Identifying the chorion gene cluster and phylogenetic analysis of chorion genes
OrthoFinder found that chorion genes were tandemly arrayed on chromosome 1 (Chr. 1). For more detailed information, we performed BLASTP search against non-redundant protein database, in addition to the Uniprot database (Bateman, 2019) with an e-value less than 1e-5, using the predicted gene models within and around the chorion gene region. As a result, 80 chorion genes were found on Chr. 1 and these genes made a gene cluster. Within this cluster, five non-chorion gene models (evm.model.Sr_HGAP_JL_scaf_2.1123,1128,1135,1136 and 1137) were also identified (Table S6). Phylogenetic analysis of chorion genes was conducted with 80 S. ricini chorion genes, 121 B. mori chorion genes, 21 P . xylostella chorion genes, 29P . xuthus chorion genes, 24 D . plexippuschorion genes registered at the Uniprot and NCBI database and one non-chorion gene (evm.model.Sr_HGAP_JL_scaf_2.1135) as outgroup. Muscle was used to generate alignments of protein sequences (Edgar, 2004). Aligned sequences were subjected to phylogenetic analysis by maximum likelihood and ultrafast bootstrap methods (Minh, Nguyen, & Haeseler, 2013) with 1000 replicates using IQ-TREE ver. 1.5.5 (Nguyen, Schmidt, Haeseler, & Minh, 2015). The phylogenetic tree was constructed based on PMB+F+R5 model.
In order to check whether S . ricini has high-cysteine chorion gene or not, amino acid sequences of 38 high-cysteine chorion protein of B. mori was aligned to deduced amino acid sequences of 80 S. ricini chorion genes via BLASTP program.