Combining SNP discovery from next-generation sequencing data with bulked segregant analysis (BSA) to fine-map genes in polyploid wheat

Abstract

BackgroundNext generation sequencing (NGS) technologies are providing new ways to accelerate fine-mapping and gene isolation in many species. To date, the majority of these efforts have focused on diploid organisms with readily available whole genome sequence information. In this study, as a proof of concept, we tested the use of NGS for SNP discovery in tetraploid wheat lines differing for the previously cloned grain protein content (GPC) gene GPC-B1. Bulked segregant analysis (BSA) was used to define a subset of putative SNPs within the candidate gene region, which were then used to fine-map GPC-B1.ResultsWe used Illumina paired end technology to sequence mRNA (RNAseq) from near isogenic lines differing across a ~30-cM interval including the GPC-B1 locus. After discriminating for SNPs between the two homoeologous wheat genomes and additional quality filtering, we identified inter-varietal SNPs in wheat unigenes between the parental lines. The relative frequency of these SNPs was examined by RNAseq in two bulked samples made up of homozygous recombinant lines differing for their GPC phenotype. SNPs that were enriched at least 3-fold in the corresponding pool (6.5% of all SNPs) were further evaluated. Marker assays were designed for a subset of the enriched SNPs and mapped using DNA from individuals of each bulk. Thirty nine new SNP markers, corresponding to 67% of the validated SNPs, mapped across a 12.2-cM interval including GPC-B1. This translated to 1 SNP marker per 0.31 cM defining the GPC-B1 gene to within 13-18 genes in syntenic cereal genomes and to a 0.4 cM interval in wheat.ConclusionsThis study exemplifies the use of RNAseq for SNP discovery in polyploid species and supports the use of BSA as an effective way to target SNPs to specific genetic intervals to fine-map genes in unsequenced genomes.

A Kelch domain-containing F-box coding gene negatively regulates flavonoid accumulation in Cucumis melo L.

“…BSA has been widely used ever since, uncovering genetic markers and genes in a wide variety of plants. The rapid development of new sequencing technologies and computation systems allows a very efficient use of BSA while using next generation sequencing technologies to discover polymorphism that discriminate between the bulks (Liu et al, 2012;Trick et al, 2012). Combining BSA with RNA-Seq and detailed metabolite analyses enabled a relatively rapid and accurate isolation and characterization of CmKFB, the gene of interest in this work.…”

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

“…To directly survey SNP markers near the detected QTLs, association analysis was performed using bulk DNA from the F 2 populations [20,21] figure S1). Genotyping of 92 published SSR markers and 191 SNP markers in the BF 1 population connected our new linkage map to the published map successfully [25] (electronic supplementary material, table S4).…”

“…whether a gene-for-gene relationship is established between rice resistance and the corresponding BPH virulence [15]. Finally, to directly identify molecular markers linked to the putative virulence gene vBph1, bulked segregant analysis and next-generation sequencing (NGS) technologies were applied [20,21]. These systematic applications of genetics against BPH virulence to rice resistance contribute to understanding the nature of BPH 'biotypes' that has long been ambiguously discussed [7,12,15,16,17,22].…”

Six-Rowed Spike3 (VRS3) Is a Histone Demethylase That Controls Lateral Spikelet Development in Barley

“…This procedure is based on genome-wide resequencing of phenotypic bulks of mutant F2 individuals to fine-map the gene of interest. Mapping-by-sequencing based on whole-exome capture or RNA sequencing to reduce genome complexity has also been successfully applied for fine mapping in wheat and barley (Trick et al, 2012;Pankin et al, 2014;Liller et al, 2017). Although mapping by sequencing has proven successful to identify candidate genes, it still requires a substantial effort in generating and phenotyping large mapping populations.…”