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Open Access Highly Accessed Research article

Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

Santosh Kumar12, Frank M You1 and Sylvie Cloutier12*

Author Affiliations

1 Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M9, Canada

2 Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, Manitoba, R3T 2N2, Canada

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BMC Genomics 2012, 13:684  doi:10.1186/1471-2164-13-684

Published: 6 December 2012

Abstract

Background

Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs.

Results

Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%.

Conclusions

Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence.

Keywords:
Single nucleotide polymorphism (SNP); Genotyping-by-sequencing (GBS); Reduced representation library (RRL); Illumina; Flax; Linum usitatissimum; AGSNP