Genomics and Applied Biology 2024, Vol.15, No.5, 223-234 http://bioscipublisher.com/index.php/gab 225 Another important effort was a meta-analysis of pooled published genomics data, which highlighted the incomplete nature of current Cannabis genome assemblies and emphasized the need for coordinated efforts to improve the quality and completeness of these assemblies (Kovalchuk et al., 2020). Following these advancements, a groundbreaking study successfully established an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Cannabis sativa, providing a valuable tool for functional genomic studies (Zhang et al., 2021) (Figure 1). 3.2 Methodologies used in sequencing The methodologies employed in Cannabis genome sequencing have evolved significantly over time. Initially, second-generation sequencing (SGS) technologies were used, but these were limited by short read lengths and difficulties in resolving repetitive regions (Lu et al., 2016). The advent of third-generation sequencing (TGS) technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), has revolutionized the field. PacBio's HiFi reads offer high per-base accuracy and long read lengths, making them suitable for high-quality de novo assemblies (Murigneux et al., 2020; Nurk et al., 2020). ONT's MinION and PromethION platforms provide ultra-long reads, which are beneficial for assembling complex genomes and detecting structural variations (Lu et al., 2016; Jain et al., 2017; Murigneux et al., 2020). Figure 1 Transgenic cannabis seedlings and results of transgenic screening (Adopted from Zhang et al., 2021) Image caption: (a) Shoots regenerated from stems of the transgenic seedling. In the estimation of developmental regulator effects on shoot organogenesis, we obtained one transgenic seedling G41-1 carrying the pG41sg T-DNA fragment. Then G41-1 stem was cut into pieces and incubated in the regeneration medium containing kanamycin for 6 weeks. There are five shoots germinated from the stem explant. (b) A transgenic seedling regenerated from the G41-1 stem. All the five shoots were transferred to soil after a 5-week incubation in the root-induction medium. The first fully expanded leaves were sampled every three weeks when growing in greenhouse. Red circle: sampling in the first round of screening; white circle: sampling in the second round of screening. (c) Transgenic-specific PCR result of the chimeric plants containing mutagenesis at CsPDS1. Eleven chimeric seedlings (chim1-11) were randomly selected and transferred to soil after incubation in the root-induction medium. Since their first fully expanded leaves lost the T-DNA fragment, these plants were identified as no transgenic with primers AtU6-F1/R1 in the second round of screening. P: DNA sample of pG41sg, N: DNA sample of no transgenic plant; white arrows: specific PCR product. (d) and (e) Transgenic-specific PCR results of the five seedlings regenerated from G41-1. In the second round of screening, these plants (Cas9-1 to Cas 9-5) were identified as transgenic plants based on transgenic-specific PCR results amplified with primers AtU6-F1/R1 and CsCAS9F2/R2 (Adopted from Zhang et al., 2021)
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