Genomics and Applied Biology 2024, Vol.15, No.5, 223-234 http://bioscipublisher.com/index.php/gab 230 8 Challenges and Limitations 8.1 Limitations in genome sequencing data and accessibility Despite significant advancements in genome sequencing technologies, the current genome assemblies of Cannabis sativa remain incomplete. Approximately 10% of the genome is missing, and 10%-25% remains unmapped. Critical regions such as 45S and 5S ribosomal DNA clusters, centromeres, and satellite sequences are not represented, which hampers the accurate annotation of gene copies (Kovalchuk et al., 2020). Additionally, the high heterozygosity in wild-type varieties of C. sativa poses challenges in generating a complete and accurate genome sequence (Gao et al., 2020). The rapid generation of large amounts of sequencing data also raises issues related to data storage, processing, and accessibility, which are critical for advancing genomic research (Kircher and Kelso, 2010; Kahn, 2011). 8.2 Genetic diversity and its impact on functional gene mining The genetic diversity within Cannabis sativa, particularly between its subspecies and cultivars, complicates functional gene mining. The high heterozygosity observed in wild-type varieties introduces a wide range of genetic variations that need to be accounted for in genomic studies (Gao et al., 2020). Single nucleotide polymorphisms (SNPs) in cannabinoid synthase genes significantly affect the plant's chemotype, making it essential to understand these variations for breeding programs aimed at specific cannabinoid profiles (Singh et al., 2020). Moreover, the uncertain taxonomic classification of Cannabis subspecies further complicates the genetic analysis and breeding efforts (Hurgobin et al., 2020). 8.3 Ethical and regulatory issues in Cannabis research Cannabis research is heavily regulated due to its classification as a narcotic drug under international treaties such as the Single Convention on Narcotic Drugs of 1961. These regulations have historically restricted scientific research and cultivation of cannabis, limiting the availability of genetic resources and hindering progress in genomics studies (Hurgobin et al., 2020). Although some jurisdictions have relaxed these regulations, ethical concerns regarding the use of cannabis for recreational and medicinal purposes continue to pose challenges. Ensuring compliance with varying legal frameworks and addressing societal concerns are critical for the advancement of cannabis research (Hurgobin et al., 2020; Sirangelo et al., 2022). 8.4 Technical challenges in Cannabis cultivation for genomic studies Cultivating Cannabis sativa for genomic studies presents several technical challenges. The dioecious nature of the plant, where male and female flowers develop on separate plants, complicates breeding programs and the study of flower development (Hurgobin et al., 2020). Additionally, the need for controlled growing conditions to ensure consistent phenotypic expression adds to the complexity of cultivation. The high variability in cannabinoid content among different cultivars necessitates precise control over environmental factors to obtain reliable data for genomic studies (Sirangelo et al., 2022). Furthermore, the lack of robust SNP markers and the need for a comprehensive set of SSR markers for marker-assisted breeding programs highlight the technical limitations in current cannabis genomics research (Hurgobin et al., 2020). 9 Future Directions in Cannabis Genomics 9.1 Potential breakthroughs in gene mining and functional genomics The future of cannabis genomics holds significant promise for breakthroughs in gene mining and functional genomics. One of the key areas of focus is the identification and functional characterization of genes involved in the biosynthesis of cannabinoids and terpenes, which are critical for the plant's medicinal properties. Recent studies have demonstrated the potential of virus-induced gene silencing (VIGS) to knock down specific genes in Cannabis sativa, providing a powerful tool for reverse genetic studies to uncover unknown gene functions (Schachtsiek et al., 2019). Additionally, advancements in in silico analysis and genome editing technologies, such as CRISPR, Zinc Fingers, and TALENs, are paving the way for precise modifications of genes involved in cannabinoid biosynthesis, which could lead to the development of new cannabis strains with enhanced therapeutic properties (Matchett-Oates et al., 2021).
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