DOI: 10.5061/dryad.fttdz08vr
title: Artificial selection on cis-element of Abl contributes cocoon yield increase in domestic silkworm

description: The silkworm (Bombyx mori) is an important silk-producing domestic insect.
 The quality and yield of the silk produced by B. mori exceeds that of its
 ancestor, the wild silkworm B. mandarina. However, to date little is known
 about the molecular mechanisms underlying domestication-related increases
 in silk yield. Here, we identified a gene associated with both
 domestication and silk-related quantitative trait locus (QTL): Abelson
 tyrosine protein kinase (Abl). Population genomic data for B. mori and B.
 mandarina identified obvious signatures of artificial selection in the
 genomic region bearing the Abl gene. There were two fixed nucleotide
 substitutions (−244 and −1311) in the transcription factor binding motif
 of the upstream regulatory region of B. mori Abl. Compared to Abl in the
 wild silkworm B. mandarina, B. mori Abl exhibited significantly greater
 promoter activity and was upregulated in the silk gland from the last day
 of the 5th larval instar to pupal stage (P0). Compared to the wild type,
 CRISPR/Cas9-generated Abl loss-of-function mutants exhibited a higher
 sensitivity to diseases, shorter developmental duration, and reductions in
 economically important silk traits, including cocoon weight, pupal weight,
 and cocoon layer thickness. Comparison of silk-gland transcriptomes
 between the wild type and the mutant indicated that genes enriched in
 ribosome biosynthesis, splicing, RNA transport, and the carbon metabolism
 were significantly downregulated in the mutants. Weighted gene
 co-expression network analysis (WGCNA) confirmed that genes related to
 ribosome biosynthesis were pivotal, driving the significant
 differentiation in silk yield between B. mori and B. mandarina. Here, we
 demonstrated that artificial selection acts on the cis-elements of
 silk-trait QTL gene Abl in a novel case (the domestic silkworm), and that
 this selection pressure increased Abl promoter activity and expression
 level. By promoting protein translation and synthesis, artificial
 selection further enhanced the robustness of silkworm larvae and improved
 cocoon silk synthesis.
description: Three duplicate samples were
 set for RNA-seq of slik glands of the wild type and mutant at the
 6<sup>th</sup> day of the 5<sup>th</sup> instar
 larval stage (5L6D), wandering stage (W) and prepupae (PP), respectively. Silk glands were dissected,
 stored in dry ice and then sent to Novogene Company (Tianjin, China) for
 RNA extraction and RNA-seq. Sequencing libraries were generated using
 NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following
 manufacturer’s instructions and index codes were added to attribute
 sequences to each sample. Clustering of the index-coded samples was
 performed on a cBot Cluster Generation System using HiSeq 4000 PE Cluster
 Kit (Illumia) according to the manufacturer’s instructions. After cluster
 generation, the library preparations were sequenced on an Illumina Hiseq
 4000 platform and 150bp paired-end reads were generated.
 Sequence raw data were qualified, filtered, built, and mapped
 to the silkworm reference genome (http://www.silkdb.org/silkdb/) by HISAT2
 . HTSeq v0.6.0 was used to count the read numbers mapped to each gene and
 further normalized by DEseq2 in the R package 1.16.1 (TNLIST, Beijing,
 China) .Principal component analysis (PCA) was performed using genes
 expressed in the slikglands (Normalized
 reads &gt;5) with an online software. The expression
 value of each gene was calculated and normalized using
 fragments per kilobasesof exon per million reads mapped (FPKM).