ICR 2020 Poster - Segra International скачать в хорошем качестве

ICR 2020 Poster - Segra International

Nanopore NGS Panel for Examination of Key Genes on Cannabinoid and Terpene Synthetic Pathways Cannabis varieties are commonly bred or selected for expressed cannabinoid and / or terpenoid profiles. Allelic variation of key genes in these pathways would be expected to have phenotypic impact. Thus, an understanding of significant allelic variation in these enzymes would potentially be useful in underpinning marker assisted selection strategies for breeding novel cannabis varieties with particular cannabinoid and / or terpenoid characteristics. To investigate potential of this approach, we have applied Oxford Nanopore NGS technology adapted to a barcoded multi-sample, multiple gene target panel. Our pilot method, currently covering 10 genes from the cannabinoid synthetic pathway and 3 terpene synthase genes, has been applied to nearly 50 samples to date. Target genes from each sample are independently amplified by optimized PCR reactions, followed by pooling, barcoding, library preparation, and NGS. Bioinformatic demultiplexing allows for separation of target gene sequences from each sample to be individually analyzed at well supported depths of coverage. Comparison of these genomic sequences against extant cDNA sequences allows for determination of predicted amino acid sequences for each target. Unlike common short read NGS technologies, where allelic phasing can be unclear, the use of nanopore long (single) read methods may allow for easier resolution of individual parentally derived allelic sequences. Derived amino acid sequence(s) for each examined gene target were aligned and amino acid substitutions were examined in comparison to paired chemotypic data where available (the majority of samples tested). Specific dramatic differences in chemotypic profile were observed to clearly associate with particular amino acid substitutions in some target genes. Where possible, we have mapped these substitutions back onto available protein crystallographic structures to attempt to assess whether these observed changes are merely linked or are more likely directly mechanistically relevant. In addition to describing our methodology and presenting highlights of our pilot data with possible mechanistic relevance to chemotype, we present our bioinformatics workflows and data on direct and indirect costs of implementing this approach using the Oxford Nanopore platform. Overall, we find the method represents a relatively low cost, high yield approach to uncovering markers of utility in directed cannabis breeding programs. Segra International - http://www.segra-intl.com/