You are here: ERA-PG / File repository / Call 2006 / / MuExpress
Title Isolation of key genes for kernel development through the identification, in a collection of 300 mutant lines, of Mutator insertions in genes expressed in the maize seed
   
Acronym MuExpress
   
Duration 1 April 2007 - 1 April 2010
   
Project leader Gregorio Hueros, University of Alcalà de Henares, Spain
   

Other project participants

 Christophe Tatout, Biogemma [Company], France
Peter Rogowsky, University of Lyon, France
Udo Wienand, University of Hamburg, Germany
  
Funding National Institute for Agricultural Research (INRA), France 
   Research Centre Juelich – Project Management Juelich (FZJ-PTO) on behalf of the Federal Ministry of Education and Research (BMBF), Germany
  Ministry for Education and Science (MICINN), Spain
   
Total granted budget  € 922,522
   
Abstract  

Maize is essential for world nutrition and widely cultivated in France, Spain and Germany. The size and shape of the two seed compartments (embryo and endosperm) is determined during seed development, a process involving an estimated 1000 genes. Less than 10% are known today and the identification and characterisation of genes involved in seed development presents a major scientific and agronomic challenge.
The project aims at the identification and functional analysis of genes involved in maize seed development via the large scale molecular characterisation of transposon induced maize seed mutants. It is based on a collection of 300 mutants selected from a much larger initial collection based on a clean 3:1 segregation of the mutant phenotype and a stable phenotype over at least 3 generations. These mutants have been selected as being particularly defective in endosperm rather than embryo formation, thus enriching for mutations in genes preferentially involved in endosperm formation. After a minimum of 2 backcrosses the material is genetically quite homogenous and genomic DNA of wildtype and mutant pools is available. In a sister group of 300 mutants the bottleneck has been the identification of the transposon copy responsible for the phenotype among the roughly 100 copies present in the genome. To circumvent the very low efficiency of the AIMS technique on genomic DNA encountered in previous experiences, we propose here to decomplexify the system and to apply the technique to seed cDNA. All FSTs will be systematically sequenced opening not only the way for the subsequent co-segregation studies that will link the phenotype to a single FST but also creating a valuable inventory of Mutator insertions in coding sequences present in the material. A strong bioanalysis of the candidate sequences using all available tools in maize as well as the synteny to rice and other cereals will provide insight into the potential molecular function of the candidate genes. Among others the expression profiles of the candidates will be extracted from existing micro-array data and the in silico map position in the maize genome compared with the positions of seed QTLs.
   
Progress
Figure 1 Diagram showing the biological materials used in the project
 
Figure 2 The experimental strategy used in MuExpress as compared with conventional approaches
 
Figure 3 A Li-Cor gel showing the Mu-cDNA display obtained for 10 of the lines analyzed in the project. For each line the Mu-cDNA display obtained from a wild type sample and two mutant kernels is shown. Green arrows insertions present in many lines, not associated to the mutant phenotype. Orange arrows, insertions present in a single sample, very likely somatic. Red arrows, potential causal insertions. Figure 4 A an example of the design of PCR primers used in the FST validation process, in a first instance a gene specific primer is used in combination with a Mu-terminal inverted repeat primer. B positive and negative results obtained during the process of candidate gene validation, the PCR test is used in increasingly larger segregating populations to prove/disprove phenotype-insertion linkage.