org/10.21203/rs.3.rs-1429586/v1

Abstract

The Reeves’ turtle, Mauremys reevesii, resides in freshwater bodies of Korea, China, Japan, and Taiwan, and is designated as an endangered species in the Republic of Korea (ROK). However, international pet trade among East Asian countries has made it difficult to identify the indigenous population of M. reevesii in the ROK. In this study, a genotyping marker targeting the mitochondrial cytochrome b (COB) region that easily distinguishes the Korean population was developed for high-resolution melting (HRM) analysis and applied to 27 blood samples obtained from diverse Korean freshwater bodies and pet trade markets. The HRM analysis produced three distinct HRM curves that were designated as the Korean Type, Chinese Type, and Minor Type, whose results exactly matched those of COB sequencing. Our genotyping method will be useful for the rapid and accurate identification of the native population of M. reevesii in the ROK.

Full Text

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 the AccuPrep® 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.

Declarations

Acknowledgments

This work was supported by a grant from the National Institute of Ecology (NIE), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIE-B-2022-45).

Funding

Not applicable.

Conflict of interests

The authors do not have any conflict of interest.

Author contributions

Project management by Ju-Duk Yoon. The study conception and design by Keun-Sik Kim, Ju-Duk Yoon. Data analysis, prepared figures 1 and table 1 by Keun-Yong Kim, Jung Soo Heo, Keun-Sik Kim, and Chang-Deuk Park. Material preparation, sample collection by Hong-Shik Oh, Seon-Mi Park. The first draft of the manuscript was written by Chang-Deuk Park. Ju-Duk Yoon, Keun-Sik Kim, and Keun-Yong Kim commented on previous versions of the manuscript.

References

  1. Chen X, Li R, Wang C, Mu C, Song W, Liu L, Zhan P (2020) An effective method for identification of three mussel species and their hybrids based on SNPs. Conservation Genetics Resources 12(1):5-8
  2. Cheung SM, Dudgeon D (2006) Quantifying the Asian turtle crisis: market surveys in southern China, 2000–2003. Aquatic Conservation: Marine and Freshwater Ecosystems 16(7):751-770
  3. Er TK, Chang JG (2012) High-resolution melting: applications in genetic disorders. Clinica Chimica Acta 414:197-201
  4. IUCN (2020) The IUCN Red List of Threatened Species. Gland, Swiss: International Union for Conservation of Nature and Natural Resources. http://www.iucnredlist.org/species/170502/97431862#threats. Accessed 9 November 2021
  5. Kaiser H, Carvalho VL, Freed P, O’Shea M (2010) A widely traveled turtle: Mauremys reevesii (Testudines: Geoemydidae) in Timor-Leste. Herpetology Notes 3:93-96
  6. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of molecular evolution 6(2):111-120
  7. Lee JH, Jang HJ, Suh JH (2011) Ecological guide book of herpetofauna in Korea. National Institute of Environmental Research, Incheon
  8. Lovich JE, Yasukawa Y, Ota H (2011) Mauremys reevesii (Gray 1831) – Reeves’ turtle, Chinese three-keeled pond turtle. Chelonian Research Monographs 5:1-10
  9. Miller JM, Hallager S, Monfort SL, Newby J, Bishop K, Tidmus SA, Fleischer RC (2011) Phylogeographic analysis of nuclear and mtDNA supports subspecies designations in the ostrich (Struthio camelus). Conservation Genetics 12(2):423-431
  10. National Institute of Biological Resources (NIBR) (2018) Korean endangered species. National Institute of Biological Resources, Incheon
  11. Oh HS, Park SM, Han SH (2017) Mitochondrial haplotype distribution and phylogenetic relationship of an endangered species Reeve's turtle (Mauremys reevesii) in East Asia. Journal of Asia-Pacific Biodiversity 10(1):27-31
  12. Özvegy J, Marinković D, Vučićević M, Gajić B, Stevanović J, Krnjaić D, Aleksić-Kovačević S (2015) Cytological and molecular identification of Haemogregarina stepanowi in blood samples of the European pond turtle (Emys orbicularis) from quarantine at Belgrade zoo. Acta Veterinaria-Beograd 65(4):443-453
  13. Radvánský J, Bazsalovicsová E, Králová-Hromadová I, Minárik G, Kádaši Ľ (2011) Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants. Parasitology Research 108(1):201-209
  14. Ramón-Laca A, Gleeson, D, Yockney I, Perry M, Nugent G, Forsyth DM (2014) Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA. PLoS ONE 9(3):e92043
  15. Reed GH, Wittwer CT (2004) Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clinical Chemistry 50(10):1748-1754
  16. Ruíz-Rivero O, Garcia-Lor A. Rojas-Panadero B, Franco JC, Khamis FM, Kruger K, Pérez-Hedo M (2021) Insights into the origin of the invasive populations of Trioza erytreae in Europe using microsatellite markers and mtDNA barcoding approaches. Scientific Reports 11(1):1-15
  17. Slowinski JB, Keogh JS (2000) Phylogenetic relationships of elapid snakes based on cytochrome b mtDNA sequences. Molecular Phylogenetics and Evolution 15(1):157-164
  18. Suzuki D, Ota H, Oh HS, Hikida T (2011) Origin of Japanese populations of Reeves' pond turtle, Mauremys reevesii (Reptilia: Geoemydidae), as inferred by a molecular approach. Chelonian Conservation and Biology 10(2):237-249
  19. Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38(7):3022-3027
  20. Vossen RH, Aten E, Roos A, den Dunnen JT (2009) High‐resolution melting analysis (HRMA)—more than just sequence variant screening. Human Mutation 30(6):860-866
  21. Vun VF, Mahani MC, Lakim M, Ampeng A, Md-Zain BM (2011) Phylogenetic relationships of leaf monkeys (Presbytis; Colobinae) based on cytochrome b and 12S rRNA genes. Genetic and Molecular Research 10(1):368-381

Table

Table 1. Information on sampling sites, single nucleotide sequence (SNP) sites of mitochondrial cytochrome b gene based on the nucleotide sequence analysis, melting temperature (Tm) and genotypes based on the high-resolution melting (HRM) analysis of Mauremys reevesii in the ROK.

Sample code

Sampling site

Nucleotide sequence analysis*

HRM analysis

530

536

545

Tm (°C)

Genotype

Mre 01

Seomjin River (Gokseong-gun)

T

C

T

77.9

Chinese

Mre 02

Seomjin River (Gokseong-gun)

T

C

T

78.0

Chinese

Mre 03

Bongseo Reservoir

T

T

T

77.7

Korean

Mre 04

Gangchon (Chuncheon-si)

T

C

T

77.9

Chinese

Mre 05

Gangchon (Chuncheon-si)

T

C

T

77.9

Chinese

Mre 06

Gangchon (Chuncheon-si)

T

C

T

78.1

Chinese

Mre 07

Bongseo Reservoir

T

T

T

77.7

Korean

Mre 08

Bongseo Reservoir

T

C

T

78.0

Chinese

Mre 09

Yonggang (Gurye-gun)

T

T

T

77.7

Korean

Mre 10

Mt. Songni

T

C

T

78.0

Chinese

Mre 11

Seorim Reservoir

T

T

T

77.6

Korean

Mre 12

Seorim Reservoir

T

T

T

77.7

Korean

Mre 13

pet trade market

T

C

T

78.1

Chinese

Mre 14

pet trade market

T

C

T

78.0

Chinese

Mre 15

pet trade market

C

C

C

79.0

Minor

Mre 16

Seorim Reservoir

T

T

T

77.7

Korean

Mre 17

Sancheong-gun

T

T

T

77.7

Korean

Mre 18

Sancheong-gun

T

T

T

77.7

Korean

Mre 19

Sancheong-gun

T

C

T

78.1

Chinese

Mre 20

Sancheong-gun

T

T

T

77.7

Korean

Mre 21

Yongjeong Reservoir

T

T

T

77.7

Korean

Mre 22

Gangchon (Chuncheon-si)

T

C

T

78.1

Chinese

Mre 23

Gangchon (Chuncheon-si)

T

C

T

78.1

Chinese

Mre 24

Gangchon (Chuncheon-si)

T

C

T

78.1

Chinese

Mre 25

Korean freshwater body

T

T

T

77.7

Korean

Mre 26

Korean freshwater body

T

T

T

77.6

Korean

Mre 27

Korean freshwater body

C

C

C

79.0

Minor

*The numbers indicate the downstream positions from the start codon of the mitochondrial COB gene of M. reevesii based on the results of their nucleotide sequence analysis.