Isolation of Full-Length Coding Sequence of MaAN2

We obtained an anthocyanin-related R2R3-MYB unigene from the transcriptome of M. armeniacum flowers (Lou et al., 2014). This DNA fragment lacked the 5′- and 3′-untranslated regions; thus, we isolated full-length cDNA of MaAN2 from the flowers in M. armeniacum followed the method described by Huang et al. (2016b). The primers used for the 5′- and 3′-RACE PCR, as well as full-length gene-specific primers are listed in Supplementary Table S2. Finally, this cDNA sequence was submitted to the NCBI GenBank database with an accession number {"type":"entrez-nucleotide","attrs":{"text":"KY781168","term_id":"1211066929"}}KY781168. Besides, the R2R3-MYB was clustered with the AN2 subgroup; therefore, it was considered as MaAN2.

Multiple alignments were analyzed using DNAMAN 8.0. The R2 and R3 repeats as well as the two conserved motifs were highlighted with differently colored lines and boxes. The listed R2R3-MYBs were downloaded from the GenBank database (accession numbers listed in Figure ​Figure11). The program MEGA 6.0 was used for constructing a phylogenetic tree by maximum likelihood method.