by the production ofanother phylogenetic tree using the maximum parsimony analysis (MP), and it wasfound that almost all HaAP2/ERFmembers were located in the sametopological clusters.In addition, HaAP2/ERFs retained motifs wereanalyzed and considered. In total, 25 conserved motifs are identified and namedas motif1 to motif 25 (Figure S1). Among them, 9 subjects included motifs 1, 2,3, 4, 5, 7, 10, 11 and 12 in the AP2/ERF range, while 16 were linked to areasoutside the region DNA-binding domain, which seemed to contain eitherfunctional factors, or areas related to nuclear positioning and transcriptionregulation50. Conservedamino acids in HaAP2/ERF transcription factors family The amino acid 30G wascompletely protected in all of the 288 sequences. In 99% of the DREB and ERFsequences, 27W, 28L, 29G, 4G, 8R, 16E amino acids are completely protected.
Thesequences 37A, 38A are also highly protected.Protein sequences ofthe two AP2 domains present in a AP2, were found to have following conservedamino acid residues in most of the sequences: 9R, 12G, 34Q, 38G, 46A, 47A, inthe first AP2 domain and 1R, 8R, 9G, 11S, 15G, 26W, in the second domain in allAP2 gene. In the present study, ERF and DREB members of ERF subfamily weredistinguished based on the sequence alignment.
Conserved amino acids, 14V, 19Ein DREB subfamily and 14A, 19D in ERF subfamily distinguished the two from eachother. The domains with conserved 14V, irrespective of a residue at 19thposition were also classified as DREBs because of the importance of 14V over19E in determining the DNA-binding specificity of DREB transcription factor tothe DRE cis-element4 (Figure S1).Intriguingly, it wasobserved that in RAVs, glycine was found instead of valine, and alanine wasfound conserved at position 14 in AP2/ERF proteins.The sequences of the Soloistare sequences HLG and LYD that these sequences were found in other plants,including Arabidopsis.