DHPLC Efficiency for Semi-automated CDNA-AFLP Analyses and Fragment Collection in the Apple Scab-resistance Gene Model

Overview

Journal Planta

Specialty Biology

Date 2012 Jan 25

PMID 22270558

Citations 3

Authors

Roberta Paris

Luca Dondini

Graziano Zannini

Daniela Bastia

Elena Marasco

Valentina Gualdi

Valeria Rizzi

Pietro Piffanelli

Vilma Mantovani

Stefano Tartarini

Affiliations

Soon will be listed here.

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.

Citing Articles

Phenotyping, genetics, and "-omics" approaches to unravel and introgress enhanced resistance against apple scab () in apple cultivars ( × ).

Svara A, De Storme N, Carpentier S, Keulemans W, De Coninck B Hortic Res. 2024; 11(2):uhae002.

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Successful intergeneric transfer of a major apple scab resistance gene (Rvi6) from apple to pear and precise comparison of the downstream molecular mechanisms of this resistance in both species.

Perchepied L, Chevreau E, Ravon E, Gaillard S, Pelletier S, Bahut M BMC Genomics. 2021; 22(1):843.

PMID: 34802418 PMC: 8607633. DOI: 10.1186/s12864-021-08157-1.

Gene expression profiling by cDNA-AFLP reveals potential candidate genes for partial resistance of 'Président Roulin' against Venturia inaequalis.

Bastiaanse H, Muhovski Y, Parisi O, Paris R, Mingeot D, Lateur M BMC Genomics. 2014; 15:1043.

PMID: 25433532 PMC: 4302150. DOI: 10.1186/1471-2164-15-1043.

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