PINK PEPPER SCIENCE

Development of the New CBD Variety 'Pink Pepper' and Its Complete Genome as a Gold Standard for Cannabis Genomics

In collaboration with Kangwon National University, South Korea, the Institute of Cannabis Research (ICR) proudly announces the development of the CBD-rich cannabis variety, "Pink Pepper."

This groundbreaking research achieved the complete genome assembly of "Pink Pepper," now serving as a high-quality reference genome for Cannabis sativa L., setting a new standard for cannabis genomic studies.

The study employed cutting-edge sequencing technologies, including PacBio and ONT long-read sequencing, combined with accuracy enhancement through Illumina reads. This approach resulted in a chromosome-level genome assembly with a total length of 770 Mb, a GC content of 34.09%, and 30,459 annotated protein-coding genes. Notably, repetitive elements accounted for 77.13% of the genome. The assembly has been officially registered as a cannabis reference genome in the NCBI database (Link to Genome).

This achievement marks a significant milestone in cannabis genomics, offering a critical resource for breeding, genetic improvement, and research on cannabinoid and terpene biosynthesis. The comprehensive genome map highlights key cannabinoid synthesis pathways, particularly the genes on Chromosome 7 involved in Δ9-THCA and CBDA production. Additionally, this study represents the first-ever prediction of the centromere in the cannabis genome.

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Chromosomes of the Cannabis sativa L. cv. Pink Pepper Genome

The centromeres of the Pink Pepper genome were predicted using CentIER (Du et al., 2024). The color of each chromosome represents gene density identified in 0.1 Mbp units. THCAS: delta-9 tetrahydrocannabinolic acid synthase; CBDAS: cannabidiolic acid synthase; Chr: chromosome number. GO categorization of description describes functional annotations predicted based on protein domains, which were not identified in NCBI nr and Araport11.

Pink Pepper Genome: Setting the Gold Standard for Cannabis Genomic Research

Synteny plot of shared genes at the reference genome level among Humulus lupulus (GCF_963169125.1), Cannabis sativa (GCF_029168945.1), and Ziziphus jujuba (GCF_031755915.1). The plot displays only synteny blocks with a similarity rate of 30% or higher. hl1-hl10 represent the chromosome numbers of H. lupulus, cs1-cs10 represent the chromosome numbers of Cannabis sativa 'Pink Pepper', and zj1-zj12 represent the chromosome numbers of Z. jujuba.

Tissue-Specific Transcriptome Expression Analysis

To investigate tissue-specific gene expression patterns, RNA sequencing (RNAseq) data were processed and analyzed. Adaptor sequences and low-quality reads were first removed, after which the cleaned data were mapped to the assembled genome using RNAstar (v2.7.10b+galaxy 4). The mapping quality was assessed with MultiQC (v1.11+galaxy 1), revealing that a high proportion of reads were uniquely aligned to the genome: 89.63% from inflorescence, 92.02% from leaf, 89.81% from root, and 91.98% from stem. The distribution of mapped reads across chromosomes was as follows: Ch1 (127G), Ch2 (76G), Ch3 (70G), Ch4 (110G), Ch5 (55G), Ch6 (55G), Ch7 (49G), Ch8 (84G), Ch9 (62G), and Ch10 (85G) (Fig. b).

To quantify gene expression levels, transcripts per million (TPM) values were computed using Salmon quant(v1.10.1+galaxy2). To identify patterns of gene activity across different tissues, k-means clustering was applied, grouping genes into 10 distinct clusters based on their expression levels (Fig. c). The results showed the following patterns:

Leaf-specific gene expression: Clusters 2, 3, and 5 contained genes highly expressed in leaves (1 gene, 37 genes, and 5 genes, respectively).
Inflorescence and root-specific expression: Cluster 8 contained 20,714 genes that were highly active in both tissues.
Root-specific gene expression: Cluster 9 contained 1,365 genes strongly expressed in roots.
Low expression in leaves: Clusters 10 and 4 contained 249 and 25 genes, respectively.
Stem-specific gene expression: Cluster 1 contained 1 gene exclusively expressed in stems.
Shared expression between stems and roots: Clusters 6 and 7 contained 3 genes and 1 gene, respectively.

To better understand the functions of leaf-specific genes, Gene Ontology (GO) analysis was performed on clusters 2, 3, and 5. The analysis indicated that these genes were strongly associated with photosynthesis and pigment-related functions. The most enriched GO terms included photosynthesis, photosynthetic membrane, thylakoid, photosystem, plastid, chloroplast, and photosystem II, all of which are essential for light absorption and energy production in plants (Fig. d).

Pink Pepper Biomarker

Given the significant genetic and commercial value of the Pink Pepper variety as a CBD producer, it is imperative to establish robust methods for tracing and managing its unauthorized use and reproduction. To address this, Insertion or Deletion (InDel) Markers have been adopted to ensure the protection and identification of this variety, which is registered and patented in Korea.

What Are InDel Markers?

Insertion or Deletion Markers are tools that detect insertion or deletion regions by comparing DNA base sequences. These markers are widely utilized for variety identification and polymorphism analysis due to their advantages:

High polymorphism detection
Codominance
Cost efficiency
Ease of interpretation

Development of Pink Pepper DNA Markers

High polymorphism detection
Codominance
Cost efficiency
Ease of interpretation

Pink Pepper Biomarker Testing

The development of InDel markers for Pink Pepper identification provides a reliable and precise method for distinguishing this variety at the genetic level. These markers enable accurate breeding selection, ensuring varietal purity and consistency in cultivation.

Identification of Pink Pepper Variety Using InDel Markers

Top gel: PCR amplification with the InDel3 marker (expected band size: 333 bp).
Bottom gel: PCR amplification with the InDel4 marker (expected band size: 389 bp).

Sample descriptions:

Pink Pepper mother plant
Female progeny derived from the Pink Pepper mother plant treated with silver nitrate
Pink Pepper × Cheongsam F1 generation obtained through open pollination