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Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry

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Abstract

Mis-priming associated with uncharacterised single nucleotide polymorphisms (SNPs) may lead to failure of PCR for genotyping. This is particularly troublesome in high-throughput SNP genotyping applications relying on multiplex PCR (2–40-plex) generating many short amplicons (80–120 bp) of similar size, an approach best suited for whole genome scans. However, if the target SNPs are clustered within a few target genes one option to ameliorate this is to increase the amplicon length, effectively reducing the potential for primer/template interactions and mis-priming. We tested this approach in a diverse population of 372 Eucalyptus pilularis individuals (π = 8.11 × 10−3, H e = 0.75) using a modified Sequenom iPLEX gold assay. Four candidate genes (MYB1, MYB2, CAD and CCR) were amplified in a single long range multiplex capture PCR generating 6 long amplicons ranging in size from 907 to 2,225 bp. This contrasts with the standard approach which would have required the amplification of 98 short amplicons in 4 multiplex reactions. These 6 long amplicons provided the assay template for 98 assays (87 SNP and 11 InDel) within the 4 candidate genes. Reaction results indicated that longer amplicons could provide a suitable template for genotyping assays, with 90.8% of assays functional and 84.3% of assays suitable for downstream analysis. Additional advantages of this approach were the capacity for troubleshooting using gel electrophoresis and savings of 94% in capture primer synthesis costs. This approach will have the greatest relevance for candidate gene approaches for association testing in uncharacterised populations of organisms with high sequence diversity.

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Acknowledgments

This research was funded by the CRC for Forestry. The authors thank Forests NSW for providing leaf samples, as well as Chris Harwood, Peter Bundock, Julie Pattemore, Helen Hill, Dan Waters and Ardashir Kharabian Masouleh for many helpful comments and suggestions.

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Authors and Affiliations

  1. CRC for Forestry, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW, 2480, Australia

    Timothy R. Sexton, Robert J. Henry & Mervyn Shepherd

  2. CRC for Forestry, Forest Molecular Biology and Genetics Group, University of Melbourne, Creswick, VIC, 3363, Australia

    Luke J. McManus

  3. Southern Cross Plant Genomics, Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW, 2480, Australia

    Stirling Bowen

Authors
  1. Timothy R. Sexton
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  2. Robert J. Henry
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  3. Luke J. McManus
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  4. Stirling Bowen
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  5. Mervyn Shepherd
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Corresponding author

Correspondence to Timothy R. Sexton.

 

 

Appendix 1 Capture primers used to amplify regions of CAD2, CCR, MYB1 and MYB2 in multiplex
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Sexton, T.R., Henry, R.J., McManus, L.J. et al. Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry. Mol Breeding 25, 471–480 (2010). https://doi.org/10.1007/s11032-009-9345-0

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  • DOI: https://doi.org/10.1007/s11032-009-9345-0

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