*4.8. Detection of Gene Duplication Events and Estimation of Synonymous (Ks) and Nonsynonymous (Ka) Substitutions per Site and Their Ratio*

Duplicated gene pairs derived from segmental or tandem duplication were identified in physic nut genome based on the method described in the Plant Genome Duplication Database [52,53]. An all-against-all BLASTP comparison (*E*-value <sup>≤</sup> <sup>1</sup> <sup>×</sup> <sup>10</sup>−20) provided the gene pairs for syntenic clustering determined by MCScanX (*E*-value <sup>≤</sup> <sup>1</sup> <sup>×</sup> <sup>10</sup>−20) [54]. Tandem duplication arrays were identified using BLASTP with a threshold of *<sup>E</sup>*-value < 1<sup>×</sup> <sup>10</sup>−20, and one unrelated gene among cluster members was tolerated, as described for *A. thaliana*. Pairs from segmental and tandem duplications were used to estimate *Ka*, *Ks*, and their ratio. Coding sequences from segmentally and tandemly duplicated *Dof* gene pairs were aligned by PRANK [55] and trimmed by Gblocks. The software DnaSP (Available online: http://www.softpedia.com/get/Science-CAD/DnaSP.shtml) [56] was then used to compute *Ka* and *Ks* values for each pair following the YN model (a simple model of voting) [57]. If *Ka*/*Ks* > 1, there is positive selection pressure; if *Ka*/*Ks* = 1, there is neutral selection or natural selection pressure; if *Ka*/*Ks* < 1, there is a purification selection effect [58,59].
