|Quigley, Charles - Chuck|
|Pastor Corrales, Marcial - Talo|
Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2010
Publication Date: 8/16/2010
Citation: Hyten, D.L., Song, Q., Fickus, E.W., Choi, I., Quigley, C.V., Hwang, E., Pastor Corrales, M.A., Cregan, P.B. 2010. High-throughput SNP discovery and assay development in Common Bean. Biomed Central (BMC) Genomics. 11:475. Interpretive Summary: Molecular markers such as single nucleotide polymorphisms (SNPs) have become extremely important in helping to improve crops through gene discovery and marker assisted selection. However, the number of SNPs available for the common bean genome needs to be increased to help map genes that contribute to agronomically important traits quickly and cost effectively. A new method of SNP discovery has been applied to common bean which uses new DNA sequencing techniques and this method was developed and used to find a total of 3487 SNPs. A total of 1,050 of these SNP markers were used to create a tool which can screen the 1050 markers on many different common bean cultivars. The large number of new markers discovered will be used by crop researchers, crop breeders and seed companies to increase the efficiency of molecular marker applications in common bean for gene discovery and genetic improvement.
Technical Abstract: Next generation sequencing has dramatically increased the speed at which single nucleotide polymorphisms (SNPs) can be discovered and subsequently used as molecular markers for research. Unfortunately, for species such as common bean which do not have a whole genome sequence available the use of next generation sequencing for SNP discovery is much more difficult and costly. To this end we developed a method which couples sequences obtained from the 454-FLX system with the Genome Analyzer for high-throughput SNP discovery. Using a multi-tier reduced representation library we discovered a total of 2795 SNPs which contained enough flanking sequence and were suitable for GoldenGate SNP assay development. Using Sanger sequencing to determine the validation rate of these SNPs we found that 86% are likely to be true SNPs. Furthermore, we designed a 1,536 GoldenGate assay which contained 1050 of the 3487 predicted SNPs. A total of 827 of the 1050 SNPs produced a working GoldenGate assay (79%). Through combining two next generation sequencing techniques we have developed a method that allows high-throughput SNP discovery in any diploid organism without the need of a whole genome sequence or the creation of normalized cDNA libraries. The need to only perform one 454-FLX run and one GA sequencer run allows high-throughput SNP discovery with enough sequence for assay development to be performed in any organisms especially suited to ones that have traditionally received less funding than the model organisms.