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Open Access Highly Accessed Research article

A Toolkit for bulk PCR-based marker design from next-generation sequence data: application for development of a framework linkage map in bulb onion (Allium cepa L.)

Samantha Baldwin1, Roopashree Revanna1, Susan Thomson1, Meeghan Pither-Joyce1, Kathryn Wright1, Ross Crowhurst1, Mark Fiers1, Leshi Chen2, Richard Macknight13 and John A McCallum1*

Author affiliations

1 The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch, New Zealand

2 Department of Applied Computing, Faculty of Environment, Society and Design, Lincoln University, PO Box 84, Lincoln, 7647, New Zealand

3 Biochemistry Department, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealand

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Citation and License

BMC Genomics 2012, 13:637  doi:10.1186/1471-2164-13-637

Published: 19 November 2012

Abstract

Background

Although modern sequencing technologies permit the ready detection of numerous DNA sequence variants in any organisms, converting such information to PCR-based genetic markers is hampered by a lack of simple, scalable tools. Onion is an example of an under-researched crop with a complex, heterozygous genome where genome-based research has previously been hindered by limited sequence resources and genetic markers.

Results

We report the development of generic tools for large-scale web-based PCR-based marker design in the Galaxy bioinformatics framework, and their application for development of next-generation genetics resources in a wide cross of bulb onion (Allium cepa L.). Transcriptome sequence resources were developed for the homozygous doubled-haploid bulb onion line ‘CUDH2150’ and the genetically distant Indian landrace ‘Nasik Red’, using 454™ sequencing of normalised cDNA libraries of leaf and shoot. Read mapping of ‘Nasik Red’ reads onto ‘CUDH2150’ assemblies revealed 16836 indel and SNP polymorphisms that were mined for portable PCR-based marker development. Tools for detection of restriction polymorphisms and primer set design were developed in BioPython and adapted for use in the Galaxy workflow environment, enabling large-scale and targeted assay design. Using PCR-based markers designed with these tools, a framework genetic linkage map of over 800cM spanning all chromosomes was developed in a subset of 93 F2 progeny from a very large F2 family developed from the ‘Nasik Red’ x ‘CUDH2150’ inter-cross. The utility of tools and genetic resources developed was tested by designing markers to transcription factor-like polymorphic sequences. Bin mapping these markers using a subset of 10 progeny confirmed the ability to place markers within 10 cM bins, enabling increased efficiency in marker assignment and targeted map refinement. The major genetic loci conditioning red bulb colour (R) and fructan content (Frc) were located on this map by QTL analysis.

Conclusions

The generic tools developed for the Galaxy environment enable rapid development of sets of PCR assays targeting sequence variants identified from Illumina and 454 sequence data. They enable non-specialist users to validate and exploit large volumes of next-generation sequence data using basic equipment.

Keywords:
Marker; Onion; Genetic mapping; Next generation sequencing; SNP