Overall Project Approach

A biodiversity study should provide the reference data necessary to estimate within-breed genetic variability as well as genetic distances between breeds. This was achieved by sampling 50 individuals from each of over 60 different breeds and lines and determining diversity at DNA level. Statistical analysis of microsatellite genotypes as well as of amplified fragment length polymorphisms will provide estimates of breed relatedness and of the extent of the genetic diversity among the commercial breeds and local populations in 16 European countries. The taking into account of this goal from the start will show to the breeders the practical interest of these tools, thus ensuring that pig diversity studies keep growing well after the closure of this project.

Over the last few years (1991-1994), our groups have made major contributions to the making of genetic maps, in pig through the EC co-ordinated Pig Gene Mapping Project (PiGMaP). This has required the development and characterisation of new polymorphic markers, and the setting-up of genotyping facilities and of associated databases. More recently, in work leading to this proposal, a pilot study (PiGMaP II, 1994-1996) in pig biodiversity has been carried out, and a number of well-characterised and robust microsatellite loci have already been selected.

The present demonstration essentially follows the FAO recommendations, as outlined in the MoDAD report^* putting a strong emphasis on the use of microsatellites as the core of a genetic biodiversity study. Microsatellites (simple tandem repeat, or STR) loci consist of blocks of short repeats where the repeated motif is a mono- di-, tri, tetra -or even penta- nucleotide e.g. CACACACACACACACACACA. The advantages of these markers for studies of genetic variation are manifold. They are ubiquitous throughout mammalian genomes. There are estimated to be 65,000 - 100,000 such loci in the porcine genome. The loci are randomly distributed throughout the genome. They exhibit substantial variation in the number of repeats. Through a combination of amplification using the Polymerase Chain Reaction (PCR) and high-resolution polyacrylamide gel electrophoresis the variation in repeat numbers can be determined. The repeat lengths variants are inherited as alleles. PCR technology allows the determination of this allelic variation from very limited quantities of DNA (less than 50 ng). Automated fluorescent DNA fragment analysers (also used for DNA sequencing) will be employed to estimate the length variants efficiently with the data captured automatically and electronically. Microsatellites were also assayed using DNA bulk sampling, from which allele frequency in a breed was inferred from the signal intensity of the allele in the corresponding bulk.

As a complementary approach, the technique of Arbitrary Amplification of Fragment Length Polymorphism (AFLP) was also applied. This is a procedure to selectively amplify specific subsets of a restriction digest of genomic DNA. The polymorphisms detected are point mutations or insertion-deletion polymorphisms, which, contrary to microsatellites, cannot be assigned to specific regions of the genome.

A variety of target groups were addressed. The participation of end-users as subcontractors providing samples, the involvement of several European pig breeding companies, and co-ordination with the EC Farm Animal Industrial Platform (FAIP) and Biotechnology for Biodiversity Platform (BBP), will facilitate the demonstration of the practical use of standard assays for the European industry. The involvement of FAO as a partner in the project helped to put it in a more global perspective of maintenance of domestic animal diversity.

Full information on raw data, as well as summary information on genetic distances, inferred phylogenies and relationships, will be made available to the wider community (including breeders, policy makers), through World Wide Web servers alongside the genome databases developed through the EC (BIOTECH) Genome Mapping Informatics Infrastructure (GEMINI) project.

The main risks and difficulties in the project were in obtaining the biological material needed, performing the laboratory analyses and interpreting the results.

Collection and transportation of blood samples between countries was made difficult for administrative reasons. Those difficulties were minimized here by realizing DNA extractions from the blood samples in the country of origin. DNA exchanges between the partners in this project raised no problems. However, it should be realized that the number of individuals requested per breed (50) not reached in some breeds of very low population size. A minimum of 25 individuals, from 13 different litters, was therefore set for a breed to be eligible in the analysis.

The laboratory techniques to be used required some standardization across different laboratories. In our case, this difficulty was avoided by sharing the typing work between 3 laboratories, markerwise, i.e. one laboratory for AFLP and two laboratories for microsatellites. DNA pooling (bulk) was performed in one laboratory in order to demonstrate the reduction in costs which may achieved by bulk typing.

Finally, in the interpretation of genetic distances, it should be kept in mind that phylogenetic trees derived from genetic distances are not expected to be always appropriate for livestock populations, because of the exchanges between branches known to have occurred.

^* Measurement of Domestic Animal Diversity, FAO (1995)