One advantage of reverse genetics is that it overcomes some of the limitations of forward genetics that are caused by functional redundancy. In reverse genetics, researchers start with a known DNA sequence of interest, and try to determine the effects of a mutation on the phenotype of the organism.
Once a phenotypically abnormal individual is identified, map-based cloning or other molecular analyses must be used to identify the DNA sequence that was altered by mutation. Forward genetics aims to evaluate the phenotype of hundreds or thousands of individuals to find abnormalities in characteristics like growth or development. Two main approaches have been developed to relate genotype to phenotype in mutated populations. Therefore the frequency of Single Nucleotide Variants (SNVs) for sequence length becomes an important factor in the probability of finding a phenotypic effect. While one study showed that the frequency of EMS-induced mutations was estimated at about 1 mutation/300 kb screened, the density of mutations can vary for different plants and treatments. Ethyl methanesulfonate (EMS) is widely used for inducing point mutations in plants, and results mostly in G/C to A/T transitions that show a nearly random distribution throughout the genome. Mutations can be induced by treating individuals with physical, biological or chemical mutagens. To accelerate the development of novel germplasm and to better exploit the available DNA sequence resources for flax, we sought to develop a mutant population and a reverse genetics platform for this crop. The commercial potential of flax, as well as interesting aspects of its biology (including well-documented phenotypic and genomic plasticity of some accessions ), have led to an increase in research activity in this species, highlighted by the release of an assembly of its whole genome sequence. 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. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.įlax ( 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.
Clc genomics workbench number of reads too low torrent#
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. 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. Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement.
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