PMC:8275577 / 26407-28671 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/8275577","sourcedb":"PMC","sourceid":"8275577","source_url":"https://www.ncbi.nlm.nih.gov/pmc/8275577","text":"DNA extraction and sequencing\nTissue samples (n = 144) of morphologically identified bats of R. acuminatus (n = 10), R. affinis (n = 57), R. malayanus (n = 14), and R. shameli (n = 63) were analysed for this study. These bats were captured with mist nets and harp-traps during several field surveys in Cambodia (2010), Laos (2007) and Vietnam (2011) to promote bat conservation. In 2007, the Muséum national d’Histoire naturelle (MNHN, Paris, France) was mandated by UNESCO and the World Heritage House of Luang Prabang to conduct a mammal survey in northern Laos. In 2010, the MNHN was mandated by UNESCO and the National Authority of Preah Vihear to conduct a mammal survey in northern Cambodia. Field surveys in Vietnam were authorized by the Forest Protection Department of the Ministry of Agriculture and Rural Development, the local manager boards of different protected areas (see online supplementary Table S1), and the Institute of Ecology and Biological Resources (IEBR, Hanoi, Vietnam). The animals were handled according to guidelines and recommendations of the American Society of Mammologists27. They were measured, photographed and identified by the authors (AH, GS and VTT). Tissue samples were taken from the chest muscles of voucher specimens or from the patagium (biopsy punches; 2 mm diameter) of released bats. Samples were preserved in 95% ethanol.\nTotal DNA was extracted using QIAGEN DNeasy Tissue Kit (Qiagen, Germany) in accordance with the manufacturer’s instructions. The barcode fragment of the CO1 gene (657 bp) was amplified and sequenced using the primers UTyr and C1L70528. PCR amplifications of the CO1 gene were performed as previously published29. PCR products were purified using ExoSAP Kit (GE Healthcare, UK) and sequenced using the Sanger method on an ABI 3730 automatic sequencer at the Centre National de Séquençage (Genoscope) in Evry (France). Haplotypes were assembled with forward and reverse eletcropherograms using Sequencher 5.1 (Gene Codes Corporation, Ann Arbor, MI, USA). No gaps and stop codons were found in the CO1 sequences after translation into amino-acids. Sequences generated for this study were deposited in the GenBank database (accession numbers MW712891-MW713034) (see online supplementary Table 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