WHOLE GENOME SEQUENCING OF SEVERAL CANNABIS DERIVED POWDERY
WHOLE GENOME SEQUENCING OF SEVERAL CANNABIS DERIVED POWDERY MILDEW SAMPLES AND THE DEVELOPMENT OF FIELD PORTABLE COLORIMETRIC DETECTION TOOLS FOR INFECTING FUNGI Kevin Mc. Kernan 1, Yvonne Helbert 1, Lei Zhang 1, Wendell Orphe 1, Rino Ferrarese , Douglas Smith 1 Affiliations: 1 Medicinal Genomics Corporation, Woburn, MA; 2 CT Pharma Solutions, Portland , CT, Introduction Powdery Mildew Genome Sequenced Powdery Mildew is a frequent and destructive fungi infecting Cannabis crops. It is believed to be vascularized in plant tissue and may remain visually undetected during cloning or other vegetative stages of growth 1. It usually expresses conidia late into flowering causing more severe economic loss. Mc. Partland, Clarke and Watson 2 have suggested P. macularis and L. taurica may be responsible for cannabis derived powdery mildew. Despite this, no molecular evidence for the speciation of this fungi has been published to date. In an effort to design a simple field portable test to detect the vascularized dormant presence of powdery mildew prior to conidia propagation, we whole genome shotgun sequenced several powdery mildew samples from multiple geographic locations in the Northeastern United States. Assembled contigs were shown to have ITS similarity to Golovinomyces ambrosiae and orontii. However, significant divergence across large 100 kilobase contigs suggest a novel and diverged species infecting Cannabis. Using these sequence references we designed an assay compatible with an eight well USB portable amplification system designed by mini. PCR. This $650 amplification system is combined with a novel colorimetric DNA amplification assay that affords i. Phone based detection of the presence of powdery mildew DNA. Hemp Russet Mite Genome Sequenced We Sequenced the genome of the hemp russet mite to develop unique you. PCR primer assays for detecting Russet mites from a hole punch from a clone. Figure 4. Powdery Mildew vascularizes into the plant as described by We. Bling et al. The mycelium network may remain invisible for up to 41 days (7). We collected spores from 4 different regions in New England 3 different regions of California and sequenced their genomes. We used these sequences to design a you. PCR assay sensitive to all strains. This assay can assist in screening clones or incoming material to a grow for eggs, nymph or adult mites. Figure 7. Top Left: Russet mites on cannabis. Top Middle: Russet mites collected. Top Right: you. PCR assay for Russet mites. Bottom: High coverage and high quality assembly of the Russet mite genome. Blue hashes are paired 250 bp reads. Red and green hashes are unpaired watson and crick strand reads. Yellow reads are partially mapping reads at the end of contigs. Pink histogram is the coverage depth (10, 000 X) Type I, III, IV genotyping Figure 5. PM sampling from multiple geographic areas. Detection Before Sporulation Symptomatic (visible PM or “Infected”) and non-Symptomatic clones were collected from Resistant and Susceptible strains and tested with multiple samples of the plant. All Infected plants were detected with a single hole punch. Many of their clones exposed to the same room and environment were detected with 2 -3 4 mm Hole Punches. Many resistant lines remained negative (Pink). Figure 1. Portable USB mini. PCR with colorimetric you. PCR. Device runs on a USB laptop or Galaxy phone. Polymorphisms in THCA and CBDA synthase were mapped with Pacific Biosciences circular consensus sequencing. Multiplexed You. PCR assays were designed to capture the knock-out variants contributing to the Bt: Bd chemotype system described by de Meijer et al. This was expanded on 25 samples from the Emerald Cup, 56 hemp samples from Colorado and 11 samples from Massachusetts. de Meijer et al Table 1: Nonsymptomatic susceptible lines tested positive but require more than one 4 mm Hole punch to reliably detect the mycelium network. Hyper sampling these samples demonstrated variable mycelium density. Figure 8: Type II and Type III plant DNA was collected from across the country to validate the markers Table 2: Type. II & III plants that test positive with the CBD Assay. Future Directions – Pipeline of Assays Figure 2. you. PCR workflow starts with a hole punch in a small leaf. A simple 8 minute boil in Solution A results in DNA ready for colorimetric amplification. MGC’s current you. PCR® assays and release timelines are targeting Regulated Human Pathogens, Plant Pathogens, and valuable Genetic markers for marker assisted selection. These assays target the Clone market, Seed market and Safety markets. While 3 rd party testing will always be mandated for neutrality and reduced conflict of interests, large grows will eventually internalize testing to minimize 3 rd party failure and to reduce plant pathogen exposure. Figure 6. Hypersampling Nonsymptomatic samples shows a 5/8 positive rate showing infection at low MOI. Figure 3. Colorimetric read out enables mobile phone detection Research and Development Contact Kevin Mc. Kernan Medicinal Genomics Corporation Email: Kevin. Mc. Kernan@medicinalgenomics. com Acknowledgements Joe Baillargeon, Khem Organics for Russet mite advise Conclusions: We have developed a fast and portable DNA based detection system for Powdery Mildew infections in Cannabis. This can be used to screen mothers or individual clones to ensure incoming clones are free of disease. Combined with other pests this can provide a valuable insurance program for uninsured Cannabis grows. Sales Contact References Blair Beach Medicinal Genomics Corporation Email: info@medicinalgenomics. com 1. Mc. Kernan, K. J. , Spangler, J. , Helbert, Y. , Zhang, L. & Tadigotla, V. DREAMing of a patent-free human genome for clinical sequencing. Nat Biotechnology 31, 884 -887 (2013). 2. Mc. Kernan, K. J. et al. Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment. PLo. S One 9, e 96492 (2014). 3. Mc. Kernan K, Spangler J, Helbert Y, Lynch RC, Devitt-Lee A, Zhang L, et al. Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests. F 1000 Research. 2016; 5: 2471. Pub. Med PMID: 27853518. Pubmed Central PMCID: 5089129 4. Mc. Kernan K, Spangler J, Zhang L, Tadigotla V, Helbert Y, Foss T, et al. Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers. F 1000 Research. 2015; 4: 1422. Pub. Med PMID: 27303623. Pubmed Central PMCID: 4897766. 5. Stewart, E. J. Growing Unculturable Bacteria. Journal of Bacteriology 194, 4151 -4160 (August 2012). 6. Kusari, P. , Kusari, S. , Spiteller, M. , & Kayser, O. Endophytic fungi harbored in Cannabis sativa L. : diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity 60, 137– 151 (2013). 7. de Meijer EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P, et al. The inheritance of chemical phenotype in Cannabis sativa L. Genetics. 2003 Jan; 163(1): 335 -46. Pub. Med PMID: 12586720. Pubmed Central PMCID: 1462421. 8. Wessling R, Panstruga R. Rapid quantification of plant-powdery mildew interactions by q. PCR and conidiospore counts. Plant methods. 2012 Aug 31; 8(1): 35. Pub. Med PMID: 22937820. Pubmed Central PMCID: 3522566. 9. Twomey MC, Wolfenbarger SN, Woods JL, Gent DH. Development of partial ontogenic resistance to powdery mildew in hop cones and its management implications. Plo. S one. 2015; 10(3): e 0120987. Pub. Med PMID: 25811173. Pubmed Central PMCID: 4374666. www. medicinalgenomics. com
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