Transcriptome sequencing and assembly
To provide evidence of transcripts for genome structure annotation, we conducted RNA-seq for four developmental stages of egg, larva, pupae, and adults (male and female) reared under normal conditions as described above. To identify the differentially expressed genes between normal (ND) and diapausing larvae, we constructed another two RNA-seq libraries for the long-day (LD) and short-day (SD) induced 5thinstar larvae. In total, 7 RNA-seq libraries were constructed, including one for eggs, three for larvae, one for pupae, one for male adults and one for female adults of PFM. All libraries were prepared using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina according to the manufacturer’s instructions (Vazyme, NanJing, China) and sequenced on an Illumina Novaseq platform to obtain 150-bp paired-end reads. After removing the low quality reads with Trimmomatic v0.38 (Bolger et al., 2014), the reads were mapped to the chromosome-level genome using Hisat v2.2.0 (D. Kim, Paggi, Park, Bennett, & Salzberg, 2019) and assembled with StringTie v2.1.2 (Pertea et al., 2015). FPKM (Fragments Per Kilobase per Million) values of each annotated gene in each RNA-seq were estimated with cufflinks v2.2.1 (D. Kim et al., 2013).
Differential gene expression among larvae reared at different photoperiod (SD, ND and LD) was assessed using cufflinks v2.2.1 (D. Kim et al., 2013). Genes with a fold-change ≥ 2 and q-value ≤ 0.05 were considered significant differentially expression genes (DEGs) between samples. For significantly expressed genes, up-regulated or down-regulated genes in both comparisons (ND vs. LD and ND vs. SD) were considered as genes related to diapause, while the FPKM values specifically high in the SD or LD condition were considered as light-induced genes. Gene expression visualization of DEGs were conducted with the Pheatmap R package.