Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis

Genetic screens are a powerful approach for studying diverse processes by isolating mutants showing phenotypes directly or indirectly involved in biological pathways. Identifying the molecular lesion underlying these phenotypes is crucial towards understanding the mechanism of the process it is involved in. In order to reveal the molecular identity of the mutant, positional cloning is commonly employed to identify the mutations [1]. However, despite the availability of the Arabidopsis genome sequence, positional cloning from diverse mutant screens can be time-consuming or even hampered due to low chromosomal recombination in megabase-sized regions surrounding the mutation [1-4].Next-generation sequencing (NGS) technology for whole-genome sequencing (WGS) provides an alternative method for molecular characterization of mutations [5]. However, the copious numbers of mutations generated during the mutagenesis processes become a hindrance due to the presence of hundreds or thousands of mutations unrelated to the specific phenotype. This introduces a high degree of complexity in subsequent WGS data analysis aimed at identifying mutations responsible for the phenotypes. Specialized computational methods, hardware, and expertise, not available in most laboratories, are typically needed to accomplish the analysis. Backcrosses mutants to wild type plants for several generations can attenuate complexity by eliminating unrelated mutations [6], but this is very time consuming when using Arabidopsis. Improved approaches, SHOREmap and Next-Gen Mapping (NGM), combine integrated mapping with NGS and have led to identification of EMS (ethyl methanesulfonate)-generated mutation sites in Arabidopsis [7-9]. However, these strategies require whole genome sequencing, and so huge amounts of uninformative non-target regions are sequenced which is very costly and can be impractical for many laboratories involved in genetic studies. For example, based on published reports, characterizing one