Rapid Genome‐wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer

Abstract

Massively parallel sequencing platforms have allowed for the rapid discovery of single nucleotide polymorphisms (SNPs) among related genotypes within a species. We describe the creation of reduced representation libraries (RRLs) using an initial digestion of nuclear genomic DNA with a methylation-sensitive restriction endonuclease followed by a secondary digestion with the 4bp-restriction endonuclease DpnII. This strategy allows for the enrichment of hypomethylated genomic DNA, which has been shown to be rich in genic sequences, and the digestion with DpnII serves to increase the number of common loci resequenced between individuals. Deep resequencing of these RRLs performed with the Illumina Genome Analyzer led to the identifi cation of 2618 SNPs in rice and 1682 SNPs in soybean for two representative genotypes in each of the species. A subset of these SNPs was validated via Sanger sequencing, exhibiting validation rates of 96.4 and 97.0%, in rice (Oryza sativa) and soybean (Glycine max), respectively. Comparative analysis of the read distribution relative to annotated genes in the reference genome assemblies indicated that the RRL strategy was primarily sampling within genic regions for both species. The massively parallel sequencing of methylation-sensitive RRLs for genome-wide SNP discovery can be applied across a wide range of plant species having suffi cient reference genomic sequence.

Large‐scale transcriptome analysis in chickpea (Cicer arietinum L.), an orphan legume crop of the semi‐arid tropics of Asia and Africa

“…These technologies have been effectively used to generate large-scale transcriptome data in several plant species such as Arabidopsis (Weber et al , 2007), Medicago (Cheung et al , 2006), maize (Emrich et al , 2007), barley (Steuernagel et al , 2009) and soybean (Deschamps et al , 2010). With an objective to develop transcriptomic and genomic resources in chickpea, this study employed two NGS technologies: Roche/454 and Illumina/Solexa.…”

Transcriptome Analysis in Sheepgrass (Leymus chinensis): A Dominant Perennial Grass of the Eurasian Steppe

“…However, whole-genome sequencing is currently expensive and impractical for sheepgrass, which has a very large genome (9.65 Gb for a haploid genome). NGS technologies have been effectively used to generate large-scale transcriptome data in several plant species, such as Medicago [23], Arabidopsis [24], maize [25], barley [26], soybean [27], chickpea [28], [29], and developing oilseeds [30]. Recently, a transcriptome data was generated in sheepgrass ( Leymus chinensis ) under saline-alkaline treatment using Roche-454 massive parallel pyrosequencing technology and a large number of saline-alkaline responsive differentially expressed genes (DEGs) were obtained [31].…”

Genetic diversity analysis of yellow mustard (Sinapis alba L.) germplasm based on genotyping by sequencing

“…RRLs have been widely applied to reduce the complexity of plant genomes (cf. Gore et al 2009;Maughan et al 2009;Deschamps et al 2010;Hyten et al 2010). They are constructed by means of a restriction enzyme (RE) digest followed by fragmentsize selection, and allow for sampling diverse, but identical, genomic regions from several individuals (Altshuler et al 2000).…”