Chloroplast Genome-Based Insights into Variety Identification in Toona sinensis

Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional approaches. To address the challenge of discriminating Toona sinensis varieties, we sequenced and compared 15 complete chloroplast genomes from five varieties in northern China. Although these genomes exhibited a highly conserved structure, we identified eight variety-specific simple sequence repeats (SSRs), two unique tandem repeats, and several hypervariable regions with elevated nucleotide diversity. Phylogenetic analysis demonstrated that whole chloroplast genomes provided the highest resolution for variety identification, outperforming conventional barcodes. Furthermore, we developed 13 specific primer pairs targeting variable regions, and PCR validation confirmed their reliable amplification across varieties. In addition, sequence-level validation by Sanger sequencing of representative SSR and tandem repeat markers revealed stable, variety-specific repeat copy number differences. These results demonstrate that the identified chloroplast markers can effectively discriminate closely related T. sinensis varieties. This study confirms that despite overall conservation, the T. sinensis plastome contains sufficient variation for reliable identification, providing a robust framework for future germplasm conservation and molecular breeding.