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Combined genetic and functional genomic approaches for stress and disease resistance marker assisted selection in fish and shellfish

Final Report Summary - AQUAFIRST (Combined genetic and functional genomic approaches for stress and disease resistance marker assisted selection in fish and shellfish)

The overall aim of the project AQUAFIRST was to identify, in sea bream, sea bass, oyster, and rainbow trout, genes of which expression is associated with disease and stress resistance and, from this information, to develop genetic approaches that allow characterisation of genetic markers for marker-assisted selective breeding of disease and/or stress resistant individuals.

The originality of this approach relies on the successful association of:
(i) functional genomic approach, i.e. development of microarray technology, to characterise disease and stress-responsive genes in the four species (trout, oyster, seabream, sea bass);
(ii) improvement of the genetic map for seabream and seabass associating information from microsatellite and SNP linkage map and radiation hybrid map and development of a linkage map based on microsatellites and SNP markers in oyster;
(iii) evaluation of quantitative trait loci which have an effect on disease and / or stress resistance in the four species.

This required mobilisation of a multidisciplinary research team gathering geneticists, physiologists and immunologists and a relevant coordination between the different research groups. The scientific strategy which has been employed in this project involved four complementary parts which were developed over three and half years:
Part 1: Identification and characterisation of genes involved in functional responses to stress or pathogen exposure
Part 2: Characterisation of 'Single nucleotide polymorphism' (SNP) in the candidate genes previously identified
Part 3: Using a QTL analysis, identification of association between stress or disease resistance traits and these candidate genes and also linked microsatellites markers
Part 4: Outline operational genetic protocols incorporating identified QTL and traditional breeding approaches in oyster, sea bream, sea bass and trout.

This strategy, which aimed to associate functional genomic data and QTL analysis in order to characterise genetic markers, was developed in trout and oyster for which a QTL mating design have been based on divergent families selected for stress or pathogen resistance. In sea bream and sea bass, a similar strategy was carried out and QTL analysis was carried out on fish taken from broodstocks. For genotyping analysis in these two species, the project took advantage of the tools developed in flagship projects funded by EC 'Bridgemap' and 'Bassmap' addressing sea bass and sea bream. Based on low to medium density linkage and gene maps already obtained or under construction in other project ('Marine genomic Europe' Network of Excellence), the project aimed to increase the density of sea bream and sea bass genetic maps. EST characterised by functional genomic analysis as being involved in stress or disease responses also go onto the linkage and gene maps. With this approach and radiation hybrid technology in these species, we have the most dense gene maps of any aquaculture species and exceptional tools for QTL approaches and Marker assisted selection (MAS).

Successful accomplishment of these approaches required establishment of genomic tools which were more or less present at the start of the project. Whereas a large EST collection and microarrays was already available for trout owing to the French Agenae genomic program and to the European Stressgenes project, development of such tools in oyster, sea bass and sea bream were more limited. A significant part of the efforts of the partners were then related to the development of these functional genomic tools and also to the training of partners to the use of these tools and relevant analysis of the transcriptome data.

Finally, these studies were carried out on stress/pathogen situations related to aquaculture problems. Thus, in fish, it was proposed to work on acute confinement stress or exposure to pathogens specific of each fish species. In oyster, a major problem encountered in production plans is summer mortality which is a complex phenomenon related to possible environmental, physiological (gonad maturation) or pathological causes. For these stress situations, several biological responses (traits) were followed (for some of them, segregation between different families has already been observed and heritability measured). Finally, development of these physiological and genetic studies on similar stress situations in four marine species favoured the possibility of a comparative approach.

This project led to large development of the genomic resources and new information related to genetic in aquaculture species. An overall analysis of these research activities highlighted the following major outcomes:

- A large development of genomic tools:
This includes not only functional genomic tools (EST collections, microarrays, database for management of the sequences and the microarray data) but also genomic tools (SNP, RH panel, new microsatellite markers, high-resolution linkage and gene map). This success was observed in the 4 species, seabass, sea bream, oyster and trout. On these issues, the project also beneficiated from the strong support and scientific input from one of NoE MGE partners (Max-Planck institute, Berlin). These links have also stimulated gathering of researchers for further research efforts on sequencing of the full genome of the sea bass European sea bass mapping consortium).

- A need for sustainable resources:
This effort raises an important question related to the sustainability of these resources after the end of AQUAFIRST project. Efforts have already been made. For example, Sigenae team (INRA, Toulouse, France) has proposed to keep on managing on his own resources the AQUAFIRST genomic database for two more years. This is an important question which would deserve full consideration if the European scientific community does not want to lose all the benefits from efforts developed during the last eight years.

- A large set of new scientific information:
Beyond the development of new genomic tools, this project has also generated several major new information related to the cluster of genes involved in the stress responses or in the first identification of QTL related to disease resistance or stress resistance traits in fish. In oyster, a QTL related to summer mortality have been identified. This information will be disseminated through publication in international peer-review journal. The list of presentations and publications (accepted or in progress) of these results fully confirm the scientific success of the present project and we can anticipate that more publication will come out.

- A large scientific exchanges between physiologist using functional genomic tools and geneticists using genomic tools:
The present project has been a successful attempt to stimulate these discussions and more benefits will come later as such exchanges should be carried on. Development of genomic approaches to answer specific questions related to aquaculture species will lead to such an integration of these genetic and physiological answers. In this context, AQUAFIRST project was a fully successful project for the development of links and construction of common concepts between geneticists and physiologists.

- The future of the AQUAFIRST project will be also very important:
This will include finalisation of data analysis in order to publish them in high-ranked scientific journal. It will also include continuation of scientific discussion between partners on specific topics such as:
(1) metagenomic analysis of the transcriptome data obtained in different tissues in the same species or for the same stress situation in different species;
(2) precise localisation an identification of the QTL using gene expression information;
(3) integration of the different genetic maps including BAC end sequencing map and RH map.