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AcronymMELRIP
  
TitleUnderstanding the climacteric vs non-climacteric fruit ripening mechanisms in melon using transcriptomic, metabolomic and reverse genetic approaches
  
Duration1 April 2007 - 1 April 2010
  
Project leader Jordi Garcia-Mas (CSIC, Spain)
  

Project partners

Abdel Bendahmane, INRA - Plant Genomics Research Unit URGV, France [Company]
Danièle Hosemans, Vilmorin Clause & Cie, France
Michel Pitrat, INRA - Research Unit for Genetic Improvement of Fruit and Vegetables GAFL, France
Mark Stitt, Max Planck Institute for Molecular Plant Physiology, Germany
Fernando Nuez, Center for the Conservation and Breeding of the Agricultural Biodiversity COMAV, Spain
Torben Jahrmann, Semillas Fitó S.A., Spain [Company]
   
FundingFrance National Institute for Agricultural Research (INRA)
 GermanyResearch Centre Juelich – Project Management Juelich (FZJ-PTO) on behalf of the Federal Ministry of Education and Research (BMBF)
 Spain Ministry for Education and Science (MEC) 
  
  Total granted budget€ 922,522
  
Abstract 

Melon is an attractive model species that may help to understand the molecular differences between non-climacteric and climacteric fruit ripening because it contains both types of genotypes. This proposal involves the simultaneous use of transcriptome and metabolome analysis in climacteric and non-climacteric melon lines in order to understand the molecular differences between both types of fruit ripening. A search and analysis of melon mutants in key genes in fruit ripening will also be performed. The TILLING and EcoTILLING technologies will enable the identification of mutants that do not have a distinctive visible phenotype. The discovery of such novel variants could directly lead to the creation of new melon hybrid cultivars with commercial interest, a task that will be undertaken by the two private partners that participate in this proposal.

  
Progress

1. Workpackage 1. Transcriptomics

Fruit flesh tissue has been taken every 5 days from pollination to mature fruit from the four melon genotypes (two climacteric, two non-climacteric). After previous metabolite analysis, sucrose and malate showed changes between 25 and 35 DAP. We decided to take 15, 25, 35 and harvest date as the 4 time points for transcriptome analysis.
RNAs have been obtained for the 60 samples that will be hybridized with the microarray, and the corresponding cDNAs have been submitted to NimbleGen.

Figure 1 Melon fruit types and stages for performing transcriptome analysis
Several genes with changes in expression between 15 and 45 DAP in ‘Piel de sapo’ have been validated by qRT-PCR, confirming that the NimbleGen microarray is useful. We have changed the design of the current array, using a 4 x 70K configuration with 4-5 probes per unigene, and 300 new unigenes have been added to the previous design.
Melon unigenes in the microarray have been annotated by MPIMP to be used with MAPMAN. A previous hybridization comparing 15 and 45 DAP ‘Piel de sapo’ fruit flesh shows that MAPMAN will be a useful tool to analyse melon microarray data. New pathway displays will be added to MAPMAN, including ethylene signalling, carotene metabolism, and stachyose and raffinose metabolism.
Figure 2 MAPMAN metabolism overview comparing the transcriptome data obtained from 15d v 45d ‘Piel de sapo’ flesh fruit samples

2. Workpackage 2. Metabolomics

Preliminary metabolite measurements (sucrose, fructose, glucose, malate, amino acids, and chlorophylls) were used to decide the time points for performing transcriptome analysis.. For metabolome profiles, LC-MS/MS will be used for sugars and organic acids, GC-MS for alcohols and fatty acids and HPLC for amino acids, nucleotides and phenylpropanoids. Some enzyme activities could not be measured, as ACC oxidase or invertase. The new MPIMP technician has been trained on the robot platform, and they have already assayed quite a few of the samples.

 

Figure 3 Robotized metabolic phenotyping to be performed with the melon fruit samples

Ethylene will be measured at CSIC-IRTA in summer 2008. Aroma analysis will also be performed.

Carotene analysis has also been performed. Carotenes are accumulated between 25 and 35 DAP for all genotypes. Beta-carotene seems to be the major carotene in Dulce and Védrantais. Piel de sapo does not accumulate carotenes. PI 161375 has a profile that is a mixture of beta-carotene and green-tissue carotene.

3. Workpackage 3. TILLING and EcoTILLING

TILLING: several target genes have been chosen, and mutations have been identified for two of them. Possible mutant families are under analysis now.
New target genes will be selected after analysing the transcriptome data, and genomic sequences of these genes will be obtained.

EcoTILLING: 200 melon genotypes have been harvested for phenotypic evaluation during 2008 (Semillas Fitó: 5 plants per accession, VCo: 3 plants per accession, COMAV: 3 plants per accession). Several quality traits are being measured in the collection. COMAV will process aliquots for MPIMP to measure sugars. Aromas will be done in collaboration with a lab in Cartagena.

Figure 4 Some melon types from the core collection of 200 genotypes that will be used in MELRIP