MEGA 11

Total RNA was extracted from infected leaves using TRIzol reagent (Invitrogen, CA, USA) and the quantity and quality of total RNA were assessed using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, DE, USA). The cDNA library was constructed as described by Liu et al. [22 (link)] and Zhang et al. [23 (link)]. Subsequently, NGS was performed on an Illumina Hiseq 4000 platform (LC-Bio Technologies Co., Ltd., Hangzhou, China). The resulting raw reads were trimmed of adaptor sequences and low-quality reads and then assembled using Trinity (v.2.4.0) [24 (link)]. The assembled contigs were subsequently screened against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLASTn and BLASTx searches with standard parameters, as described previously [22 (link), 23 (link)]. For phylogenetic analysis, the genome sequences were aligned using the ClustalW alignment and the phylogenetic trees were further constructed using the MEGA 11 [25 (link)], employing a neighbor-joining method with layouts of maximum composite likelihood model and 1000 bootstrap replications, as described by Zhang et al. [23 (link)]. The complete viral genome of CVB-CN was obtained by RT-PCR and 5′- and 3′-RACE-PCR using a SMARTer RACE 5′/3′ Kit (Takara Bio Inc.) with CVB-specific primers (Additional file 1) according to the manufacturer′s instructions.

A total of 72 Arabidopsis thaliana WRKY protein sequences were downloaded from TAIR (https://www.arabidopsis.org/ (accessed on 4 April 2022)), and all protein sequences of the WRKY gene family from L. chinense var. rubrum were aligned using ClustalW. Phylogenetic trees were constructed using MEGA 11 [65 (link)] with the neighbor-joining method and bootstrap analysis (1000 replicates) [66 (link)]. The NJ tree containing the WRKY protein sequences of Arabidopsis thaliana and L. chinense var. rubrum was visualized and enhanced using the Evolview [67 (link)] online tool (https://www.evolgenius.info/evolview-v2 (accessed on 20 April 2022)). According to the WRKY conserved domain features and the phylogenetic tree for the classification of LcWRKY genes, an ML tree was constructed that contained the protein sequences of all LcWRKY genes, using MEGA7 [68 (link)]. The protein sequences of the LcWRKYs were aligned using the DNAMAN software (Ver 9.0), and Microsoft Paint was utilized to enhance the sequence alignment image; in particular, the structural features of LcWRKYs were marked.

Total genomic DNA for the new collected specimens was extracted from liver tissues with the standard extraction method (Sambrook et al. 1989 ). The mitochondrial gene NADH dehydrogenase subunit 2 (ND2) was amplified and sequenced by using published primers (Wang et al. 2019a (link)). PCR and sequencing methods followed Liu et al. (2020) (link). Sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA 11 (Tamura et al. 2021 (link)). Representative species of Pseudocalotes Fitzinger were chosen as outgroups according to Wang et al. (2022) (link). Genetic data for 32 species of Diploderma and two species of outgroup taxa were obtained from GenBank (Table 1).
Sequences were aligned using MUSCLE (Edgar 2004 (link)) integrated in MEGA 11 (Tamura et al. 2021 (link)). The best substitution model GTR + Γ was selected using jModelTest 2.1.10 (Darriba et al. 2012 (link)). Bayesian Inference (BI) was performed in MrBayes 3.2.7 (Ronquist et al. 2012 (link)), based on the selected substitution model. Two runs were performed simultaneously with four Markov chains. The chains were run for 10,000,000 generations and sampled every 1,000 generations. The first 25% of the sampled trees was discarded as burn-in and then the remaining trees were used to estimate Bayesian posterior probabilities (BPP); nodes with BPP values of 0.95 and higher being considered well-supported (Huelsenbeck et al. 2001 (link); Wilcox et al. 2002 (link)). Maximum Likelihood (ML) analysis was performed in IQ-TREE 1.6.12 (Nguyen et al. 2015 (link)) using the selected substitution model. One thousand bootstrap pseudoreplicates via the ultrafast bootstrap (UFB) approximation algorithm were used to construct a final consensus tree, nodes with UFB values of 95 and above being considered significantly supported (Minh et al. 2013 (link)). Uncorrected genetic pairwise distances (p-distances) between species were calculated in MEGA 11 (Tamura et al. 2021 (link)) with the pairwise deletion option for handling alignment gaps and missing data.

N-metabolic genes were selected, and alignment of these genes was performed. The top three similar gene sequences of nitrate assimilation and denitrification were retrieved after doing BLASTP against the NCBI Nr database for the sequence alignment. Sequence Manipulation Suite version 2 was used for alignment and polished the protein sequences [20 (link)]. All the protein sequences of N-metabolism genes (assimilatory and respiratory nitrate reductase, nitrite reductase, nitric oxide reductase, hydroxylamine reductase, and glutamine synthetase) of Lelliottia amnigena and their similarities genes were analyzed by BLASTP and saved in FASTA format as an input file. To investigate the phylogenetic relationship of selected nitrogen metabolism genes was performed with the help of the MEGA 11(Mega Evolutionary Genetic Analysis version 11) tool. First, the protein sequence was aligned with MUSCLE and phylogenetic tree was constructed based on neighbor-joining [21 (link)]. The percentage of bootstrap [22 (link)] values were shown at the nodes. The evolutionary distances were computed using the Jones Taylor Thornton method [23 (link)] and are in the units of the number of amino acids substitutions per site. Branch length are given below the node. It defines the genetic changes i.e., longer the branch more genetic changes.

Amplified PCR products (n = 5) were purified using Wizard® SV Gel and PCR product purification kit (Promega, Germany) according to the instructions supplied by the manufacturer. Concentration of extracted total DNA was quantified using micro-volume spectrophotometer (Nanodrop 2000c, USA) and sequenced by LGC Genomics (Germany). The sequences were edited, and contig was formed using Vector NTI 11.5 software (Invitrogen). For comparative phylogenetic analysis, blastn was used to collect additional sequence data of ICP4 gene from GenBank. All sequence analysis was performed in MEGA version 11 [31 (link), 32 (link)]. Multiple sequence alignments were executed using MUSCLE program. Evolutionary relationship among the current isolates and isolates from other geographical areas was estimated based on phylogenetic trees constructed using the Maximum Likelihood with Kimura 2-parameter method with bootstrap replicates set at 1000.

The forward and reverse nucleotide contigs were merged using BioEdit 7.2 to reconstruct the full 16S rRNA genes, and aligned with CLUSTAL W. The phylogenetic tree, which contains PSB sequences of 16S rRNA gene and sequences with high similarity scores from the GenBank database, was constructed with MEGA 11.0 using the neighbor-joining method [36 (link)] with 1000 bootstrap analysis. Sequences (Supplementary Materials available here) were searched against the nonredundant nucleotide BLAST database for microbial identity. The sequences were then submitted to the NCBI GenBank database, and accession numbers were allocated as follows: ON931237, ON931235, ON931236, ON931234, ON931238, ON931233, and ON931239.