DOI: 10.6084/m9.figshare.23553015.v2
title: A major endogenous glycosidase mediating quercetin uptake in <em>Bombyx mori</em>

description: <br><b>S1 Text. Determination and phylogenetic characterization of the amino acid sequence of KWMTBOMO12227.</b><br><b>S1 Table. Information of the F2 individuals, parents, and phenotypic score used for the QTL analysis.</b><br><b>S2 Table. QTLs associated with flavonoid content in cocoon.</b><br><b>S3 Table. Composition of the semi-synthetic diets supplemented with quercetin or rutin.</b>*Amount added per 100 g dry diet: K2HPO4, 2.25 g; CaCO3, 1.0g; KCl, 0.50 g; MgSO4, 0.3 g; FePO4・4H2O, 0.10g; ZnCl2, 0.01 g.** This mixture includes cellulose powder (61.76 g/100 g). Amount added per 100 g dry diet: biotin, 0.2 mg; choline-HCl, 150 mg; folic acid, 0.2 mg; inositol, 200 mg; nicotinic acid, 10 mg, Ca-pantothenate, 15 mg; pyridoxine-HCl, 3 mg; riboflavin, 2 mg; thiamine-HCl, 2 mg.*** Amount added per 100 g dry diet: propionic acid, 0.75 mL; chloramphenicol, 15 mg.<br><b>S4 Table. Summary of the Japanese and Chinese local silkworm strains examined.</b><br><b>S5 Table. Orthogroups of the 12 species of Lepidoptera and numbers of detected gene duplication events.</b><br><b>S6 Table. Domestic silkworm glycosidases.</b><br><b>S7 Table. Primers used in the experiments.</b><br><b>S1 Fig. Photographs of representative cocoons of the wild silkworm, </b><i><b>Bombyx mandarina</b></i><b>.</b>A fresh mulberry leaf and cocoons of the wild silkworm in a bright field (left) and those irradiated with ultraviolet light A in a dark field (right). The cocoons under ultraviolet irradiation exhibit fluorescence that is characteristic of quercetin-5-<i>O</i>-glucoside and quercetin-5,4´-di-<i>O</i>-glucoside, the major quercetin metabolites in the silkworm tissues and cocoon [14,15,28]. The cocoons were collected in June 2023 at Tsukuba, Ibaraki, Japan. Bar = 10 mm.<br><b>S2 Fig. Expression profile of the candidate genes within the</b><i><b> Gd </b></i><b>locus in third-day final instar larva of the p50T strain.</b>All organs and tissues, except for ovaries, were taken from male larvae. Data are means ± SD. SG, silk gland; TPM, transcripts per million.<br><b>S3 Fig. Total flavonoid content in the cocoons of the p50T strain and the </b><i><b>KWMTBOMO12227</b></i><b>-knockout lineages reared on fresh mulberry leaves.</b>Data are means ± SD.<br><b>S4 Fig. Prediction of a transmembrane domain in the amino acid sequence of KWMTBOMO12227 by using the SOSUI tool.</b><br><b>S5 Fig. Weight of pupa and cocoon of p50T and the knockout mutants of </b><i><b>LQGH1</b></i><b>.</b>(A) Weight of the pupa. (B) Weight of the cocoon. The values above the graph are <i>p</i>-values calculated by Student’s <i>t</i>-test. The crosses inside the boxes indicate the means.<br><b>S6 Fig. Alignment of the amino acid sequences of LQGH1 from the p50T and J01 strains.</b>Amino acid sequences were aligned using Clustal Omega.<br><b>S7 Fig. Alignment of LQGH1-like proteins in Macroheterocera species.</b>(A) Whole alignment of 101 LQGH1-like proteins in Macroheterocera species. Amino acid residues identical to LQGH1 at positions with &gt;99% conservation are highlighted by blue. The 100 homologous proteins were collected by a BLASTp search using the amino acid sequence of LQGH1 from each protein sequence data of <i>Bombyx mori</i> (KWMT), <i>Antheraea yamamai </i>(Ayam), <i>Deilephila porcellus</i> (Dpor), <i>Dendrolimus punctatus</i> (Dpun) and <i>Pheosia gnoma</i> (Pgno) with a cutoff criterion e-value of &lt;1E-100. The sequence data of <i>B. mori</i> were obtained from KAIKObase (https://kaikobase.dna.affrc.go.jp/index.html) and those of <i>A. yamamai</i>, <i>D. porcellus</i> and <i>P. gnoma</i> were obtained from InsectBase 2.0 (http://v2.insect-genome.com/). The sequences were aligned using Clustal Omega. (B) Magnified view of J01-type deletion region of the alignment. Descriptions of the consensus symbols indicating the conservation level of amino acid residues are available at the Clustal Omega FAQ (https://www.ebi.ac.uk/seqdb/confluence/display/THD/Help+-+Clustal+Omega+FAQ). In brief, ‘:’ indicates residues that harbor a strongly similar physicochemical property to that in LQGH1, and ‘.’ indicates residues that harbor a weakly similar physicochemical property to that in LQGH1.<br><b>S8 Fig. Genotyping of the</b><i><b> Gd</b></i><b> locus of Japanese or Chinese local white-cocoon strains.</b>Haplotypes of the strains were determined by PCR genotyping. P/P, only the p50T genotype was detected; P/J/K, all p50T, J01, and Kosetsu genotypes were detected; J/J, only the J01 genotype was detected; J/K, both the J01 and Kosetsu genotypes were detected; K/K, only the Kosetsu genotype was detected; U, unknown, nothing detected. The primer set amplifying the genomic region from exons 1 to 2 of <i>KWMTBOMO14639</i> (<i>rp49</i>) was used as the control (predicted fragment length = 1548 bp). The grey dotted lines are straight lines connecting band markers of known size (M) that were applied at both ends of the samples. These lines represent sizes of 4000, 200, 500, and 1500 bp, from top to bottom. “JL” and “CL” stand for “Japanese local” and “Chinese local”, respectively. The colors indicate the color of the cocoon.<br><b>S9 Fig. RNA-seq read alignment to the genomic sequence of </b><i><b>KWMTBOMO12227.</b></i>(A) Mapping result of the midgut-derived RNA-seq reads and <i>KWMTBOMO12227</i> transcript isoforms visualized with Integrative Genomics Viewer. Coverages on each exon and splice junctions of three replicates are illustrated on tracks R1–3. (B) Coverage mean of each base of exon 10 and the 15-bp extension region. Data are means ± SD.<br><b>S10 Fig. Phylogenetic tree of insect and rat proteins related to KWMTBOMO12227.</b>Values on the nodes represent the bootstrap score (trials = 100). Unreliable nodes with bootstrap values under 50 are shown as multi-branching nodes. Arabidopsis thioglucoside glucohydrolase 1 (TGG1) was used as the outgroup.<br><b>S11 Fig. Phylogenetic tree of </b><i><b>Gd</b></i><b> glycosidase orthologs in Lepidoptera.</b>(A) The phylogenetic tree was constructed by the maximum likelihood method. Values on the nodes represent the bootstrap score (trials = 100). Unreliable nodes with bootstrap values under 50 are shown as multi-branching nodes. Rat LPH was used as the outgroup. Red branches represent clades including the silkworm <i>Gd</i> glycosidases. Genes of the domestic silkworm are marked in bold. (B) Species tree of Lepidoptera with reference to the report by Kawahara et al. [71]. Red circles indicates species harboring an LQGH1 ortholog.