A survey of genes involved in Arachis stenosperma resistance to Meloidogyne arenaria race 1

Abstract

Abstract. Root-knot nematodes constitute a constraint for important crops, including peanut (Arachis hypogaea L.). Resistance to Meloidogyne arenaria has been identified in the peanut wild relative Arachis stenosperma Krapov. & W. C. Greg., in which the induction of feeding sites by the nematode was inhibited by an early hypersensitive response (HR). Here, the transcription expression profiles of 19 genes selected from Arachis species were analysed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), during the early phases of an A. stenosperma-M. arenaria interaction. Sixteen genes were significantly differentially expressed in infected and non-infected roots, in at least one of the time points analysed: 3, 6, and 9 days after inoculation. These genes are involved in the HR and production of secondary metabolites related to pathogen defence. Seven genes encoding a resistance protein MG13, a helix-loop helix protein, an ubiquitin protein ligase, a patatin-like protein, a catalase, a DUF538 protein, and a resveratrol synthase, were differentially expressed in all time points analysed. Transcripts of two genes had their spatial and temporal distributions analysed by in situ hybridisation that validated qRT-PCR data. The identification of candidate resistance genes involved in wild peanut resistance to Meloidogyne can provide additional resources for peanut breeding and transgenic approaches.

Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

“…Following qPCR reactions, raw fluorescence data was directly used by the Miner statistical algorithm [ 25 ] for Cq and primer efficiency calculations ( Table 1 ). This tool has proven to be accurate, simple and user-friendly as it does not require the establishment of a standard curve, as previously observed [ 24 ]. All tested primer pairs showed very high efficiency, ranging from 0.95 to 1.10 ( Table 1 ) and were used to adjust Cq values in subsequent qPCR analysis.…”

“…qPCR reactions were carried out in 96-well plates on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described [ 24 ]. Reactions were conducted in three technical replicates for each sample.…”

“…PCR amplification efficiency and optimal cycle of quantification (Cq) values of all samples were calculated from raw fluorescence data using the online real-time PCR Miner tool [ 25 ], as previously described [ 24 ]. These data were used to assess reference genes stability by three statistical programs: geNorm PLUS [ 16 ], NormFinder version 0.953 [ 17 ] and BestKeeper version 1 [ 18 ].…”

“…To validate the use of the reference genes in all experimental conditions studied, the expression profile of two target genes ( NECRO and HR-Li ) was normalized using the two most stable candidate reference genes and the two least stable, as determined by the combined ranking order outputs from geNorm, NormFinder and BestKeeper algorithms. The relative expression of NECRO and HR-Li target genes after virus infection in all pathosystems here studied was estimated and statistically tested using REST 2009 software version 2.0.13 [ 26 ], as previously described [ 24 ].…”

Legume biology: the basis for crop improvement

“…The research article by Morgante et al (2013) focuses on the identification of candidate genes involved in peanut resistance to root-knot nematodes. Quantitative real time PCR (qRT-PCR) approach has been used in this study to look into the transcription expression profiles of selected candidate Arachis species genes.…”