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dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model

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Abstract

cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant–microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE® System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus–Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant–pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants.

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Abbreviations

ACN:

Acetonitrile

cDNA-AFLP:

Amplified fragment length polymorphism on cDNA

dHPLC:

Denaturing high pressure liquid chromatography

hpi:

Hours post inoculation

HR:

Hypersensitive reaction

PCD:

Plant cell death

qRT-PCR:

Real time quantitative reverse transcription PCR

R gene:

Resistance gene

ROS:

Reactive oxygen species

TEAA:

Triethylammonium acetate

TDF:

Transcript-derived fragment

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Acknowledgments

The authors thank the Centre for Applied Biomedical Research (CRBA) of Bologna for valuable contributions to the dHPLC analyses. The authors also thank Fondazione Cassa di Risparmio in Bologna for supporting CRBA. The authors are also grateful to Dr. Stavros Papadimitriou from Transgenomic LTD. for technical advice. This work was supported by the MIUR FIRB project “http://DNA”.

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Authors and Affiliations

  1. Department of Fruit Tree and Woody Plant Science, University of Bologna, viale Fanin 46, 40127, Bologna, Italy

    Roberta Paris, Luca Dondini, Graziano Zannini & Stefano Tartarini

  2. Centre for Applied Biomedical Research (CRBA), S. Orsola-Malpighi Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy

    Daniela Bastia, Elena Marasco & Vilma Mantovani

  3. Genomics Platform, Parco Tecnologico Padano, Via Einstein, 26900, Lodi, Italy

    Valentina Gualdi, Valeria Rizzi & Pietro Piffanelli

Authors
  1. Roberta Paris
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  2. Luca Dondini
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  3. Graziano Zannini
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  4. Daniela Bastia
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  5. Elena Marasco
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  6. Valentina Gualdi
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  7. Valeria Rizzi
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  8. Pietro Piffanelli
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  9. Vilma Mantovani
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  10. Stefano Tartarini
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Corresponding author

Correspondence to Roberta Paris.

Additional information

The authors R. Paris and L. Dondini contributed equally to the work.

Electronic supplementary material

ESM1, Online Resource 1 List of the TDF sequences collected after dHPLC separation of cDNA-AFLPs.

ESM2, Online Resource 2 List of 51 TDF with their corresponding predicted function based on FASTA similarities.

ESM3 Online Resource 3 List of 32 selected TDFs for qRT-PCR analyses, primer sequences and amplification efficiencies calculated with respect to the actin reference gene.

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Supplementary material 1 (PDF 81 kb)

Supplementary material 2 (PDF 88 kb)

Supplementary material 3 (PDF 91 kb)

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Paris, R., Dondini, L., Zannini, G. et al. dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model. Planta 235, 1065–1080 (2012). https://doi.org/10.1007/s00425-012-1589-y

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