Sequencing-based variant detection in the polyploid crop oilseed rape

BMC Plant Biology

Methodology Article

Jérôme Pauquet¹  Martin Trick²  Rachel Wells²  Colin Morgan²  Eleni Soumpourou²  Ian Bancroft³  Leah Clissold⁴  Fiona Fraser⁴ 

[1] Chemin de Panedautes, Domaine de Sandreau, BIOGEMMA S.A.S., 31700, Mondonville, France;John Innes Centre, Norwich Research Park, NR4 7UH, Norwich, UK;John Innes Centre, Norwich Research Park, NR4 7UH, Norwich, UK;Department of Biology, University of York, Wentworth Way, Heslington, YO10 5DD, York, UK;John Innes Centre, Norwich Research Park, NR4 7UH, Norwich, UK;The Genome Analysis Centre, Norwich Research Park, NR4 7UH, Norwich, UK;

关键词: SNP;    Mutation;    Polyploid;    Crop;   

DOI  :  10.1186/1471-2229-13-111

 received in 2013-07-22, accepted in 2013-07-23,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsA 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.ConclusionsThe 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.

【 授权许可】

Unknown   
© Wells et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.