| Summary: | Plant R2R3-MYBs comprise one of the largest transcription factor families; however, few <i>R2R3-MYB</i> genes in pecan have been functionally analyzed due to the limited genome information and potential functional redundancy caused by gene duplication. In this study, 153 <i>R2R3-MYB</i> genes were identified and subjected to comparative phylogenetic analysis with four other plant species. Then, the pecan R2R3-MYB gene family was divided into different clades, which were also supported by gene structure and motif composition results. Fifty-two duplication events including 77 <i>R2R3-MYB</i> genes were identified in this gene family, and <i>Ka/Ks</i> values showed that all of the duplication events were under the influence of negative selection. Expression levels of pecan <i>R2R3-MYB</i> genes during the graft union formation process were further investigated using RNA-seq with four different timepoints after grafting, namely, 0, 8, 15 and 30 d. Sixty-four differentially expressed <i>R2R3-MYB</i> genes were identified and showed different expression patterns after grafting. Co-expression networks were further constructed to discover the relationships between these genes. The co-expression networks contained 57 nodes (<i>R2R3-MYB</i> genes) and 219 edges (co-expression gene pairs) and <i>CIL1528S0032</i> contained the maximum number of edges. Fifteen genes contained more than 10 edges; the majority of these were up-regulated during graft union formation and verified by qRT-PCR. This study provides a foundation for functional analysis to investigate the roles that R2R3-MYBs play in graft union formation in pecan and identify the key candidate genes.
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