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Disease and stress resistant common Carp: Combining quantitative, genomic, proteomic and immunological makers to identify high performance strains, families and individuals

Final Report Summary - EUROCARP (Disease and stress resistant common Carp: Combining quantitative, genomic, proteomic and immunological makers to identify high performance strains, families ...)

The common carp is the third most important farmed freshwater fish species in the world. Eastern European carp genebanks have been responsible for the selective improvement of carp for intensive and semi-intensive pond culture in Europe and their dissemination worldwide. Several serious disease problems such as Koi herpesvirus (KHV), and erythrodermatitis (Aeromonas salmonicida and A. hydrophila) threaten carp farming in many countries. Selection in carp has tended to develop high performing but inbred strains for crossbreeding. The inclusion of disease and stress resistance as traits within breeding programmes will require the use of modern quantitative and molecular genetic tools. Disease resistance has proved to be a difficult trait to assess and improve in fish, direct challenges on potential broodstock run the risk of turning such fish into carriers. Functional genomics, proteomics and gene mapping has the potential to identify candidate genes and quantitative trait loci (QTL) for resistant fish without a challenge.

The EUROCARP project made possible the recent development of the carpBASE series of databases and the corresponding carpARRAY microarray chips. A significant advance that benefited the project substantial was the improvements in the annotation of carp ESTs based on two runs of the new 454 FLX high-throughput DNA sequencing system. This has generated a very large increase in the number of annotated genes. As a result, we have now generated a new carpBASE (version 7.1) containing the entire dataset, with a new and much more complete annotation. Two major experiments have been completed, designed to define the genes responding to bacterial or viral exposure, and to identify those responding genes that differentiate infected from control animals, and tolerant from susceptible phenotypes. The work focused on head kidney tissues due to its central complex interwoven loop design in which we compared six disease tolerant families with six disease susceptible families. This maximises the statistical power for a given level of effort and cost. We used 6-fold biological replicates (i.e. tissue from 6 different specimens) on 72 arrays. This experiment was run separately for both bacterial and viral exposures requiring a total of 144 arrays. The hybridisations were performed at 42 degrees Celsius in Liverpool, followed by washing, imaging on a Genepix scanning fluorescence microscope and image extraction on Bluefuse software (BlueGnome, Cambridge). The data was spatially normalised, LOWESS corrected and statistically analysed all using scripts developed in Matlab following the ideas on ANOVA developed by Wit and McClure. Multiple sampling (FDR) corrections were employed throughout, and we then extracted genes that displayed a significant response (< 0.05) in at least one ANOVA contrast. Overall responses and quality of the data were assessed using volcano plots, and P-value distributions. We clustered responding genes across all ANOVA comparisons and explored which clusters were linked to phenotypic responses of interest. The EUROCARP project made possible the development of the carpBASE series of databases and the corresponding carpARRAY microarray chips. A significant recent advance that benefited the project substantially was the improvements in the annotation of carp ESTs based on two runs of the new 454 FLX high-throughput DNA sequencing system. This has generated a very large increase in the number of annotated genes. As a result, we have now generated a new carpBASE (version 7.1) containing the entire dataset, with a new and much more complete annotation.

High heritabilities were estimated for harvest weight (circa 1 kg) and pond survival and suggest that considerable genetic gain can be obtained for production performance by selective breeding. Gene sequences for all known carp transcripts have been collated into a searchable database, and the description of this collation has been published in the Journal of Fish Biology. Latter phases of EUROCARP incorporated the results from an additional 'nextgeneration' sequence collation which has dramatically extended our classification of carp genes. This data has been co-assembled with the pre-existing EUROCARP collation and the resulting database is presented at http://www.agf.liv.ac.uk/cb7pathways. These data sets were modelled to identify new optimised approaches to selective improvement of common carp strains worldwide. This information will be disseminated through the established international breeding and carp producer networks.

Methodical requirements on compiling fish breeds catalogue were prepared and the initial materials on carp breeds officially registered in seven countries of Central and Eastern Europe (Hungary, Czech Republic, Poland, Russia, Ukraine, Belorussia and Moldova) were collected and analysed. On the basis of this data the catalogue of carp breeds Cyprinus Carpio L. of the countries of Central and Eastern Europe was prepared for publishing. This catalogue describes 72 carp breeds rearing in different nature-climatic zones of Central and Eastern Europe. For each breed there are morphological, exterior, genetic, reproductive and fish-breeding characteristics by which breeds differ from each other. The catalogue content is exclusive and includes photos of cultivated carp breeds. For the first time in the world practice the catalogue of such level is publishing both in English and Russian.

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