2b-RAD: a simple and flexible method for genome-wide genotyping

Genotyping based on restriction site7ndash;associated (RAD) sequencing around type IIB enzyme recognition sites is reported. The streamlined reduced-representation approach features even and tunable genome coverage and enables large-scale genotyping studies by maximizing the amount of genotypic information that can be obtained from individuals for a given amount of sequencing. We describe 2b-RAD, a streamlined restriction site–associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the method's accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations.