DOI: 10.5061/dryad.8w9ghx3gt
title: Data from: Chromosome-level reference genome assembly and gene editing of the dead-leaf butterfly Kallima inachus

description: The leaf resemblance of Kallima (Nymphalidae)
 butterflies is an important ecological adaptive mechanism that
 increases survival. However, the genetic mechanism underlying ecological
 adaptation remains unclear owing to a dearth of genomic information.
 Herein, we revealed the karyotype (n = 31) of the dead-leaf
 butterfly Kallima inachus, assembled its high-quality chromosome-level
 reference genome (568.92 Mb; contig N50: 19.20 Mb), and
 identified its Z and candidate W chromosomes. To our knowledge, this is
 the first study to report on these aspects of this species. In the
 assembled genome, 15,309 protein-coding genes and 49.86% repeat elements
 were annotated. Phylogenetic analysis showed that K.
 inachus diverged from Melitaea cinxia (no leaf resemblance), both
 of which are in Nymphalinae, around 40 million years ago.
 Demographic analysis indicated that the effective population size of K.
 inachus decreased during the last interglacial period in
 the Pleistocene. The wings of adults with the pigmentary gene
 ebony knocked out using CRISPR/Cas9 showed phenotypes in which
 the orange dorsal region and entire ventral surface darkened,
 suggesting its vital role in the ecological adaption of dead-leaf
 butterflies. Our results provide important genome resources for
 investigating the genetic mechanism underlying protective resemblance in
 dead-leaf butterflies and insights into the molecular basis of protective
 coloration.
description: Pupae of K. inachus from Jingdong County, Puer City, Yunnan
 Province, China, were collected and reared until eclosion in garden
 conditions (25-27 ℃, 80% relative humidity, 16 h/8 h light/darkness).
 Around 100 female and male adults were kept in a greenhouse of 20 m × 20 m
 × 3 m (length × width × height) with spoiled fruits as adult food and with
 Strobilanthes as egg-laying and larval host plants. The fifth instar
 larvae were collected as samples for karyotype analysis and for Hi-C
 sequencing, while female adults were used for Illumina sequencing for
 genome survey and PacBio sequencing for de novo genome assembly. Newly
 laid eggs were used for gene-editing experiment. In addition, one male
 collected from Yunnan Province was sequenced using Illumina platform for
 identifying the W chromosome. Genomic DNA was isolated
 from the head and thorax tissues of a female individual using TreliefTM
 Animal Genomic DNA Kit (TsingKe, China). Paired-end libraries (insert
 size: 350 bp) were generated using NEB Next® Ultra DNA Library Prep Kit
 for Illumina HiSeq4000 platform at Novogene (Tianjin, China). The female
 raw reads that have more than 90% bases with quality &lt; Q20 were
 filtered, and the rest were employed to estimate the genome size based on
 the 17 k-mer size using kmerfreq (Liu et al., 2013). These female Illumina
 reads were also used to correct errors of de novo assembled genome at the
 base level. In addition, genomic DNA was also isolated from the thorax
 tissues of two male individuals using a phenol-chloroform DNA extraction
 protocol, and paired-end libraries (insert size: 350 bp) were generated
 using KAPA® Hyper Prep Kit for Illumina HiSeq Xten platform at Novogene
 (Tianjin, China). These male reads were used to validate the
 identification of the W chromosome. For PacBio SMRT long-read sequencing,
 genomic DNA from another female adult was isolated to construct one 20 kb
 library (NextOmics, China). DNA templates and enzyme complexes were
 transferred to the zero-mode waveguide for SMRT sequencing on a PacBio
 Sequel instrument at the Genome Center of Nextomics (Wuhan,
 China). We carried out sample (four fifth instar
 larvae) treatment and library construction for Hi-C sequencing following
 previous protocols (Belton et al., 2012; Erez et al., 2009; Ma et al.,
 2015; Nagano et al., 2015) with some improvements. Firstly, the isolated
 cells from sliced tissues were cross-linked, lysed and digested by
 restriction enzyme Dpn II overnight. Secondly, the cohesive ends were
 blunted, reversed and marked with biotin-14-dATP. Thirdly, DNA was
 purified by removing of biotin from un-ligated ends, then sheared to
 fragments of 200~300 bp by Covaris M220 (Covaris, Woburn, MA). After DNA
 size selection with AMPure XP beads, point ligation junctions were pulled
 down by Dynabeads® MyOneTM Streptavidin C1 (Thermofisher). Finally, the
 Hi-C library was sequenced on the Illumina NovaSeq sequencing platform at
 Novogene (Tianjin, China).
description: <strong>Kin_Hic.fasta</strong>:  the chromosome-level
 genome data of Kallima inachus
 12species_ortholog_align_cds.fa: the alignment sequences of 12
 species orthlog genes