Dnaman

Based on the transcriptome data, the primers TroIRF3-F/TroIRF3-R were used to clone the open reading frame (ORF) of TroIRF3 by polymerase chain reaction (PCR) amplification (Table S1). Sequencing was performed following gel purification of the PCR products into the pEASY^®-T1Simple Vector (Transgen, Beijing, China). A homology search of the TroIRF3 protein sequence was conducted using BLAST programs on NCBI (http://www.ncbi.nlm.nih.gov/blast). Signal peptide prediction was performed using SignalP 5.1. Thereafter, DNAMAN (Lynnon Biosoft, USA) was used to align the amino acid sequences. SWISS-MODEL was used to predict the TroIRF3’s tertiary structure. A phylogenetic analysis was performed using neighbour-joining methods through MEGA 6.0.

Sequence alignments, translations, comparisons and the construction of phylogenetic tree were carried out using DNAMAN (version 5.2.10, Lynnon BioSoft, Vaudreuil, Canada). The BLAST algorithm was used to search the NCBI GenBank (www.ncbi.nlm.nih.gov/) databases for homologous sequences.

Three strains of B. bassiana were randomly selected from each different collection site confirmed to be infected by virus BbCV2 by dsRNA extraction and RT-PCR. Three pairs of primers (Additional file 2: Table S2) were designed for full-length amplification of nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene of virus BbCV2 (GenBank no. MW314841.1). Products obtained by RT-PCR amplification were extracted from 1% agarose electrophoresis gels, cloned using a TA/Blank Zero Cloning Kit (Vazyme, Nanjing, China), transformed into competent Escherichia coli DH5-α cells (Vazyme) by heat shock, and sequenced by Sangong Bioengineering Co. Ltd. (Shanghai, China). The complete sequence of the BbCV2 RdRp gene was obtained by splicing using DNAstar7.1 (DNASTAR, Inc., Madison, USA) and DNAMAN version 9 (Lynnon Biosoft, Vaudreuil, Canada). Modification and splicing of the measured cDNA gene sequence were assessed using Snap Gene 6.0.2 (Dotmatics Ltd, Windhill, UK) and DNAMAN (LynnonBiosoft, Vaudreuil, Canada) 9. MEGA11 (Mega Limited, Auckland, New Zealand) and DNAsp5 (University of Barcelona, Barcelona, Spain) were used to construct a phylogenetic tree of virus BbCV2 in the selected B. bassiana strains collected from different locations in Jilin Province, and the genetic distance was calculated.

The individual dsRNA segments were separated by gel electrophoresis, and each segment was excised and purified using a gel extraction kit. The purified dsRNA was used to construct libraries by random PCR amplification and for the termini of each dsRNA segment, the phosphorylated 5’-end oligonucleotide (RACE-OLIGO) was ligated to the 3’-terminus of each strand of dsRNA as previously described [47 (link)]. All sequences were assembled and aligned with DNAMAN (version 5.2.9, Lynnon Biosoft). The sequence information of previously reported reoviruses referenced in this paper was retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genome/). On the basis of multiple sequence alignments with ClusterX2, neighbor-joining phylogenetic trees were constructed using MEGA version 6.0 programs [74 ].

We deposited the obtained sequences to the NCBI database for basic local alignment search tool (BLAST) searches, following the online comparative and bioinformatic analyses of the Da-dio genes (Available online: http://www.ncbi.nlm.nih.gov). The amino acid sequences of the five Da-dios were aligned with homologous sequences downloaded from NCBI using the DNAMAN (Lynnon Biosoft, Quebec, QC, Canada) program. Additionally, the neighbor-joining method of the MEGA6 program was used to construct a phylogenetic tree. The molecular weights and theoretical pI (isoelectric point) values of the dioscorins were calculated with the ProtParam online tool (Available online: http://www.expasy.ch/tools/protparam.html). Potential conserved domains and signal peptides were analyzed using the SMART program (Available online: http://smart.embl-heidelberg.de/).

Multiple sequence alignment of the deduced amino acid sequences of repeats I and IV from Arabidopsis AnnAt1 to 8 was performed using DNAMAN (Lynnon Biosoft). A homology model of Ann5 was produced by the Swiss-Model program (http://www.expasy.ch/) and then colored using PyMOL software. The model was based on template 2zocA (2.00 A), and the QMEAN Z-Score was −1.018.