Race system

To characterize CP genes of TFMs and NFMs, we amplified a segment of CPs via nested PCR using primers shown in S1 Table. Primers were designed based on nucleotide sequences of a conserved region of CPs of Dermanyssus gallinae (HZ459284, KR697573) and other mite species including Varroa destructor (XM 022808259, XM 022835169), and Metaseiulus occidentalis (XM 018640260). Amplified fragments were cloned into a pGEMT easy vector (Promega, Madison, WI, USA) and nucleotide sequences were analyzed using a Beckman CEQ GeXP automated sequencer (Beckman Coulter Inc., Brea, CA). Based on partial sequences of CPs of TFMs and NFMs, we designed primers for 3′ and 5′ RACE to amplify the open reading frames (ORFs) of CPs. We performed 3′ and 5′ RACE PCR using the RACE system (Invitrogen) in accordance with the manufacturer’s protocol. 5′ and 3′ RACE PCR products were separated via agarose gel electrophoresis, purified, cloned into the pGEMT-Easy vector (Promega), and transformed into competent DH5α Escherichia coli cells.

The RACE procedures were performed using the RACE System according to the manufacturer's instructions of Invitrogen, Life Technologies. Both 3’ and 5’-RACE reactions utilized the PCR master mix reagent kit (Fermentas, Singapore) with Taq DNA polymerase. The PCR consisted of 30 cycles, each comprising 30 s at 94 °C, 1 min at 58 °C, and 1 min at 72 °C. A final polymerization step was carried out for 7 min. For Nested PCR, the amplicon from the initial PCR reaction served as the template. The resulting PCR products, which possessed a single A overhang at the 3’ end, were cloned into a vector with compatible T-overhangs (pGEM-T Easy vector, Promega, USA) for Edman degradation sequencing.

In order to complete the inchoate 5′ ends of the five almost full-length scaffolds, a rapid amplification of cDNA ends (RACE) was performed. This 5′ RACE was carried out with a RACE-System (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer’s instructions except of the usage of SuperScript™ III Reverse Transcriptase (Invitrogen™, Carlsbad, CA, USA) instead of SuperScript™ II Reverse Transcriptase (Invitrogen™, Carlsbad, CA, USA). Nested PCR was performed with gene specific primers (Table S1) and Taq DNA Polymerase with Standard Taq Buffer (New England BioLabs, Inc., Ipswich, MA, USA) as described by the manufacturer and an annealing temperature of 55 °C.
After visualization on a 1% agarose gel, PCR-products were excised of the gel and purified using NucleoSpin Gel and PCR Clean-up system (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol.
Each 3 μL of the purified PCR products was Sanger-sequenced using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Sequence data were analyzed using the Geneious software package (Biomatters, Auckland, New Zealand, Version 10.2.6), trimmed with an error probability limit of 0.05 on both ends and aligned to the respective scaffolds.