Genomics and Applied Biology 2024, Vol.15, No.5, 223-234 http://bioscipublisher.com/index.php/gab 226 3.3 Assembly and annotation of Cannabis genomes The assembly and annotation of Cannabis genomes have seen significant improvements with the use of TGS technologies. For instance, the assembly of wild-type C. sativa varieties achieved scaffold and contig N50 sizes of 83.00 Mb and 513.57 kb, respectively, with 98.20% of the protein-coding genes functionally annotated (Gao et al., 2020). The use of hybrid sequencing strategies, combining long reads from TGS with short reads from SGS, has further enhanced the accuracy and completeness of genome assemblies (Rhoads and Au, 2015). Tools like HiCanu have been developed to leverage the high accuracy of PacBio HiFi reads, resulting in superior assembly continuity and accuracy (Nurk et al., 2020).Higher assembly quality is achieved by integrating PacBio SMRT long read length and HiCanu plotting (Wei et al., 2024). 3.4 Comparative genomics of Cannabis species Comparative genomics of different Cannabis species, such as Cannabis sativa and Cannabis indica, has provided insights into their evolutionary history and genetic diversity. Studies have shown that current genome assemblies are incomplete, with significant portions of the genome missing or unmapped, which complicates accurate annotation and comparison (Kovalchuk et al., 2020). The use of advanced sequencing technologies and improved assembly methods is essential for generating high-quality reference genomes that can facilitate comparative studies and the identification of species-specific genetic traits (Li et al., 2017; Lang et al., 2020). 4 Functional Gene Mining in Cannabis 4.1 Key genes identified for Cannabinoid biosynthesis Cannabinoid biosynthesis in Cannabis sativa is a complex process involving several key genes. The primary enzymes responsible for the production of major cannabinoids include tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS). These enzymes are crucial for the synthesis of Δ 9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Studies have shown that the expression levels of these genes vary significantly between different cannabis strains, with THCAS being predominantly expressed in drug-type strains like Purple Kush, while CBDAS is more common in hemp varieties such as 'Finola' (Bakel et al., 2011; Fulvio et al., 2021) (Figure 2). Additionally, the presence of cannabichromenic acid synthase (CBCAS) has been noted, although its role in cannabinoid biosynthesis is less clear and requires further investigation (Fulvio et al., 2021). 4.2 Genes involved in resistance to diseases and environmental stress Cannabis sativa has evolved various genetic mechanisms to resist diseases and environmental stress. Recent genomic studies have identified several candidate genes associated with these traits. For instance, genes involved in the synthesis of cellulose and lignin have been linked to structural integrity and resistance to pathogens (Ren et al., 2021). Genes related to regulation of salt stress are involved in plant response to salt stress through expression (Liu et al., 2022). Moreover, the identification of single nucleotide polymorphisms (SNPs) in genes related to stress responses provides insights into the plant's ability to adapt to different environmental conditions (Zhao et al., 2021). These genetic markers are crucial for breeding programs aimed at developing disease-resistant and environmentally resilient cannabis strains. 4.3 Functional genes related to fiber production and plant growth The production of high-quality fiber in hemp varieties of Cannabis sativa is governed by specific genes that influence fiber content and plant growth. Research utilizing specific length amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) has identified several genes that are highly correlated with fiber content. These include genes involved in transcription regulation, auxin transport, and sugar metabolism (Zhao et al., 2021). Additionally, the genetic differentiation between drug-type and fiber-type cannabis has been linked to variations in the THCAS and CBDAS genes, which also affect plant growth and fiber quality (Cascini et al., 2019).The first cannabis spike-type gene CsMIKC1, cloned from cannabis, reveals the molecular mechanism driving the development of female cannabis flowers and is the starting point for elucidating the functions of many homologous genes involved in inflorescence development (Xu et al., 2024).
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