High-throughput methods to identify male Cannabis sativa using various genotyping methods

Area of Science:

• Plant genetics
• Molecular biology
• Agricultural science

Background:

• Cannabis sativa exhibits sexual developmental plasticity, with male-associated DNA regions (MADC) linked to staminate flower development.
• MADC2 is a specific PCR-based genetic marker correlating with male phenotypes in Cannabis.

Purpose of the Study:

• To develop and demonstrate cost-effective, high-throughput male genotyping assays for Cannabis sativa.
• To validate the accuracy and commercial viability of novel sex determination methodologies.

Main Methods:

• Real-time quantitative polymerase chain reaction (qPCR) assay development for MADC2 detection.
• Assay development for high-resolution melting analysis (HRM), loop-mediated isothermal amplification (LAMP), and recombinase amplification (RPA).
• Analysis of 8200 leaf samples for MADC2 detection using qPCR.

Main Results:

• A robust high-throughput duplex TaqMan qPCR assay for male-specific genomic signatures was demonstrated.
• The qPCR assay showed >98.5% accuracy in detecting MADC2, with 3156 males and 5047 non-males identified.
• Proof of concept for alternative assays using MADC2 and other commercially viable sex determination methods was established.

Conclusions:

• High-throughput genotyping assays enable efficient screening and selection of desired Cannabis sex for industrial (fiber, grain) and medicinal (cannabinoids) applications.
• The developed methodologies provide scalable and accurate sex determination for the commercial Cannabis industry.