This overview of progress in legume improvement in Europe was done in conjunction with GLIP research in 2004-2005, and was presented in October 2005 in Madrid at a GLIP Dissemination event (go to Madrid-05 for more details including slides).

EU GRAIN LEGUMES Integrated Project: to boost production and maintain quality
by T.H. Noel ELLIS (John Innes Centre, Norwich, United Kingdom, GLIP coordinator)

The Grain Legumes Integrated Project (GLIP) is a large multinational project, co-funded by the 6th RDT Framework Programme of the European Union, striving to develop new strategies to enhance the use of grain legume crops in food for human consumption and animal feed in Europe and beyond. The principle objective of the project is to mobilise and integrate the European research effort on grain legumes to address the major agricultural constraints affecting the production of grain legume crops in Europe. Emphasis will be placed on the use of state-of-the-art methodologies including genomics and bioinformatics.
The first year of the project has brought significant progress in the development of genomic and genetic resources for both the model plant Medicago truncatula (the wild barrel medic) and a crop species, Pisum sativum: sequencing of the Medicago genome (within the framework of an international consortium with the USA), plant mutant collections with high-throughput targeted analysis, oligo micro-arrays and bioinformatics data integration tools. These resources are being used to analyse the factors determining seed protein quality, plant growth, and plant reaction to diseases, soil salinity and drought conditions. Comparative genomics with cross-species molecular markers will facilitate the transfer of information among legume species.
In addition to this genetic and genomic research, the role of grain legumes in animal feed and their environmental and economic impact on agricultural systems are being investigated. Innovative systems such as intercropping, where several crops (e.g., pea and barley) are grown together as a mixture, are being assessed, with results showing a positive impact on plant nutrition and growth, soil improvement and disease control.
This project is unique in having set up a Grain Legumes Technology Transfer Platform to assist the transfer of the combined results from the different research investigations into commercial products. Membership of this platform is open to any interested parties, such as scientific institutes, European plant breeders, food and feed companies.

Current progress in the research for disease resistance in grain legumes
by Diego Rubiales (CSIC, Instituto de Agricultura Sostenible, Córdoba, Spain) and Ana M. Torres (CIFA "Alameda del Obispo"- IFAPA, Córdoba, Spain)

A major limitation on legume production in Europe is the unreliability of yield during cultivation. This is predominantly caused by susceptibility to diseases. A better understanding of the plants’ responses to these stresses will allow the development of tools to tackle these problems, ultimately ensuring stable yields for farmers cultivating legume crops.
The EU project 'GLIP' focuses on the main legume biotic stresses, of which the major culprits are fungal diseases. The most important diseases in grain legumes in Europe are Aphanomyces euteiches, Orobanche crenata, Mycosphaerella pinodes and Erysiphe pisi in pea, Botrytis fabae and Ascochyta fabae in faba bean and and A. rabiei and Fusarium oxysporum in chickpea, respectively. GLIP undertake genetic studies and gene expression profiles to identify key regulators of resistance to some of these diseases. As a first step pathogenic diversity of major diseases is studied providing information on resistance breaking strains and allowing to target key strains for control. Plants have evolved a diverse array of resistance specificities to the majority of the diseases they encounter. Sources of resistance to the major pea diseases is being sought in both pea and Medicago truncatula by assessing natural diversity in defined ecotypes. The mechanisms of action of these resistance specificities is being characterised and the genes/QTLs responsible mapped. In addition expression profiling during both compatible and incompatible interactions is being undertaken in M. truncatula utilising microarrays. These studies should provide target genes for developing disease control mechanisms in pea.
The studies in chickpea and faba bean will expand on studies already undertaken in these species. A number of QTLs have been identified in faba bean and chickpea that provide resistance to O. crenata and A. fabae. A candidate gene approach will be taken, utilising the knowledge of resistance gene structure, in an attempt to identify the genes responsible for these resistances. In addition a superSAGE analysis will be undertaken in chickpea infected with A. rabiei. This approach will allow the distinction between transcripts from the pathogen and those from the host plant, and thus on the identification of genes induced in the host plant.
In the talk of GLIP-Madrid05, we provide a brief summary of the current status regarding genetic improvement of different legume crops against several important biotic stresses, focussing on the application of marker technology, and Quantitative Trait Loci (QTL) analysis for Markers-Assisted Selection (MAS) developed by our group. The information obtained so far, will facilitate the strategy for gene pyramiding and the future development of lines and cultivars with multiple resistance traits.

Contribution of GLIP to improve abiotic stress tolerance in legumes
by Thierry Huguet (LIPM, CNRS – INRA, Castanet Tolosan, France) and Martin Crespi (CNRS - ISV, Gif Sur Yvette, France), on behalf of GLIP-WP41 partners

Knowing the pattern of activation of gene expression during environmental stress will lead to a better understanding of the interrelationships of the multiple signalling systems that control stress-adaptive responses in legumes. Therefore, in order to dissect the mechanisms dealing with abiotic stress tolerance in the agriculturally important grain legumes, we are analysing gene expression patterns and metabolomic changes induced by diverse environmental stresses in pea, chickpea and M. truncatula using various genomic approaches. This is coupled with detailed genetic mapping of crosses between salt tolerant and sensitive varieties in chickpea and Medicago truncatula. Comparing transcriptome responses in an integrative way in model and grain legume species coupled with QTL characterisation at the genetic level in M. truncatula and chickpea, will serve to evaluate the control mechanisms exerted by the QTLs on gene expression patterns. This will serve to identify regulators of gene expression and metabolic adaptation using cultivars showing differential responses in Medicago truncatula and chickpea. These genes will then be used in reverse genetic approaches in the model legume (e.g. TILLING, deletion and insertional mutagenesis) to asses their role in abiotic stress tolerance. 

We have already constructed various SSH cDNA libraries in Medicago, chickpea and pea to identify genes linked to salt and drought stress environmental constraints that yield several thousands of genes. In parallel, we contributed to the development of a massive quantitative RT-PCR platform to monitor 1000 transcription factor genes in Medicago truncatula. At least 120 of these genes were found up or down regulated during salt stress. Dissection of the regulatory networks affected by environmental stresses will lead to the production of a cDNA microarray carrying diagnostic, stress-responsive cDNAs from the Galegoid legumes to ensure transmission of the results to the legume breeding industry and will serve to define a rational approach for grain legume improvement.

The main deliverables we expect to produce during the project are:
1. Candidate genes induced by salt, drought or cold stress in M. truncatula, pea, chickpea.
2. SSH cDNA libraries of pea, chickpea and M. truncatula submitted to drought and salt stress conditions.
3. Molecular markers controlled by QTLs linked to abiotic stress tolerance in M. truncatula and chickpea.
4. Metabolome adaptations in pea and M. truncatula submitted to drought/salt stress.
5. Fine map position of M. truncatula and chickpea QTLs involved in salt tolerance.
6. Functional relevance of a few selected regulatory genes in environmental tolerance in M. truncatula through reverse genetics.
7. Evaluated and patented marketable LeguStressChipTM ready for sale.

Genomics of model and crop legumes, a resource for legume breeding
by Jean Denarié (LIPM, CNRS- INRA, Castanet-Tolosan Cedex, France)

The model legume Medicago truncatula (Mt) is closely related to the major European legume crops (pea, faba bean, chickpea, alfalfa and clover), and a strong conservation of genome organisation has already been shown between Mt, pea and alfalfa. These findings are the basis of the organisation of the GLIP modules concerned with genetic and genomic approaches. The objectives are to contribute to the development of Mt resources and to develop tools to transfer the information gained from the model systems (also including Arabidopsis thaliana and Lotus japonicus) to European legume crops.

How are genes organised on the legume genomes?
Identifying and ordering most genes on the Mt genome is addressed by the international project of Mt genome sequencing which, is progressing well. In parallel, efforts are being made to generate high density maps for European legume crops. Comparison of the genetic maps of Mt and legume crops will facilitate the transfer of genetic (gene order) information from the model to crops.

Which genes control important agronomic traits?
Technological platforms have been developed in Europe that allow large throughput studies of legume functional genomics in both Mt and pea. Large-scale gene expression studies, using microarray technologies, allow the identification of genes that are expressed in different physiological and agronomic conditions (seed formation, resistance to stress, etc.). In parallel, large collections of mutants are being created by different means (point mutations, deletions, transposon insertions) to determine the function of individual genes. Frequently, important agricultural traits are controlled by more than one gene, the so-called quantitative trait loci (QTLs). Information of gene order on the chromosomes and on the function of individual genes should greatly facilitate the identification of QTLs.

The need for bioinformatics
Bioinformatics is required to manage the huge amount of genomic (DNA sequences, genome annotation) and functional information (gene expression analysis, proteome, metabolome) produced by GLIP and other national and international projects. The development of databases to structure, analyse and integrate the data is in progress together with the development of web-based search and browse interfaces to communicate and distribute data. Special emphasis is being placed on the development of methods to make these data easily available via query interfaces to plant breeders and other legume experts.

Comparative genetics to gain a better understanding from model plant species – The example of lentils
by T.H. Noel Ellis (John Innes Centre, Norwich, United Kingdom) and Marcelino Pérez de la Vega (Campus de Vegazana, Facultad de Biologia, Leon, Spain)

In the Grain Legumes Integrated Project, comparative genetic mapping is being carried out for pea, chickpea, faba bean, lentil, lupin, Phaseolus and clover. The genome information from Medicago truncatula is central to this because of its close taxonomic relationship to several species, but information from Lotus japonicus is also contributing to these studies, especially for Phaseolus. In this scheme several hundreds of gene-specific markers are being located on genetic maps of the crop species to identify syntenic regions in sequenced genomes. The idea of this is to allow the genome information from the sequenced genomes to provide useful information about gene content and order for these crop species.
Lentil (Lens culinaris Medik.) is a self-pollinated diploid (2n=14) legume species with a relative large genome of 4,063 Mbp. Lentil is an important food crop grown in many temperate areas and is valued for its relatively high amounts of healthy and good-quality plant proteins. Despite their economic and nutritional value, lentils have received less research attention than many other legume crops.
To date, our genetic lentil map includes 161 markers grouped into ten linkage groups covering about 2,000 cM. To date, we have identified 29 (out of 116 tested) gene specific markers that are polymorphic in our lentil population. In addition more than 30 different resistance genes analogues have been identified in Lens. Together these markers provide the basis for the inclusion of lentil in the emerging comparative genetic map of legumes.