SSR Diversity and Mutational Patterns in the ndhF–rpl32 Spacer of Cannabis cpDNA

Year 2025, Volume: 9 Issue: 4, 1374 - 1378, 26.12.2025

Saber Delpasand Khabbazi

https://doi.org/10.31015/2025.4.37

Abstract

Cannabis is a multipurpose plant widely utilized for industrial, medicinal, and recreational purposes. Accurate differentiation between hemp and drug-type varieties is essential due to legal regulations, breeding strategies, and economic value. Although genes involved in cannabinoid biosynthesis are located in the nuclear genome, chloroplast DNA (cpDNA) markers represent a complementary and stable genetic resource for discrimination and evolutionary studies because of their conserved structure and uniparental inheritance. In the present study, fifteen cannabis accessions representing diverse geographic origins were analyzed to investigate sequence variation and simple sequence repeat (SSR) composition within the ndhF–rpl32 intergenic spacer of the chloroplast genome using in silico approaches. Comparative sequence analysis indicated that this region is largely conserved; however, detectable variation was observed in the form of single nucleotide polymorphisms (SNPs) and insertion/deletion events (InDels). A total of seventeen SNPs were identified, with transversion mutations occurring more frequently than transitions. Notably, A↔T substitutions predominated and were mainly associated with A/T-rich regions, suggesting relaxed selective constraints within this non-coding spacer. InDels were distributed across several positions, further contributing to sequence variability among accessions. SSR analysis revealed the presence of exclusively mononucleotide and tetranucleotide repeat motifs. Among these, the A and AAAT motifs were the most abundant, while several accessions lacked detectable SSRs within the target region. The observed diversity in SSR composition and mutational patterns highlights the discriminatory potential of the ndhF–rpl32 intergenic spacer. These findings demonstrate that the ndhF–rpl32 region harbours informative cpDNA variation and represents a promising molecular marker for distinguishing cannabis varieties. This study enhances current knowledge of chloroplast genome diversity in cannabis and provides a valuable framework for future phylogenetic, breeding, and forensic investigations.

Keywords

Cannabis , cpDNA , Intergenic spacer , Mutation , SSR

References

• Barcaccia, G., Palumbo, F., Scariolo, F., Vannozzi, A., Borin, M., & Bona, S. (2020). Potentials and challenges of genomics for breeding cannabis cultivars. Frontiers in Plant Science, 11, 1472.
• Birky, C. W., Jr. (2001). The inheritance of genes in mitochondria and chloroplasts: Laws, mechanisms, and models. Annual Review of Genetics, 35, 125–148.
• Byrne, M., & Hankinson, M. (2012). Testing the variability of chloroplast sequences for plant phylogeography. Australian Journal of Botany, 60, 569–574.
• Cheng, Y. C., & Houston, R. (2021). Evaluation of the trnK-matK-trnK, ycf3, and accD-psaI chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa. International Journal of Legal Medicine, 135(4), 1235–1244.
• Datwyler, S. L., & Weiblen, G. D. (2006). Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms. Journal of forensic sciences, 51(2), 371-375.
• Di Nunzio, M., Barrot-Feixat, C., & Gangitano, D. (2024). Characterization and evaluation of nine Cannabis sativa chloroplast SNP markers for crop type determination and biogeographical origin on European samples. Forensic Science International: Genetics, 68, 102971.
• Dong, W. L., Wang, R. N., Zhang, N. Y., Fan, W. B., Fang, M. F., & Li, Z. H. (2018). Molecular evolution of chloroplast genomes of orchid species: insights into phylogenetic relationship and adaptive evolution. International Journal of Molecular Sciences, 19(3), 716.
• Ellegren, H. (2004). Microsatellites: Simple sequences with complex evolution. Nature Reviews Genetics, 5, 435–445.
• Hudak, J. (2020). Marijuana: A short history. Brookings Institution Press.
• Khabbazi, S. D. (2026). Comprehensive analysis of the chloroplast genome in Cannabis sativa L. reveals variations in simple sequence repeats among cultivars. Scientific Reports DOI 10.1038/s41598-025-28205-0 (In Press).
• Kim, J. H., Jung, J. Y., Choi, H. I., Kim, N. H., Park, J. Y., Lee, Y., & Yang, T. J. (2013). Diversity and evolution of major Panax species revealed by scanning the entire chloroplast intergenic spacer sequences. Genetic resources and crop evolution, 60(2), 413-425.
• Levinson, G., Gutman, G. A. (1987). Slipped-strand mispairing: A major mechanism for DNA sequence evolution. Molecular Biology and Evolution, 4(3), 203–221.
• Liu, H., Su, Z., Yu, S., Liu, J., Yin, X., Zhang, G., … Li, B. (2019). Genome comparison reveals mutation hotspots in the chloroplast genome and phylogenetic relationships of Ormosia species. BioMed Research International, 2019(1), 7265030.
• Malabadi, R., Kolkar, K., Brindha, C., Chalannavar, R., Abdi, G., Baijnath, H., Munhoz, A., & Mudigoudra, B. (2023). Cannabis sativa: Autoflowering and hybrid strains. International Journal of Innovative Science and Research Review, 5, 4874–4877.
• Matielo, C. B., Lemos, R. P. M., Sarzi, D. S., Machado, L. D. O., Beise, D. C., Dobbler, P. C. T., … Stefenon, V. M. (2020). Whole plastome sequences of two drug-type Cannabis: Insights into the use of plastid in forensic analyses. Journal of Forensic Sciences, 65, 259–265.
• McPartland, J. M., Hegman, W., & Long, T. (2019). Cannabis in Asia: Its center of origin and early cultivation, based on a synthesis of subfossil pollen and archaeobotanical studies. Vegetation History and Archaeobotany, 28, 691–702.
• Olson, M. E. (2002). Intergeneric relationships within the Caricaceae–Moringaceae clade (Brassicales) and potential morphological synapomorphies of the clade and its families. International Journal of Plant Sciences, 163, 51–65.
• Roman, M. G., Gutierrez, R., & Houston, R. (2022). Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing. Journal of Cannabis Research, 4, 13.
• Rotherham, D., & Harbison, S. A. (2011). Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay. Forensic Science International, 207(1-3), 193-197.
• Shi, Y., Huang, J., Wan, X., Shi, J., Chen, Z., & Zeng, W. (2025). The population chloroplast genomes of Populus reveal the phylogenetic relationship between three new taxa of sect. Leucoides and their parents. BMC Genomics, 26(1), 1–14.
• Small, E., & Cronquist, A. (1976). A practical and natural taxonomy for Cannabis. Taxon, 25, 405–435.
• Weng, L., Jiang, Y., Wang, Y., Zhang, X., Zhou, P., Wu, M., ... & Chen, S. (2023). Chloroplast genome characteristics and phylogeny of the sinodielsia clade (apiaceae: apioideae). BMC Plant Biology, 23(1), 284.
• Zhou, C. Y., Lin, W. J., Li, R., Wu, Y., Liu, Z. J., & Li, M. H. (2024). Characterization of Angraecum (Angraecinae, Orchidaceae) plastomes and utility of sequence variability hotspots. International Journal of Molecular Sciences, 25(1), 184.