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<p><i>Cannabis sativa</i> L. produces cannabinoids such as delta-9 tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), synthesized by THCA synthase (THCAS) and CBDA synthase (CBDAS), respectively. Understanding the genetic basis of these traits is essential for breeding industrial hemp with low THC content (hereafter, THC and CBD content refer to the sum of their acidic and decarboxylated forms). However, characterization of each hemp cultivar remains limited due to inaccessibility. We analyzed four cannabis cultivars—Spectrum 303, Superwoman S1, Biscotti Pippen, and Pillsbury Droboy—classified as chemo-type I or III. Cannabinoid accumulation (based on dry weight) was quantified across flower development stages using high-performance liquid chromatography, while <i>THCAS-like</i> and <i>CBDAS</i> transcript levels were quantified using quantitative PCR. Gene sequencing and amino acid analysis were performed to assess functionality. Chemo-type I cultivars (Biscotti Pippen, Pillsbury Droboy) exhibited high THC (approximately 160 mg g<sup>−1</sup>) and negligible CBD (&lt; 1.3&#xa0;mg&#xa0;g<sup>−1</sup>). Genomic analysis revealed truncating mutations in the <i>CBDAS</i> gene, leading to non-functional proteins. In contrast, chemo-type III cultivars (Spectrum 303, Superwoman S1) expressed putatively functional <i>CBDAS</i> and accumulated high levels of CBD (approximately 55 and 177&#xa0;mg&#xa0;g<sup>−1</sup>, respectively), while <i>THCAS-like</i> transcripts were detected at relatively low levels. Sequence analysis showed that the <i>THCAS-like</i> gene in chemo-type III cultivars was identical to known <i>cannabichromenic acid synthase</i> (<i>CBCAS</i>) sequences, which is consistent with the low THC content despite gene transcription. These results suggest that functional mutations in <i>CBDAS</i> underlie negligible CBD content in chemo-type I cultivars, whereas the presence of <i>CBCAS</i> instead of <i>THCAS</i> may explain the minimal THC levels in chemo-type III cultivars. Our findings clarify the genetic basis of cannabinoid variation and provide molecular markers for distinguishing chemo-types. This knowledge supports marker-assisted selection for developing THC-free industrial hemp.</p>

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Integrative analysis of cannabinoid profiles, gene expression, and functional variants of THCAS and CBDAS distinguishes chemo-type I and III Cannabis sativa cultivars

  • 期刊论文

  • 发表日期：

    2026年6月15日

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  • Juyoung Kim,
  • Tae Hyun Ha,
  • Jaihyuk Ryu,
  • Woon Ji Kim,
  • Sang Hoon Kim

摘要

Cannabis sativa L. produces cannabinoids such as delta-9 tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), synthesized by THCA synthase (THCAS) and CBDA synthase (CBDAS), respectively. Understanding the genetic basis of these traits is essential for breeding industrial hemp with low THC content (hereafter, THC and CBD content refer to the sum of their acidic and decarboxylated forms). However, characterization of each hemp cultivar remains limited due to inaccessibility. We analyzed four cannabis cultivars—Spectrum 303, Superwoman S1, Biscotti Pippen, and Pillsbury Droboy—classified as chemo-type I or III. Cannabinoid accumulation (based on dry weight) was quantified across flower development stages using high-performance liquid chromatography, while THCAS-like and CBDAS transcript levels were quantified using quantitative PCR. Gene sequencing and amino acid analysis were performed to assess functionality. Chemo-type I cultivars (Biscotti Pippen, Pillsbury Droboy) exhibited high THC (approximately 160 mg g−1) and negligible CBD (< 1.3 mg g−1). Genomic analysis revealed truncating mutations in the CBDAS gene, leading to non-functional proteins. In contrast, chemo-type III cultivars (Spectrum 303, Superwoman S1) expressed putatively functional CBDAS and accumulated high levels of CBD (approximately 55 and 177 mg g−1, respectively), while THCAS-like transcripts were detected at relatively low levels. Sequence analysis showed that the THCAS-like gene in chemo-type III cultivars was identical to known cannabichromenic acid synthase (CBCAS) sequences, which is consistent with the low THC content despite gene transcription. These results suggest that functional mutations in CBDAS underlie negligible CBD content in chemo-type I cultivars, whereas the presence of CBCAS instead of THCAS may explain the minimal THC levels in chemo-type III cultivars. Our findings clarify the genetic basis of cannabinoid variation and provide molecular markers for distinguishing chemo-types. This knowledge supports marker-assisted selection for developing THC-free industrial hemp.