Computational protocol: A new species of the water mite genus Sperchon Kramer, 1877 from China, with identifying Sperchonrostratus Lundblad, 1969 through DNA barcoding (Acari, Hydrachnidia, Sperchontidae)

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Protocol publication

[…] For molecular examination, each mite was transferred in individual 1.5ml tubes, and washed several times with sterile deionized water. Non-destructive DNA extraction was done on the whole mite. The genomic DNA was extracted by using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Then, the mites were fixed in absolute ethanol and stored at -20°C for morphological analysis.The standard COI barcoding fragments (658bp) were amplified with the universal primers LCO 1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO 2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (). Primers were synthesized by Shanghai Sangon Biotechnology (Shanghai, China). All amplification reactions were done in a total volume of 25?l, containing 1–5?l DNA; 12.5?l 2?Taq PCR MasterMix (Tiangen, Beijing, China) and deionized water. The PCR amplification was performed with the following profile: 5 min at 94°C;35 cycles of 30 sec at 94°C, 30 sec at 51°C, 45 sec at 72°C; final extension 10 min at 72°C. PCR products were purified by using QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). The pure segments were ligated into the pGEM-Teasy vector (Promega, Madison, WI, USA) and introduced into Escherichia coli DH5a cells. Bacteria were cultured in LB medium after blue/white selection, and then inserts were sequenced with M13 primers. Each insert was sequenced twice with ABI 3730 automated DNA sequencer by Shanghai Sangon. All sequences were submitted to BOLD and GenBank. The BOLD process ID and the GenBank accession numbers are provided in Table .All the sequence data were analysed by using MEGA (ver. 6; ), and were aligned by ClustalW. Genetic distances within and between species were calculated with a K2P model. Phylogenetic trees were constructed with neighbur-joining (NJ) and maximum-likelihood (ML) using K2P model. The sequence of Sperchonopsis echphyma was used as the outgroup. Bootstrap values were obtained from 1000 replicates. […]

Pipeline specifications

Software tools MEGA, Clustal W
Application Phylogenetics