II. Laboratory methods
Genomic DNA was extracted from tissue samples using the DNeasy Blood
Tissue Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s
protocols. Seven molecular markers were amplified and sequenced in this
study: two mtDNA (Cytochrome b [cytb ], ND2 ) and five
nuDNA (Rhodopsin intron 3 [Rho-3 ], Sex determining region Y
box containing gene 9 intron 2 [Sox9-2 ], Cyclin B2 intron 3
[CCNB2-3 ], Diazepam binding inhibitor intron 2
[DBI-2 ], Ubiquitin carboxyl-terminal hydrolase intron 2
[UCH-2 ]). Polymerase chain reaction (PCR) was performed in 30
μL reactions with 3 μL 10X Buffer (1X/μL), 2.4 μL dNTP (0.2 pm/μL), 1.5
μL each primer (0.5 pm/μL), 0.2 μL Taq polymerase (1U/μL), 20.4 μL
distilled water, and 1 μL template DNA (10 ng/μL). The 10X Buffer, dNTP,
and Taq polymerase were from the i-star TaqTM DNA
polymerase kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Korea).
Detailed PCR conditions for each marker are in Table S3 , and
primer information is in Table S4 .
The size of PCR products was confirmed with electrophoresis on a 1%
agarose gel. When multiple bands were found on the gel, PCR products
were run again on a 2% agarose gel and the correct-sized band was
excised. The PCR products and gel fragments were purified using DNA
purification columns (Zymo Research, Irvine, CA, USA). Sequencing was
performed in the both directions using the PCR primers on an Applied
Biosystems 3730XL machine and Big Dye® Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) at the
National Instrumentation Center for Environmental Management of Seoul
National University (Seoul, Korea). Due to the large size of cytb(>1000 bp), an additional primer (Bufo3-Inner1) was used
for sequencing (Fong et al., 2020; Table S4 ).