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.