Comparative chloroplast genome analysis of 10 Ilex species and the development of species-specific identification markers

The genus Ilex L. (Aquifoliaceae) includes economically important species of ornamental and medicinal value. However, germplasm identification and phylogenetic analysis of Ilex are limited due to morphological similarities and insufficient genomic resources. In this study, the complete chloroplast genome sequences of I. intermedia, I. dabieshanensis 2, and I. vomitoria were described, and along with seven published chloroplast genomes of Ilex—all of which are widely used in mid-southern China—for comparative genomics analysis. The 10 chloroplast genomes were 157,216–157,988 bp in length and displayed a typical quadripartite structure. A total of 133 genes were annotated, including 88 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Genome structure, gene content, and genome arrangement were conserved, with some variations in the IR/SC (inverted repeats/single-copy) boundary regions and divergent hotspot regions. Phylogenetic analysis among 39 genus members clarified evolutionary relationships within Ilex based on complete chloroplast genome sequences. I. intermedia, I. dabieshanensis 2, and I. vomitoria were clustered in section Aquifolium in clade C. This study also developed 11 molecular markers as DNA barcodes for discriminating among the 10 Ilex species based on hypervariable regions and validated successfully. This study provides resources for further investigations on the identification, genetic engineering, evolution, and phylogeny of Ilex species.