2.2 Genomic and transcriptomic sequencing
Genomic DNA (gDNA) was isolated from 200 individual female and male using the DNeasy Blood & Tissue Extraction Kit (Qiagen Inc., Valencia, CA, USA), following the manufacturer’s instructions. After quality and quantity measurements, the gDNA was used to construct a 150-bp paired-end sequencing library for Illumina platform. A 20 kb long-read sequencing library was constructed by gDNA isolated from 200 fundatrigeniae for PacBio Sequel II platform. For Hi-C analysis, 200 fundatrigeniae were soaked in 1% formaldehyde for 10 min at room temperature and in a 2.5 M-glycine solution to terminate the isolation and cross linking of aphid cells. The Hi-C assays and the sequencing procedures were performed via a commercial contract with Annoroad Gene Technology Co., Ltd. (Beijing, China) (Rao et al., 2014).
Transcriptomes were generated from RNA samples extracted from different stages including fundatrix, fundatrigeniae, autumn migrants, nymphs, spring migrants (sexuparae), male and female sexuales, separately. RNA quantity, purity and integrity were determined on a NanoPhotometer and an Agilent 2100 Bioanalyzer. cDNA libraries were subsequently constructed following the chain specific method. The libraries were initially quantified by the qubit 2.0 fluorometer and diluted to 1.5 ng/ul. Later, different libraries were pooled according to the requirements of effective concentration and target data volume for Illumina sequencing. Low-quality bases in the RNA-Seq raw reads were filtered using Trimmomatic (version 0.36) (Bolger, Lohse, & Usadel, 2014). Clean reads were mapped to the genome assembly using Hisat2 (version 2.1.0.5) (Kim et al., 2015), so as to obtain the putative transcripts. Transcript levels were analyzed using cufflinks (version 2.2.1) (Ghosh, & Chan, 2016).