Iris species, commonly known as rainbow flowers because of their attractive flowers, are extensively grown in landscape gardens. A few species, including Belamcanda chinensis, the synonym of I. domestica and I. tectorum, are known for their medicinal properties. However, research on the genomes and evolutionary relationships of Iris species is scarce. In the current study, the complete chloroplast (CP) genomes of I. tectorum, I. dichotoma, I. japonica, and I. domestica were sequenced and compared for their identification and relationship. The CP genomes of the four Iris species were circular quadripartite with similar lengths, GC contents, and codon usages. A total of 113 specific genes were annotated, including the ycf1 pseudogene in all species and rps19 in I. japonica alone. All the species had mononucleotide (A/T) simple sequence repeats (SSRs) and long forward and palindromic repeats in their genomes. A comparison of the CP genomes based on mVISTA and nucleotide diversity (Pi) identified three highly variable regions (ndhF-rpl32, rps15-ycf1, and rpl16). Phylogenetic analysis based on the complete CP genomes concluded that I. tectorum is a sister of I. japonica, and the subgenus Pardanthopsis with several I. domestica clustered into one branch is a sister of I. dichotoma. These findings confirm the feasibility of superbarcodes (complete CP genomes) for Iris species authentication and could serve as a resource for further research on Iris phylogeny.
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