Background

Gene duplication, including whole genome duplication or polyploidy, segmental duplication, and tandem duplication, is a very important process that increases gene copy number and thus enhances genetic diversity in many organisms. Because of this, gene duplication is thought to be a major force in evolution (Ohno, 1970; Otto and Whitton, 2000; Blanc and Wolfe, 2004; Jiao et al., 2011). After duplication, duplicated genes are subject to a variety of changes, such as accumulation of point mutations, insertions and deletions, gene conversion and transposon insertions (Ilic et al., 2003; Gu et al., 2005; Sémon and Wolfe, 2007). Theoretically, two or multiple copies of the duplicated genes have undergone different levels of selective constraint, which makes the functional divergence of the duplicated genes. This can be reflected on the sequence divergence of the homologous genes, such as non-synonymous substitution (Ka) and synonymous substitution (Ks), the latter of which the produced amino acid sequence is not modified. Because it is neutral with respect to selection, Ks can be used to determine rough divergence time. The ratio of Ka/Ks can be used to estimate the selection pressure on genes. A Ka/Ks ratio equal to one indicates a lack of selection, as is observed in pseudogenes. The ratio of Ka/Ks higher and lower than one implies positive and purifying selection, respectively. The values of Ka/Ks for the vast majority of genes are < 1.0 due to purifying selection to maintain function (Makalowski and Boguski, 1998; Nekrutenko et al., 2002). When comparing duplicate genes, differences in Ka/Ks suggest different levels or kinds of selection.

In order to determine the evolutionary distance, we first need to identify the homologous genes within or between different species. Here, we will mainly focus on the homologous genes that show syntenic relationships between different species. Syntenic genes are those genes that retain an ancestral position on a given region of a chromosome. We refer to these syntenic homologous genes as “syntelogs” (Zhao et al., 2017). The advantage of focusing on syntelogs is that if they are the result of polyploidy, all syntelogs arose at the same time, so groups of syntenic gene pairs can be compared with high confidence. Here, we describe a detailed pipeline for the identification and comparison of the evolutionary distance of syntelogs by using soybean (Glycine max) and common bean (Phaseolus vulgaris) as example species (Figure 1). It has been proposed that soybean has experienced a recent whole genome duplication event roughly 5 to 13 million years ago (MYA, Schmutz et al., 2010), occurring after the split with its close relative common bean roughly 19 MYA (Lavin et al., 2005; McClean et al., 2010). In this protocol, we will estimate the evolutionary divergence of the duplicated gene pairs in soybean by comparing them with the orthologous genes in the common bean genome.