2.7 ∣ Phylogenetic reconstruction
The phylogenetic tree was reconstructed by using shared single-copy
genes. Protein sequences for these single-copy genes were aligned using
MUSCLE, then protein sequence alignments were transformed back to CDS
alignments. CDS alignments of single-copy genes were then concatenated
into a super matrix. Based on this super matrix, A phylogenetic tree was
constructed using the ML (maximum likelihood) algorithm as implemented
in
RAxML
software (version 7.2.3) and in MEGA Version 5 software
(Alexandros & Stamatakis, 2006;
Tamura et al., 2011).
2.8 ∣ RNA sequencing and transcriptome
assembly
Total RNA was extracted, separately, from female nymphs of first instar
(FF), female nymphs of second instar (SF), , male first instar larvae
(FM), and male second instar larvae (SM) using the TRIzol kit (Life
Technologies, Camarillo, USA) following the procedure provided. A total
amount of 3 μg RNA per sample was used as input material for the RNA
sample preparations. Libraries were constructed and sequenced according
to the Illumina protocol. Sequencing libraries were generated using
NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following
manufacturer’s recommendations and index codes were added to attribute
sequences to each sample. Briefly, mRNA was purified from total RNA
using poly-T oligo-attached magnetic beads. Fragmentation was carried
out using divalent cations under elevated temperature in NEBNext First
Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized
using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH).
Second strand cDNA synthesis was subsequently performed using DNA
Polymerase I and RNase H. Remaining overhangs were converted into blunt
ends via exonuclease/polymerase activities. After adenylation of 3’ ends
of DNA fragments, NEBNext Adaptor with hairpin loop structures were
ligated to prepare for hybridization. To select cDNA fragments of
preferentially, the library fragments were purified with AMPure XP
system (Beckman Coulter, Beverly, USA). Then 3 μlUSER Enzyme (NEB, USA)
was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min
followed by 5 min at 95 °C before PCR. Then PCR was performed with
Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index
(X) Primer. At last, PCR products were purified (AMPure XP system) and
library quality was assessed on the Agilent Bioanalyzer 2100 system. The
clustering of the index-coded samples was performed on a cBot Cluster
Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia)
according to the manufacturer’s instructions. After cluster generation,
the library preparations were sequenced on an Illumina Hiseq 2500
platform and paired-end reads were generated.