Mega x software

The multiple sequence alignments were performed by MEGA-X software (www.megasoftware.net/ accessed on 23 October 2021). The Clustal W tool in Multiple sequence comparisons of protein sequences was performed using ClustalW2 with parameters set to Gap Opening Penalty = 10, Gap Extension Penalty = 0.2, and Delay Divergent Cutoff = 30%. The evolutionary tree was then constructed using the maximum likelihood method with parameters set to Bootstrap = 1000 [30 (link),31 (link)].

To illustrate the evolutionary relationships of these four new viruses with other viruses, phylogenetic trees based on the genomic sequences, CP, RdRp, and some functional proteins aa sequences were constructed. The phylogenetic analyses were performed using the neighbor-joining method in the MEGA X software (https://www.megasoftware.net/, accessed on 11 June 2022)with 1000 bootstrap replicates. The related viruses were downloaded from the GenBank database, and their detailed information is presented in Tables S4–S6.

Genotyping data for each Japanese domestic cultivar and modern tobacco varieties K326 (flue-cured variety) and TN90 (burley tobacco) as an outgroup were obtained from 30 simple sequence repeat (SSR) markers and 31 loci. The sequences of SSR markers used are provided in Table 1. Each forward primer was labeled with a FAM, VIC, TET or NED fluorescent dye (Applied Biosystems) at the 5ʹ end, and a tail sequence (5ʹ-GTGTCTT-3ʹ) was added to each reverse primer. PCR amplification was performed using a QIAGEN Multiplex kit, modified to three set for multiplex reactions in accordance with Komatsu et al. (2020) (link). The PCR products were diluted 50-fold, and 1 μl of Hi-Di formamide (Thermo Fisher Scientific) was added to 10 μl of diluted solution. The mixture was heated for 5 min at 96°C, cooled rapidly on ice, electrophoresed using a 3730xl DNA Analyzer (Life Technology, Applied Biosystems) and analyzed using GeneMapper ver. 4.0 software (Applied Biosystems). A dendrogram was constructed by the neighbor-joining method (Nei et al. 1983 (link)) using Populations 1.2.3 (Langella 1999 ), and a phylogenetic tree was constructed using MEGA X software (megasoftware.net). The reliability of each node was evaluated by 1000 trials of the bootstrap method.

A blast analysis identified that the sequences most closely related to FR-HuHEVF3f were HEPAC-15, TLS09-3, SW8a24 Spain and JAO-SpaTok12. Alignments were performed with ClustalW using the complete or near-complete genome sequences of these HEV-3f sequences as well as the reference sequences of the different genotypes of HEV (HEV-1 to -8) and subtypes of HEV-3 [43 (link)]. A phylogenetic tree was then constructed by using the Maximum Likelihood method and Tamura-Nei model with a bootstrap of 1000 replicates. The initial tree was obtained using the Neighbor-Joining method. This analysis was conducted using the MEGA X software (http://www.megasoftware.net, accessed on 5 February 2021).

The neighbor-joining (NJ) tree based on filtered SNPs was constructed using MEGA-X software (www.megasoftware.net) (p-distance model) [16 (link)]. The population subdivision pattern was preliminarily classified in the principal component analysis (PCA) by the software GCTA (version 1.25.2) [17 (link)].

To classify the R2R3-MYB genes, we constructed a rooted phylogenetic tree for TcMYB proteins and Arabidopsis thaliana MYB (AtMYB) proteins using the MEGA X software (https://www.megasoftware.net/ (accessed on 5 June 2022)) [38 (link)]. The TcMYB gene family was classified based on the members’ phylogenetic relations with Arabidopsis thaliana R2R3-MYB members. We aligned all protein sequences using ClustalW with the default parameters. The phylogenetic tree was constructed by the neighbor-joining method [39 (link)]. The phylogenetic tree of TcMYB genes was beautified using the Interactive Tree of Life (iTOL, https://itol.embl.de/ (accessed on 6 June 2022)) [40 (link)].

To study evolutionary relationships, the full-length amino acid sequences of chitinase proteins from M. domestica, M. sieversii, and Arabidopsis thaliana were aligned using Clustal X2 (http://www.clustal.org/). A phylogenetic tree was generated using MEGA-X software (https://www.megasoftware.net/) with the maximum likelihood method and topological support was assessed by means of a bootstrap analysis with 1000 replicates. The exon–intron organization of the chitinase genes was visualized using TBtools (Chen et al., 2020 (link)). Conserved motifs and domains were identified with the MEME Suite (Bailey et al., 2006 (link)) and SMART database (Letunic et al., 2021 (link)), respectively, and were visualized using TBtools (Chen et al., 2020 (link)). The domain signatures were identified using the PROSITE database (Sigrist et al., 2013 (link)). The SignalP 5.0 online server (Almagro Armenteros et al., 2019 (link)) was used to identify signal peptides in the chitinase proteins of M. sieversii and M. domestica.