The commercialĭepartment of BiologicalSciences, University of Alberta, Edmonton, AB, Canada, T6G 2E9įulllist of author information is available at the end of the article bast fibers) of flax are remarkably long and strong and are used for textiles and increasingly as substitutes for fiberglass in composite materials. linseed) is rich in polyunsaturated fatty acids including alpha-linolenic acid, which has purported health benefits and is also useful as a drying oil in manufacture of resins, finishes, and flooring.
Keywords: Linum usitatissimum, Reverse genetics, Ion Torrent, EMSįlax (Linum usitatissimum L.) is cultivated as a source of either oil or fiber, both of which have distinct properties that make flax a valuable crop. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.
#GENE CONSTRUCTION KIT TORRENT TORRENT#
Conclusions: The methodology presented here demonstrates the utility of Ion Torrent technology in detecting mutation variants in specific genome regions for large populations of a species such as flax. Out of 29 potential mutations identified after processing the NGS reads, 16 mutations were confirmed using Sanger sequencing. We then selected eight genes for which we wanted to discover novel mutations, and applied our approach to screen 768 individuals from the EMS population, using either the Ion 314 or Ion 316 chips. A pilot experiment with known SNPs showed that they could be detected at a frequency > 0.3% within the pools. Barcodes were incorporated during PCR, and the pooled amplicons were sequenced using an Ion Torrent PGM. Results: We used an amplicon-based approach in which DNA samples from an ethyl methanesulfonate (EMS)-mutagenized population were pooled and used as template in PCR reactions to amplify a region of each gene of interest. We developed this method in flax (Linum usitatissimum), to demonstrate its utility in a crop species. As an alternative to these genome or exome-scale methods, we sought to develop a scalable and efficient method for detection of induced mutations that could be applied to a small number of target genes, using Ion Torrent technology. exome capture, whole genome resequencing). Many reverse genetics approaches have been developed to identify mutations in genes of interest within a mutagenized population, including some approaches that rely on next-generation sequencing (e.g. Leonardo Galindo-Gonzalez1*1, David Pinzôn-Latorreu, Erik A Bergen1, Dustin C Jensen2 and Michael K Deyholos3īackground: Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement. Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome
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