Mutation Detection In Brassicaceae Species Using Illumina Sequencing

The utility of high throughput sequencing platforms for the identification of new variation in mutagenized populations of plants has received significant attention in recent years.  Approaches can utilize multi-dimensional pooling of extracted genomic DNA from individuals derived from mutant populations to screen for variant nucleotides.  Overlapping amplicons generated to cover target genes of interest can be pooled and barcoded to allow for sufficient resolution of potential mutant lines.  Sequencing reads are partitioned according to the barcodes, each read is subsequently aligned to a reference gene sequence and mutation frequencies at each base calculated from reads with high quality scores.  Attempts to carry out such screening in polyploid and mesopolyploid genomes of species within the Brassicaceae family are problematic due to the complexities of resolving the lower frequency of induced point mutations at single loci from variation that already exists between other very closely related loci.  Efforts to establish robust approaches for mutation detection in different Brassicaceae crop species will be described.