For phylogenetic analysis of the E2-6K-E1 region or complete genome, maximum-likelihood trees were constructed using MEGA version 6 (www.megasoftware.net ), with the Kimura-2 parameter corrections of multiple substitutions. Reliability of nodes was assessed by bootstrap resampling with 5,000 replicates. A sequence of Ivory Coast (West Africa lineage) isolate (HM045818) was used as an outgroup to root the tree. Nucleotide sequences of the E2-6K-E1 region and the complete genome of isolates used in this study were submitted to EMBL-EBI and their accession numbers are shown in Table 1 .
Mega version 6
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MEGA version 6.06 is a software package for lab equipment. It provides core functionality to manage and operate various lab instruments and devices.
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12 protocols using mega version 6
1
Phylogenetic Analysis of E2-6K-E1 and Complete Genomes
2
Phylogenetic Analysis of HSP101 Orthologs
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Protein sequences of putative orthologs of HSP101 were obtained from Gramene (http://www.gramene.org) and then aligned with ClustalW using default parameters. Evolutionary analysis was performed with the neighborjoining method based on the Poisson model by MEGA version 6 (http://www. megasoftware.net/) using the bootstrap method with 1,000 replications. The alignment and tree are provided as Supplemental Files S1 and S2.
3
Multi-species Phylogenetic Analysis of Legumes
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The different PLCPs were applied for multi-species phylogenetic analysis including 97 GmPLCPs, 26 PLCPs from A. thaliana, 33 PLCPs from M. trunctula, and 25 from L. japonicus. Clustal W program was used to conduct the full-length peptide sequence alignments. MEGA6 software [64 (link)], Neighbor-Joining (NJ) method and 1000 bootstrap replicates analysis with the p-distance model were used to perform the multi-species phylogenetic tree. The programs RAxML, MEGA version 6.0 and MrBayes 3.2 (http://www.megasoftware.net ) [64 (link)–66 (link)] were used to perform the phylogenetic of 97 GmPLCPs. RAxML 8.0.0 [67 ] was used to perform the Maximum likelihood (ML) analysis, the 1000 bootstrap replicates convergence test using the extended majority-rule consensus tree criterion (auto MRE) in RAxML was used to perform rapid 1000 bootstrap replicates analysis, and mixed model was used to construct the MrBayes analysis.
4
Phylogenetic Analysis of LraI Restriction-Modification System
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Sequence searches on the NCBI nucleotide and protein databases were conducted with BLAST [22 (link)] using lraIR/LraIR and lraIM/LraIM sequences. The phylogenetic inferences between restriction and methylase enzymes were obtained by MEGA version 6.0 (http://www.megasoftware.net/ ). The first 30 protein reference sequences of EcoRI-like endonuclease or methyltransferase enzymes chosen according to results of BLASTP search and LraI restrictase and LraI methylase sequences separately were trimmed and aligned using Clustal W [23 (link)] with default parameters. The phylogenetic trees were constructed by the maximum-likelihood (ML) method using a Tamura-Nei model. Bootstrapping of 1000 replicates was used to infer confidence levels of ML trees.
The nucleotide sequences of DNA fragments carrying genes encoding LraI restriction-modification system and 16S rRNA from L. raffinolactis BGTRK10-1 were submitted to ENA GenBank under accession numbers LT222052 and LT854837, respectively.
The nucleotide sequences of DNA fragments carrying genes encoding LraI restriction-modification system and 16S rRNA from L. raffinolactis BGTRK10-1 were submitted to ENA GenBank under accession numbers LT222052 and LT854837, respectively.
5
Phylogenetic Analysis of Parvovirus Genomes
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Sequences alignments were performed using the ClustalX (Ver.1.81) program. A neighbor-joining (NJ) tree was constructed using MEGA version 6 software (www.megasoftware.net ). Reliability of the NJ tree was calculated using 1,000 bootstrap replicates. In addition to the PPV6 viruses, the complete sequences of various other parvoviruses were obtained from GenBank.
6
Phylogenetic Analysis of ZmRCL1 Homologs
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Homologs of ZmRCL1 in various species were obtained using the NCBI protein BLAST tool. The phylogenetic tree was constructed using the neighbor-joining method within the MEGA version 6 software (www.megasoftware.net ). Default parameters are Poisson correction, pairwise deletion, and bootstrap (1,000 replicates; random seed). Full multiple sequence alignments were generated using Bio-Edit software and ClustalW program (Thompson, et al., 1994 (link)). A graphic view with a 100% or 70% threshold was displayed as black and grey boxes, indicating identical or conserved amino acids, respectively.
7
Comparative Genomic Analysis of GVI-1 Viruses
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To thoroughly compare the complete genomes of our six GVI-1 viruses, phylogenetic trees were constructed using a dataset, including the complete genomes of our six GVI-1 strains and those of five reference viruses of the GVI-1 lineage and 99 reference strains of other lineages (Supplementary Table 1) (Chen et al., 2017 (link); Jiang et al., 2017 (link); Ma et al., 2019 (link); Valastro et al., 2016 (link)) from GenBank (www.ncbi.nlm.nih.gov/genbank/ ). The complete genomic sequences of our six IBV strains and the 104 reference viruses were aligned using Clustal W, and phylogenetic trees were constructed using both Mega version 6.0 software (http://www.megasoftware.net/ ) using the maximum likelihood method and the Clustal V method using DNAStar software (Liu et al., 2006 (link)), respectively; the topology of the two methods was similar. In addition, the complete genomic sequences of 11 GVI-1 viruses, including the six in this study and five from the GenBank database were analyzed using MAFFT version 6. The percentage identities between the 11 GVI-1 viruses were calculated.
8
Phylogenetic Analysis of Avian Coronavirus S1 Genes
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The full S1 gene sequence generated in this study was subjected to BLAST searches using the National Center for Biotechnology Information database, and then analyzed phylogenetically using a dataset consisting of 73 sequences (Supplemental Table 1), including representative sequences for each genotype and lineage, as recently recommended (Valastro et al., 2016 (link); Chen et al., 2017 (link); Jiang et al., 2017 (link)), and the GX-NN130021 strain, which was closely related to I0636/16 by BLAST analysis. The S1 gene sequences of the IBV strain I0636/16 and the selected viruses were aligned using ClustalW, and phylogenetic analysis was carried out using Mega version 6.0 software (http://www.megasoftware.net/ ) using the maximum likelihood method with the Tamura–Nei substitution model and 1000 bootstrap replicates to assess the robustness of the branches. Only one IBV representative was selected from each lineage based on the phylogenetic trees to calculate the percentage identities between strain I0636/16 and the representative strains at both the nucleotide and amino acid levels.
9
Phylogenetic Analysis of DENV and CHIKV
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The sequences obtained were aligned with other DENV/CHIKV sequences available in the GenBank database (accession numbers are reported in the phylogenetic trees), by using the ClustalW program (www.clustal.org ) [37 (link)]. Alignments were manually edited with the Bioedit program [38 ]. Nucleotide Tamura-Nei model and the Neighbour-Joining method was used to construct the phylogenetic trees [39 (link)]. The Neighbour-Joining method was implemented by using MEGA version 6.06 (www.megasoftware.net ) [40 (link)]. The robustness of branching patterns was confirmed with a bootstrap analysis using 1000 replicates.
10
Phylogenetic Analysis of Anaplasma and Ehrlichia
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A phylogenetic analysis, which included DNA sequences obtained from foxes from this study, was carried out to compare these sequences to other sequences from Anaplasma spp. and Ehrlichia spp. that had previously been deposited in GenBank®. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6.06 (http://www.megasoftware.net ) [32 (link)] and a phylogenetic tree was constructed by the Maximum-Likelihood algorithms using the Kimura-2-Parameter model. Bootstrap replicates were performed to estimate the node reliability, and values were obtained from 1000 randomly selected samples of the aligned sequence data.
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