• Open Access

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Authors

  • Peter C. Bundock,

    Corresponding author
    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia
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  • Frances G. Eliott,

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia
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  • Gary Ablett,

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia
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  • Adam D. Benson,

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia
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  • Rosanne E. Casu,

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Qld 4067, Australia
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  • Karen S. Aitken,

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Rd, St Lucia, Qld 4067, Australia
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  • Robert J. Henry

    1. Co-operative Research Centre for Sugar Industry Innovation through Biotechnology, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia
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* Correspondence (fax +61 266222080; e-mail peter.bundock@scu.edu.au)

Summary

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 × Q165). The sequencing yielded 96 755 (IJ76-514) and 86 241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP – an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent – with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

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