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Todd Michael

Long-read nanopore cDNA sequencing and direct DNA methylation detection resolves copy number debate in Cannabis

Marijuana and THC are generally defined by their levels of THC – marijuana is high in THC, hemp is low in THC. Both plants can produce high levels of CBD.

Sequencing the Cannabis sativa genome revealed THC acid and CBD acid synthase genes are nested in tandem repeat clusters.

Nanopore sequencing plant lines with high and low levels of THC and CBD + genetic map data enabled anchoring into chromosomes.

This revealed a major chromosomal rearrangement affecting the synthase clusters in the high-THC line.

2 paralog types in plants: TD and WGD. They were ‘able to differentiate the expression of these closely-related paralogs using nanopore full-length cDNA sequencing’ where this was not visible using short reads.

Only one synthase copy expressed in lines high in THC or CBD; both expressed in lines high in both.

Evaluated cytosine methylation in all 3 contexts using data originally generated for genome assembly: ‘DNA methylation is a very powerful tool, even in this very repetitive genome.’

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