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