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Open Access Highly Accessed Methodology article

Sequencing-based variant detection in the polyploid crop oilseed rape

Rachel Wells1, Martin Trick1, Fiona Fraser12, Eleni Soumpourou1, Leah Clissold12, Colin Morgan1, Jérôme Pauquet3 and Ian Bancroft14*

Author Affiliations

1 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

2 Present address: The Genome Analysis Centre, Norwich Research Park, Norwich NR4 7UH, UK

3 BIOGEMMA S.A.S., Chemin de Panedautes, Domaine de Sandreau, 31700, Mondonville, France

4 Present address: Department of Biology, University of York, Wentworth Way, Heslington, York YO10 5DD, UK

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BMC Plant Biology 2013, 13:111  doi:10.1186/1471-2229-13-111

Published: 6 August 2013

Abstract

Background

The detection and exploitation of genetic variation underpins crop improvement. However, the polyploid nature of the genomes of many of our most important crops represents a barrier, particularly for the analysis of variation within genes. To overcome this, we aimed to develop methodologies based on amplicon sequencing that involve the incorporation of barcoded amplification tags (BATs) into PCR products.

Results

A protocol was developed to tag PCR products with 5’ 6-base oligonucleotide barcode extensions before pooling for sequencing library production using standard Illumina adapters. A computational method was developed for the de-convolution of products and the robust detection and scoring of sequence variants. Using this methodology, amplicons targeted to gene sequences were screened across a B. napus mapping population and the resulting allele scoring strings for 24 markers linkage mapped to the expected regions of the genome. Furthermore, using one-dimensional 8-fold pooling, 4608 lines of a B. napus mutation population were screened for induced mutations in a locus-specific amplicon (an orthologue of GL2.b) and mixed product of three co-amplified loci (orthologues of FAD2), identifying 10 and 41 mutants respectively.

Conclusions

The utilisation of barcode tags to de-convolute pooled PCR products in multiplexed, variation screening via Illumina sequencing provides a cost effective method for SNP genotyping and mutation detection and, potentially, markers for causative changes, even in polyploid species. Combining this approach with existing Illumina multiplexing workflows allows the analysis of thousands of lines cheaply and efficiently in a single sequencing run with minimal library production costs.

Keywords:
SNP; Mutation; Polyploid; Crop