High-throughput genotyping by whole-genome resequencing

The first use of DNA-based markers decades ago laid the groundwork for gene discovery through forward and reverse genetics. The types of markers and methods for constructing genetic maps have evolved rapidly with advances in molecular biology techniques. The development of PCR triggered the burst of a generation of markers that considerably simplified experimental procedures for marker designing and scoring. However, these markers, although still widely used, have shown growing limitations in chromosomal coverage, time, and cost effectiveness. The development of genomics concepts and tools has set the stage for replacing the marker-based mapping approach with genome-based high-throughput strategies. The availability of genome sequences opened the door to high-throughput genotyping. This was initially accomplished by adopting microarray technology, which detects single nucleotide polymorphisms (SNPs) through hybridizing genomic DNA to oligonucleotides spotted on gene chips. This genotyping method substantially improved the efficiency of marker collection by allowing the detection of hundreds to thousands of markers in a single hybridization (Winzeler et al. 1998). It has been applied to model systems such as human, Arabidopsis, and rice (Meaburn et al. 2006; Singer et al. 2006; Jeremy et al. 2008). Although the goal of high-throughput was achieved, serious limitations remain for the array-based method. It is laborious, time-consuming, and expensive to design, produce, and process microarrays suited for specific mapping populations. The advent of the next-generation sequencing technology holds the promise for a methodological leap forward in genotyping and genetic mapping. The new sequencing techniques not only increase sequencing throughput by several orders of magnitude but also allow simultaneously sequencing a large number of samples using a multiplexed sequencing strategy (Craig et al. 2008; Cronn et al. 2008). These recent technical advances have paved the way for the development of a sequencing-based high-throughput genotyping method that combines advantages of time and cost effectiveness, dense marker coverage, high mapping accuracy and resolution, and more comparable genome and genetic maps among mapping populations and organisms. Here we describe the first high-throughput genotyping method that uses SNPs detected by whole-genome resequencing. This type of SNP data differs from traditional genetic markers primarily in two aspects. First, it is often not the case that all members of a recombinant population can be scored at a given SNP site. Second, an individual SNP site is no longer a reliable marker or locus for genotyping due to several potential sources of sequence errors. To deal with these unique features of the SNP data generated by the next-generation sequencing, we developed a new analytical framework, that is, a sliding window approach for evaluating SNPs collectively rather than individually. The method was applied to analyzing 150 rice recombinant inbred lines (RILs) derived from a cross between indica and japonica rice cultivars using sequences generated on the Illumina Genome Analyzer (GA).