Chapter 5 196 super mix dye (Thermo Fisher Scientific). Relative transcript abundance was determined using the comparative 2ΔΔCt method, with ACTIN2 serving as the reference gene. Statistical analysis of the qPCR data was performed using R, involving an ANOVA followed by a Tukey post hoc test. Data visualization was accomplished using GraphPad software (Version 9.5.1, released on January 24, 2023). 5.5.7 Homolog analysis We conducted an homolog search involving several plant species from distinct families and utilized the default BLAST search various websites: for cDNA sequences of Arabidopsis, we sourced them from TAIR (The Arabidopsis Information Resource, 2015), while soybean Pisum sativum and Glycine max sequences were obtained from Phytozome (Goodstein et al., 2012). Additionally, we accessed Solanaceae sequences from Solgenemics (Solgenomics.net) (Mueller et al., 2005) with ITAG4.0 for Solanum lycopersicum, Capsicum annuum UCD 10X genome chromosome v1.0 for Capsicum annuum, eggplant V4 for Solanum melongena. Orthologs for Brassica nigra were obtained from http://brassicadb.cn/#/BLAST/ as part of our comprehensive ortholog search methodology. We keep the highest score of similarity over 70%, only similarity difference less than 10% would lead to keeping multiple homologs 5.5.8 Alignment, sub-sampling, and phylogenetic analyses Homologs of all transcription factors (TFs) were aligned using Muscle v3.8.31 (Edgar, 2004). Subsequently, the phylogeny were generated using the Maximum likelihood constructed by software Mega X (Kumar et al., 2016), with a Tamura Nei model generating the ML tree. Bootstrap analysis utilized 1000 rapid bootstraps. The consensus tree method was then applied to select the genes for each species. Resulting output file was subsequently employed to construct a consensus tree using a majority rule consensus, ensuring that only nodes with bootstrap support values exceeding 50 were incorporated. The outcomes of the ML analysis were visualized and adjusted using Figtree v1.4.3 (Rambaut, 2012). 5.5.9 Conservation analysis We carried out conservation analysis with mVista (Mayor et al., 2000; Frazer et al., 2004), alignment was carried out by LAGAN (Brudno et al., 2003), and conservation area was denoted with red color.
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