Research on the evolutionary fate of duplicated genes in recurrent polyploids is scarce due to the difficulties in disentangling the different homeologs and alleles of duplicated genes. This chapter describes the detailed procedures to identify different homeologs and alleles of duplicated genes, to analyze their molecular characteristics, and to reveal their functional divergence by gene editing with CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9). Using the gene editing approach, we efficiently constructed multiple knockout mutant lines with single or simultaneously disrupted different homeologs or alleles in a recurrent polyploid fish, demonstrating its usability for targeting and mutating multiple divergent homeologs and alleles in recurrent duplicated genomes.
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