Kenevir Çeşitlerinde clpP Geninin Karakterizasyonu ve Mikrosatelit Analizi

Öz

Kenevir, lifleri, tohumları ve kanabinoidleri nedeniyle endüstriyel, tıbbi ve eğlence amaçlı kullanılır. Kenevir çeşitlerinin doğru bir şekilde tanımlanmasına duyulan ihtiyaç arttıkça, moleküler belirteçler çeşitlerin ayırt edilmesi, evrimsel çalışmalar ve adli analizler için önemli araçlar haline gelmiştir. Mikrosatellitler, bitki genomunun tamamında, kloroplast DNA da dahil olmak üzere, yüksek değişkenliğe sahip ve ko-dominant DNA tekrarlarıdır. Kloroplast genleri arasında, ATP’ye bağımlı Clp proteazın önemli bir alt birimini kodlayan clpP geninin, mikrosatellit varyasyonu açısından zengin bir bölge olduğu bildirilmiştir. Bu çalışma, farklı tipleri ve coğrafi kökenleri temsil eden on beş kenevir örneği üzerinde clpP genini inceleyerek, genetik belirteç olarak kullanılabilecek mikrosatellit motiflerini tanımlamayı amaçlamıştır. Bu gen, üç ekzon ve iki introndan oluşmakta ve ekzon bölgeleri tamamen korunmuştur. Tüm dizilim farklılıkları, SNP ve Indel’ler dahil olmak üzere, intron bölgelerinde yer almaktadır. Mikrosatellit analizinde, çoğunlukla A, T ve TA motiflerinden oluşan mono- ve dinükleotid tekrarlar belirlenmiştir ve bunlar çeşitler arasında uzunluk ve dizilim açısından farklılık göstermiştir. Bazı motifler belirli genotiplere özgü olup, bu durum çeşitlerin coğrafi farklılıkları yansıtmaktadır. clpP geninde tanımlanan mikrosatellit profilleri, bu gen bölgesinin kenevir genetiği için bilgilendirici olduğunu ve clpP tabanlı belirteçlerin ıslah, filogenetik çalışmalar ve adli analizlerde kullanılma potansiyeline sahip olduğunu göstermektedir.

Anahtar Kelimeler

• Kenevir
• kloroplast DNA
• genetik çeşitlilik
• genetik belirteç
• mikrosatellitler

Characterization and Microsatellite Analysis of the clpP Gene in Cannabis Cultivars

Öz

Cannabis is used for industrial, medicinal, and recreational purposes due to its fibers, seeds, and cannabinoids. As the need for identification of cannabis varieties increases, molecular markers have become important tools for distinguishing cultivars, studying evolution, and supporting forensic analyses. Microsatellites are highly variable, co-dominant DNA repeats found throughout plant genomes, including chloroplast DNA. Among chloroplast genes, clpP, which encodes a key subunit of the ATP-dependent clp protease, has been reported as a region rich in microsatellite variation. This study investigated the clpP gene in fifteen cannabis accessions representing different crop types and geographical backgrounds, with the aim of identifying microsatellite motifs useful as genetic markers. The gene consisted of three exons and two introns, with the exon regions completely conserved. All sequence differences, including SNPs and Indels, were located in the introns. Microsatellite analysis detected mono- and dinucleotide repeats mainly A, T, and TA motifs that differed in length and organization among cultivars. Some motifs are specific to certain genotypes, reflecting the geographical differences of the varieties. The microsatellite patterns identified in the clpP gene indicate that this region is informative for cannabis genetics and highlight the potential of clpP-based markers for use in breeding, phylogenetic studies, and forensic analyses.

Anahtar Kelimeler

• Cannabis
• chloroplast DNA
• genetic diversity
• genetic marker
• microsatellites

Kaynakça

1. Amiteye, S., 2021. Basic concepts and methodologies of DNA marker systems in plant molecular breeding. Heliyon 7(10).
2. Amom, T., Nongdam, P., 2017. The use of molecular marker methods in plants: a review. Int. J. Curr. Res. Rev. 9(17): 1–7.
3. Barcaccia, G., Palumbo, F., Scariolo, F., Vannozzi, A., Borin, M., Bona, S., 2020. Potentials and challenges of genomics for breeding Cannabis cultivars. Front. Plant Sci. 11: 1472.
4. Bidyananda, N., Jamir, I., Nowakowska, K., Varte, V., Vendrame, W.A., Devi, R.S., Nongdam, P., 2024. Plant genetic diversity studies: insights from DNA marker analyses. Int. J. Plant Biol. 15(3): 607–640.
5. Byrne, M., & Hankinson, M. 2012. Testing the variability of chloroplast sequences for plant phylogeography. Australian Journal of Botany, 60: 569–574.
6. Chen, L., Pan, T., Qian, H., Zhang, M., Yang, G., Wang, X., 2021. Genetic diversity and population structure revealed by SSR markers on endemic species Osmanthus serrulatus. Forests 12: 1365.
7. Cheng, Y.C., Houston, R., 2021. Evaluation of chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa. Int. J. Legal Med. 135(4): 1235–1244.
8. Dolatabadian, A., Fernando, W.G.D., 2022. Genomic variations and mutational events associated with plant–pathogen interactions. Biology 11(3): 421.

9. Dong, W., Liu, J., Yu, J., Wang, L., Zhou, S., 2012. Highly variable chloroplast markers for evaluating plant phylogeny. PLoS ONE 7: e35071.
10. 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 orchids. Int. J. Mol. Sci. 19(3): 716.
11. Erixon, P., Oxelman, B., 2008. Whole-gene positive selection and indel evolution of chloroplast clpP1. PLoS ONE 3: e1386.
12. Ellegren, H., 2004. Microsatellites: simple sequences with complex evolution. Nat. Rev. Genet. 5: 435–445. Hudak, J. 2020. Marijuana: A short history. Brookings Institution Press.
13. Jin, Y., Wang, J., Bachtiar, M., Chong, S.S., Lee, C.G., 2018. Architecture of polymorphisms in the human genome. Hum. Genomics 12: 13.
14. 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).
15. Liu, J., Qi, Z.C., Zhao, Y.P., Fu, C.X., Xiang, Q.Y., 2012. Complete cpDNA genome sequence of Smilax china. Mol. Phylogenet. Evol. 64(3): 545–562.
16. Liu, Q., Li, X., Li, M., et al., 2020. Comparative chloroplast genome analyses of Avena. BMC Plant Biol. 20: 406. 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.
17. Oliveira, E.J., Padua, J.G., Zucchi, M.I., Vencovsky, R., Vieira, M.L.C., 2006. Origin, evolution and genome distribution of microsatellites. Genet. Mol. Biol. 29: 294–307.
18. 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.
19. Roman, M.G., Gangitano, D., Houston, R., 2019. New chloroplast markers for origin and crop type of Cannabis sativa. Int. J. Legal Med. 133: 1721–1732.
20. Shermann-Broyles, S., Bombarely, A., Grimwood, J., Schmutz, J., Doyle, J., 2014. Plastome sequences from Glycine syndetika and relatives. G3 Genes Genom. Genet. 4(10): 2023–2033.
21. Shikanai, T., Shimizu, K., Ueda, K., Nishimura, Y., Kuroiwa, T., Hashimoto, T., 2001. The chloroplast clpP gene is indispensable for chloroplast development. Plant Cell Physiol. 42(3): 264–273.
22. Sloan, D. B., Triant, D. A., Forrester, N. J., Bergner, L. M., Wu, M., Taylor, D. R. 2014. A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae). Molecular phylogenetics and evolution, 72: 82-89.
23. Small, E., & Cronquist, A. 1976. A practical and natural taxonomy for Cannabis. Taxon, 25, 405–435.
24. Wang, L., Liu, B., Yang, Y., Zhuang, Q., Chen, S., Liu, Y., Huang, S. 2022. The comparative studies of complete chloroplast genomes in Actinidia (Actinidiaceae): Novel insights into heterogenous variation, clpP gene annotation and phylogenetic relationships. Molecular Genetics and Genomics, 297(2): 535-551.
25. Xu, J., Kong, L., Wu, W., Li, C., Ren, W., Ma, W., 2025. Complete chloroplast genome sequence data of Cannabis sativa. Data Brief 60: 111632.