Background

Evolution of plant genes is inextricably coupled with various evolutionary events, including endosymbiotic events, whole-genome duplication/triplication (WGD/T), gene loss, and horizontal gene transfer (Zhang et al., 2022). Archaeplastida, including green plants (Viridiplantae), glaucophytes (Glaucophyta), and red algae (Rhodophyta), originate anciently and most of them have experienced multiple WGD/T events, resulting in dramatic changes in copy numbers and complicated evolutionary trajectories of their homologous genes (Qiao et al., 2019). Homologs, orthologs, and paralogs are important concepts for the evolutionary classification of genes, being prevalent in recent comparative genomic studies. Homologs are genes sharing a common origin; orthologs and paralogs are two types of homologous genes, which separately evolved via speciation and gene duplication (Thornton and DeSalle, 2000; Koonin, 2005). Homologous genes generally have a relatively higher degree of sequence similarity than non-homologous genes. Sequence similarity–based searching and phylogenetic analyses are useful tools for identifying homologous sequences of genes and reconstructing their evolutionary routes.

Although the definition of homology/orthology has nothing to do with biological functions, there are major functional connotations (Koonin, 2005). Homologous/orthologous genes among different plants typically perform similar or equivalent functions, which is theoretically plausible and empirically supported. Thus, for a newly identified gene in non-model plants, identifying its homologs/orthologs in model plants or crops that have well-documented functional annotations is very useful to assign its possible functions. Phylogenetic analyses can reconstruct the evolutionary trajectories of homologs/orthologs among various species, which can facilitate the understanding of the molecular mechanisms underpinning its biological functions. Here, taking the acetyltransferase like protein HOOKLESS1 (HLS1) as an example (Lehman et al., 1996; Li et al., 2004), we provide a detailed procedure for homologs/orthologs identification using large-scale genomic and transcriptomic data of distantly related plants. This protocol includes generalized steps and parameters for evolutionary analyses of plant genes, and some of these steps and parameters can be customized based on the genes of interest.