High-throughput SNP discovery and assay development in common bean.
BACKGROUND
Next generation sequencing has significantly 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 (Phaseolus vulgaris L.) 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 Roche 454-FLX system (454) with the Illumina Genome Analyzer (GA) for high-throughput SNP discovery.


RESULTS
Using a multi-tier reduced representation library we discovered a total of 3,487 SNPs of which 2,795 contained sufficient flanking genomic sequence for 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 GoldenGate assay which contained 1,050 of the 3,487 predicted SNPs. A total of 827 of the 1,050 SNPs produced a working GoldenGate assay (79%).


CONCLUSIONS
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 run and one GA sequencer run allows high-throughput SNP discovery with sufficient sequence for assay development to be performed in organisms, such as common bean, which have limited genomic resources.
Hyten
David L
DL
Soybean Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, USA. david.hyten@ars.usda.gov
Song
Qijian
Q
Fickus
Edward W
EW
Quigley
Charles V
CV
Lim
Jong-Sung
JS
Choi
Ik-Young
IY
Hwang
Eun-Young
EY
Pastor-Corrales
Marcial
M
Cregan
Perry B
PB
D030561:Databases, Nucleic Acid; D018745:Genome, Plant; D027805:Phaseolus / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*