Crop Genomics

Group Leader:
Abdelhafid Bendahmane
DR2 INRA
bendahm@evry.inra.fr
+33 1 60 87 45 02

Abdel Ihafid Bendahmane Portrait

Table of Contents
Projects:
Cloning of Vat and Pmw loci in melon
Cloning of Rfo locus
Cloning of virus resistance genes
Cloning of two sex determination genes
Tilling:
Tomato TILLING platform: TOMATILL
TILLING platform: TOMATILL, PETILL
Rapeseed TILLING platform: RAPTILL

Aurélie Andrieu

Aurélie Andrieu
ATER
andrieu@evry.inra.fr
+33 1 60 87 45 24

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Adnane Boualem Portrait

Adnane Boualem
Research Assistant (IE)
boualem@evry.inra.fr
+33 1 60 87 45 24

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Christian Clepet Portrait

Christian Clépet
(CR1) CNRS
clepet@evry.inra.fr
+33 1 60 87 45 12

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Marion Dalmais Portrait

Marion Dalmais
(IE) INRA
dalmais@evry.inra.fr
+33 1 60 87 45 23

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Fatima Dahmani Portrait

Fatima Dahmani
 
dahmani@evry.inra.fr
+33 1 60 87 45 31

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Eleblu John

Eleblu John
eleblu@evry.inra.fr
+33 1 60 87 45 27

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Sebastien Fleurier

Sébastien Fleurier
fleurier@evry.inra.fr
+33 1 60 87 45 24

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Camille Foucart

Camille Foucart
Post doctoral fellow
foucart@evry.inra.fr
+33 1 60 87 45 24

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Germain Gourdon

Germain Gourdon
(PhD)
gourdon@evry.inra.fr
+33 1 60 87 45 27

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Farhaj Izhaq

Farhaj Izhaq
(PhD)
Farhaj.Izhaq@evry.inra.fr
+33 1 60 87 45 27

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Delphine Jublot Portrait

Delphine Jublot
(AI) CNRS
jublot@evry.inra.fr
+33 1 60 87 45 31

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Bertrand Lasseur

Bertrand Lasseur
Post doctoral fellow
lasseur@evry.inra.fr
+33 1 60 87 45 24

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Aymeric Leveau Portrait

Aymeric Leveau
PhD
leveau@evry.inra.fr
+33 1 60 87 45 27

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Fabien Marcel Portrait

Fabien Marcel
Post Doc
marcel@evry.inra.fr
+33 1 60 87 45 24

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Benjamin Pouvreau

Benjamin Pouvreau
 
pouvreau@evry.inra.fr
+33 1 60 87 45 27

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Benedicte Sturbois Portrait

Bénédicte Sturbois
MdC
sturbois@evry.inra.fr
+33 1 60 87 45 01

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Christelle Troadec Portrait

Christelle Troadec
(AI) INRA
troadec@evry.inra.fr
+33 1 60 87 45 24

Introduction

We use forward and reverse genetics to isolate genes of agronomic importance. Two main tools are exploited: Positional Cloning and TILLING.
Three distinct research areas are at the core of the Positional Cloning project:

  1. development of a positional cloning platform

  2. identification of target genes of agronomic importance

  3. establishment of collaborations necessary for the isolation of the target loci

The key developments of this strategy include the establishment of high-throughput systems for identification of DNA markers and recombination events tightly linked to the target loci as well as construction and manipulation of BAC libraries. In the CFG group, most of these steps have been automated and thus far used in the cloning of loci controling resistance to bitic and abiotic stresses as well as loci controlling plants developments.

In the TILLING project we invested in the creation of reverse genetics platforms in 3 main crops: rapeseed, tomato and pea. Four distinct research areas were at the core of our project:

  1. the production and management of large collections of mutant populations

  2. the development of HTP tools for rapid and systematic identification of mutations

  3. A list of target genes of agronomic importance to be TILLED in each crop

  4. the creation of interactive and evolving databases

 

Cloning of Vat and Pmw loci in melon

Aphids cause serious damage to number of crops throughout the word, both directly through feeding and indirectly by transmitting several viruses. The Vat locus confers resistance to Aphis gossypii in cotton and many Cucurbitus species including melon. The out come of this cloning project was the identification of Vat protein as a CC-NBS-LRR type of protein. The transgenic expression of Vat in susceptible plants conferred complete resistance to A. gossypii.

Pmw locus confers resistance to powdery mildew in melon caused by Sphaerotheca fulginea. In this work we showed that the Pmw locus was allelic to the Vat locus. The Pmw and Vat proteins differ only by one deletion and three amino acid substitutions in the LRR domain.

gossypii

A1-A. gossypii on melon
A2- A. gossypii on on cotton
A3-Melon plant infected by S. fulginea.

Cloning of Rfo locus

Ogura cytoplasmic male sterility in radish is caused by aberrant mitochondrial gene, Orf138 that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the posttranscriptional level. The Ogura cms-Rfo two-component system represents an attractive model for investigating nuclear-cytoplasmic interactions as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis between Arabidopsis and radish we identified Rfo as a PPR type of proteins. This family of proteins consist of over 450 genes in Arabidopsis. The functions of these genes are largely unknown, although most of them are predicted to be targeted to mitochondria and chloroplasts and may have roles in organellar gene expression. Transgenic expression of Rfo in male sterile rapeseed restored male fertility.

Transgenic Expression

Figure: Transgenic expression of the Male fertility restoration gene, Rfo, in rapseed.
B1, male fertile transgenic plant.
B2, male sterile non transgenic plant.

 

Cloning of virus resistance genes

The pvr2 locus in pepper confers recessive resistance against different potyviruses. In this work we demonstrated that Pvr2 encodes for eIF4E. Sequence analysis of eIF4E from resistance and susceptible plants identified two point mutations as the cause of the resistance. Several lines of evidence indicate that these two mutations impair the interaction between the potyvirus genome-linked protein (VPg) and eIF4E.

The nsv locus confers immunity to a seed-transmitted carmovirus, melon necrotic spot virus. Using a combination of positional cloning and candidate gene approach we showed that NSV encodes for eIF4E. Sequence analysis of eIF4E from more than 30 resistant and susceptible germoplasms identified a single point mutation as the cause of the resistance. Understanding how this mutation confers resistance to an uncapped and an unpolyadinilated virus RNA without affecting the translation machinery is a priority.

eIF4E

Figure: left, comparison of the eIF4E in various plant species (see Nieto et al 2006).
Right, localization of the amino acid conferring resistance to MNSV on eIF4E 3D

Cloning of two sex determination genes

Cucumus melo is an outstanding model for sex determination studies. The sex expression in this species is determined by two major genes, A and G. Combination of alleles at these two loci lead to a wide range of sexual phenotypes. Plants of aa gg are hermaphrodites. Plants of A- G- genotypes are monoecious, bearing both pistilate and staminate flowers. Plants of aa G- genotypes are andromonoecious, bearing both hermaphrodite and staminate flowers. Plants of A- gg genotypes are gynoecious, bearing only pistilate flowers (Rosa 1928; Poole and Grimball 1939; Kenigsbuch and Cohen 1990). The sex of the plant could be also modified by hormone treatment. Treatment of monoecious plants with ethylene or its precursors leads transiently to plants only with female flowers. In contrast, treatment of gynoecious plants with ethylene inhibitors leads transiently to hermaphrodite flowers (Risser et Rode, 1979; Owens et al., 1980).

The A and G loci were recently cloned using positional cloning strategy. The goal of this project is to use the information from the sequence analysis of the A and G loci as a starting point to understand how the combination of alleles at these two loci determine the sex of the plant.

Sex Determination in Melon

Figure: Sex determination in melon. From the left to right are indicated hermaphrodite, male and female flowers.

Tomato TILLING platform: TOMATILL

Over the last two decades, knowledge about plant growth, development and the molecular compositions of plant organs has increased tremendously. The genes that control the function of the biological mechanisms involved are in many cases identified and well characterised. Unfortunately, development of technologies to manipulate crop genomes did not match that progress.

To bridge the gap between structural genomics and functional genomics in crop plants, we invested on the development of two complementary reverse genetics tools, TILLING and ECOTILLING. TILLING is based on production of EMS-mutagenised plant collections and rapid systematic identification of mutations in target sequences. ECOTILLING is based on the analysis of a collection of germoplasms to identify natural alleles of a given gene. Mutation detection is carried out using a mismatch specific endonuclease: Endo1.

scheme

Figure : TILLING and ECOTILLING platforms represent the gateway for gene function validation in crop plants.

TILLING platform:

Tomato Tilling Platform: TOMATILL

High quality EMS M2 population of 5000 M2 families created from a processing field tomato is exploited in this project. This far DNA was extracted from 5000 M2 families, pooled and used routinely for TILLING. Two additional mutant populations are also exploited in TILLING. Red Setter mutant population consist of more then 6000 M3 families. Money Maker mutant population consist of 5000 M2 families.

Pea Tilling Platform: PETILL

In PETILL we are exploiting a mutant population of 8000 M2 families produced previously in Therese background Genomic DNA was extracted from 3000 M2 families, pooled and used routinely for TILLING in the lab. In parallel a new mutant population of 5000 M2 families was produced from Cameor. This population is also routinely used for TILLING.

Rapeseed TILLING platform: RAPTILL

RAPTILL platform exploits a mutant population of 5000 M2 families. This far DNA was extracted from 3000 M2 families. This population is also routinely used for TILLING.