Abstract
Mutagenesis followed by phenotypic screenings is a widely used approach for characterizing genes of Arabidopsis. However, identification of the genetic basis of mutant phenotypes by conventional map-based cloning relying polymorphism markers is laborious and slow. To overcome this problem, we introduce next-generation sequencing machines for rapid, accurate and low-cost positional cloning. Next-generation DNA sequencers, which produce hundred millions of short reads (50-100 bases in length), are well suited for genome re-sequencing and SNP (single nucleotide polymorphism) detection. Taking advantage of this feature, we set up some mutant screening experiments using EMS, which introduce point mutations through the genome, and then attempted to identify causative mutations by a combination of deep-sequencing and rough mapping. We successfully identified causative mutations in some experiments much more rapidly at lower cost compared to conventional approaches, while we failed in some cases. Based on our experiences, we here propose best practices for mutant screening of Arabidopsis using next-generation DNA sequencer.
