Identified proteins were multiple sequence aligned with Clustal omega56 (link) and opened in MEGA X v 10.2.457 (link) to construct neighbor-joining phylogenetic tree with JTT (Jones-Taylor-Thornton) + G (Gamma distributed) model and 1000 bootstrap repeats58 (link). A comparative tree consisting of three Brassica species, one non-Brassicaceae (O. sativa), and a model plant (A. thaliana) was constructed to explore the evolutionary relationship of KCS and ELO proteins.
Nucleotide substitutions causing amino acid changes are known to be non-synonymous (Ka) and those that do not cause changes are termed synonymous (Ks)59 (link). The nature and magnitude of selection pressure occurring on coding sequences can be estimated through the ratio of Ka and Ks. The mode of selection and divergence time can also be determined through the Ks value60 (link),61 (link). A value of more than 1 (Ka/Ks > 1) indicates adaptive/positive selection62 (link), less than one (Ka/Ks < 1) indicates negative selection63 (link), whereas a value equal to one (Ka/Ks = 1) indicate neutral selection64 .
Nucleotide substitutions causing amino acid changes are known to be non-synonymous (Ka) and those that do not cause changes are termed synonymous (Ks)59 (link). The nature and magnitude of selection pressure occurring on coding sequences can be estimated through the ratio of Ka and Ks. The mode of selection and divergence time can also be determined through the Ks value60 (link),61 (link). A value of more than 1 (Ka/Ks > 1) indicates adaptive/positive selection62 (link), less than one (Ka/Ks < 1) indicates negative selection63 (link), whereas a value equal to one (Ka/Ks = 1) indicate neutral selection64 .
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