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Development of genetic and physical marker maps of the bovinegenome (Bovine Gene Mapping project)


The general objective of the project was to map existing markers and to produce additional markers to construct a low resolution map of the bovine genome. The following tasks were addressed: 1) constitution of a panel of reference families, permitting the construction of a genetic map 2) production of a large number of highly polymorphic markers and analysis of their segregation in the reference families 3) development of a physical marker map, using a panel of somatic cell hybrids to assign markers to syntenic groups and in situ hybridization to determine precise chromosomal locations of 'landmark' markers 4) implementation of new technologies 5) development of statistical and computational facilities. The BovMap project, coordinated under the auspices of the International Society for Animal Genetics (ISAG) brought together 33 laboratories belonging to 14 countries (10 EC and 4 EFTA) and has been contributing to the global bovine genome mapping effort. The cattle breeding industry has contributed in making available full-sib families to establish a reference family panel, the first priority task in the project. A Management system was established, with a Committee of 7 Members and 6 Scientific Coordinators in charge of the coordination in the following activities: constitution of reference families, production of markers, genetic mapping, physical mapping, new technologies and database.
The ultimate goal was to establish a marker map for mapping and subsequent cloning of quantitative trait loci (QTL), using comparative mapping or positional cloning approaches, prior to the elaboration of marker assisted selection procedures. Three phases were required to achieve these objectives, of which the project comprised only the first, 1) construction of a low resolution map 2) development of technologies and resource populations or families 3) practical implementation of results in breeding strategies.
During the first year (1993), the material and methodologies required to construct the genetic and physical marker maps were set up. At the 3rd BovMap meeting (Oslo, 4-6 February 1994), three additional laboratories jointed the BovMap European Laboratory Without Walls (EWLL) and the second year period activities were planned: 1) typing of reference families, mainly using randomly produced microsatellites, and 2) directed production of microsatellite markers from physically mapped cosmids. Progress was evaluated at the XXIV International Conference on Animal Genetics (Prague, 23-29 July 1994) and, during the following period, the first priority was given to genetic and physical mapping of markers obtained from cosmids in order to assign and determine the orientation of linkage groups. At the 4th BovMap meeting (Dublin, 17-18 February 1995), the priority for 1995 was confirmed and, during the last year period, activities focused on completing the integration of physical and genetic maps in mapping selected microsatellite containing cosmids.
In order to ensure the best international coordination with other bovine genome mapping initiatives as well as with the european PiGMaP project, representatives from Australia, Israel, Kenya and USA national programmes and PiGMaP Coordinators, were invited to the BovMap meetings.
For each area of the project, the activities and the final situation, evaluated at the BovMap closure meeting (Tours, 20 July 1996), can be summarized as follows:
1) Reference Families. Eighty DNA samples, from animals belonging to seven of the twelves full-sib families collected in Europe, were included in the International Reference family Panel (IBRP) (composed of a total of 330 DNA samples, from 21 families). Since the end of 1993, DNA from the families has been distributed to 24 BovMap laboratories by the european regional node (INRA, Jouy-en-Josas; there are two other nodes, one in Australia and one in USA). The laboratories having received the IBRP samples actively typed either their own markers or those produced by other groups.2) Marker Production. During the first year, the dinucleotide repeat microsatellite markers were mainly produced from plasmids. In order to demonstratively contribute to the integration of the physical and genetic maps, it was decided in 1994 to focus the activities of the group on the production of microsatllite markers from cosmids, which can be precisely localized on the chromosomes by fluorescent in situ hybridization (FISH). Several laboratories made large efforts and the group isolated more than 400 microsatellite containing cosmid clones, of which 250 were distributed to other participants in charge of characterizing the genetic markers or in charge of the FISH work. In addition, other tools or methodologies (phage, cDNA, chromosome-specific libraries, SINE-PCR) were used to produce markers. Genetic variations in coding sequences were also investigated using single strand conformation polymorphisms (SSCP). A total of 218 plasmids and 416 cosmids have been isolated and up to 250 microsatellites have been obtained. Finally, two labs have worked at the characterization of microsatellites containing trinucleotide or tetranucleotide repeats, and the production of markers targeted at particular chromosomal regions was initiated.3) Genetic Mapping. 238 new microsatellites, characterized mainly from plasmid (125) or cosmid (86) libraries, 9 microsatellites derived from EMBL/Genbank bovine gene sequences, and 30 polymorphisms of coding sequences have been typed in the reference families and placed on the linkage map by the BovMap group. Genotyping results are compiled by the Cattle Genotype Database (CGD) maintained in Australia.4) Physical Mapping. About 250 loci (201 cosmids or 8 phages containing microsatellites, and 41 coding genes) were localized by FISH and 10 new assignments of synteny groups to chromosome have been established. The first integrated cytogenetic and meiotic maps of the bovine genome were presented at the Prague ISAG Conference and have now been published. Today, 90 of the in situ mapped loci (83 cosmids and 7 genes) are placed on the linkage map, and therefore, as a main result of the European work, every linkage group is assigned to a chromosome and all the linkage maps are oriented. The 3 available somatic hybrid cell panels (Jouy-en-Josas, Zaragoza, Cambridge) have been evaluated using more than 500 markers or genes now assigned to each of the 31 syntenic groups, and the labs involved continue to offer their service for rapid mapping of new markers or genes.
5) New Technologies. Sperm segregation analysis was reported very early. Due to the small difference in size between bovine chromosomes, their successful isolation by cell sorting was considered unlikely. The bovine bivariate flow karyotype was established and the different peaks identified by chromosome painting with PCR-generated probes; 22 chromosomes have been sorted as pure fractions. Chromosome microdissection has also been reported, and targeted production of markers either from sorted chromosomes or from chromosome fragments has been investigated for several chromosomal regions. Many efforts were devoted to the cloning of large DNA fragments, and the successful use of Pulse Field Gel Electrophoresis (PFGE), of Yeast Artificial Chromosomes (YAC) or of Bacterial Artificial Chromosomes (BAC) has also been reported.
(6) Database. A close collaboration between BBSRC (Roslin) and INRA (Jouy-en-Josas), to develop the statistical and computational facilities necessary for the pig gene mapping project (PigMaP) and the BovMap project respectively, was maintained through the European Genome Mapping Informatics Infrastructure (GEMINI) project (CEE BIO2-CT92-0451). The BOVMAP is a public summary database, which contains information on mapped genes and markers in cattle. The users can retrieve information on loci, alleles, genetic or physical maps, polymorphisms, homologies (comparative mapping), probes, primers, contacts and bibliography. BOVMAP has been open since October 1993 and has been regularly improved, to give access to the information through graphical and user friendly front end, and, to permit comparison of the various published genetic and physical maps. The database is now accessible via the World Wide Web ( on a mosaic server at INRA (Jouy-en-Josas). By May 1996, BOVMAP contained information for nearly 1560 loci.
1) Reference Families and Genetic Mapping. DNA from the IBRP was largely distributed in Europe and most of the laboratories which contributed to CGD over the past 3 years (22 out 36) were BovMap participants, thus demonstrating their active contribution to the global effort to construct an international, public domain, bovine linkage map. From November 1993 to May 1996, the BovMap group provided genotype information for 262 markers newly produced in Europe, which represent 44% of the 593 loci placed on the map during this period. A new version of the map (updated in February 1996), containing nearly 750 DNA polymorphisms, will be published soon, and it will represent a substantial improvment on previously published maps. It covers 95% of the bovine genome, has a total length of 3650 cM, and the average interval between markers that can be unambiguously ordered is 8.5 cM. No interval greater than 40 cM exists and less than twenty 20-40 cM intervals remain.
2) Integration of the Physical and Genetic Maps. During the first year, the microsatellite markers were mainly produced from plasmids, and in 1994, it was decided to focus the activities of the group on the production of markers from cosmids which can be precisely localized on the chromosomes by fluorescent in situ hybridization (FISH). Cooperative work enabled several laboratories to develop skills in physical mapping with the support of the most experienced lab (Zürich) which distributed fully characterized and mapped cosmids as reference material. Today, eight laboratories are able to perform FISH and a close collaboration with other participants in charge of characterizing the genetic markers has been organized. As a result of the European work, all linkage groups are assigned to chromosomes and all of the linkage maps are oriented with respect to the centromere. This constitutes the main achievement of the BovMap group, and a joint paper, describing 79 cosmid-derived markers anchoring the bovine genetic map to the physical map will be published by the BovMap consortium. It is the best integrated map of the bovine genome and the coordinated contribution of the European laboratories has been essential.
3) New Technologies. The development of new methodologies has been included in the BovMap activities, with the aim of improving map resolution and constructing more detailed regional maps to develop marker assisted selection or positional cloning. The bovine bivariate flow karyotype has been established. This result is of major interest because the existence of such new material will permit the initiation of the targeted production of markers, and already several microsatellites have been produced. Nevertheless, the experience of the laboratories which used chromosome-specific libraries of various origins to produce markers has shown that the present efficiency of these methods should be improved. Obviously, the recent success reported in using microdissection and DOP-PCR amplification to clone bovine specific chromosome fragments into a plasmid vector could constitute a major opening. The location of the chromosome fragment specific libraries has been confirmed by FISH and both type I and type II markers have been isolated from these libraries. The fact that the European laboratories, now have new possibilities to perform the targeted production of markers or to isolate coding sequences from genomic material covering a region of interest (where a gap still exists on the genetic map or where a QTL or a major gene lies) is noteworthy.
Many efforts have been devoted to the cloning of large DNA fragments, and the establisment of YAC cloning methodologies has been reported, leading to the elaboration of the BOREALIS project, coordinated by R. Miller (BBSRC, Cambridge) and funded by the EU Framework IV Biotechnology programme.
4) Database. A close collaboration between BBSRC (Roslin) and INRA (Jouy-en-Josas), through the GEMINI project led to the implementation of the PiGBase and ChickBase databases in Roslin, and of the BOVMAP database in Jouy. They are being regularly improved and updated, to give access to the published information and to permit comparison of the various genetic and physical maps. One major advance was the confirmation at the ISAG conference of the decision made during the BovMap meeting in Oslo (February 1994), and BOVMAP is now definitively considered as the only public database to be permanently updated by the scientific community working on the cattle genome. It was agreed at the BovMap Dublin meeting that information should be regularly transferred to the US BOVMAP database.
5) Comparative Mapping and Gene Mapping. More than 5000 genes have been mapped in man and in order to benefit from this information it is essential to develop comparative mapping activities. When QTLs are mapped to a chromosomal region, comparative mapping information help to identify candidate genes. Except for closely related species (ie cattle, sheep and goat), microsatellite sequences have diverged during evolution, making them essentially species specific. Thus, comparison of maps in various species has to be based on gene localizations.
The main contibution of the BovMap group in comparative mapping is the demonstration of the existence of 56 large chromosomal segments conserved between man and cattle, based on heterologous chromosome painting experiments, carried out by three groups using commercially available human chromosome-specific DNA libraries as probes. The results are highly concordant and will permit a very efficient use of the human map to improve the information available on the location of genes in cattle. Furthermore, the BovMap laboratories have intensified their efforts to precisely map genes and 75 genes have now been mapped (on the physical (52), or on the genetic map (30), or on both (7)), thus contributing to the construction of an `anchor' loci map. Several cDNA libraries have been constructed from various tissues or organs (brain cortex, liver, lymphocytes, mammary gland, placenta) and will be used to investigate and map expressed genes or expressed sequence tags (ESTs).
6) Use of the Bovine Map.
Genetic resources. Several laboratories have taken advantage of the existence of numerous genetic markers to examine the genetic variability in cattle breeds, using microsatellite loci or investigating genes controlling the coat colors. The BovMap participants have decided to coordinate their efforts through a new project coordinated by J. Williams (BBSRC, Roslin). A first set of markers, recommended for use, has been chosen and in the long term perspective, the accumulation of information with common tools could lead to new developments and help to refine the genetic ressource management policies.
Parentage control. Several laboratories have evaluated the use of microsatellites for the identification of animals and paternity testing. This constitutes an outcome of the mapping effort and could lead in the near future to the improvement of routine typing procedures proposed to the animal breeding industry.
Mapping in other bovidae. The high degree of homology between the cattle, sheep and goat genomes has encouraged a rapid transfer of results obtained in the bovine species to others; for example, about 50% of the bovine microsatellites produced in cattle can be used either for genetic mapping or parentage control in sheep and goat, thus lowering the research cost for small ruminant species. Therefore, several groups have taken advantage of these homologies to develop fundamental or applied research programmes.
QTL mapping. The genetic map has been used first to successfully map monogenic traits: the mh gene causing double-muscling in cattle has been mapped on chromosome 2. Three genetic diseases have also been mapped (the uridine monophosphate synthase deficiency (DUMPS), the White Heifer disease and the syndactyly locus).
Many laboratories have now engaged or initiated quantitative trait loci (QTL) detection programmes (either for beef or milk production traits under polygenic control). The resource populations for this work have been, either created, or more often provided by the cattle breeding industry. These programmes necessitate the genotyping of very large numbers of animals. For that purpose, automated procedures have been investigated to analyse the polymorphism of microsatellites with the use of robots, to perform PCR reactions with dye labelled primers, and of automatic sequencers, to determine the size of PCR products, and to automatically transfer the information in databases. During the Dublin meeting, the BovMap laboratories decided to collaborate to avoid duplication of efforts, and the fact that ten labs are now equipped and will share their skills and experience to select markers which are suitable for use on an automatic sequencer, in order to develop large typing facilities, can be mentioned as a main breakthrough in the perspective of industrial applications. A panel of markers to test in QTL studies has been selected and, primers will be synthesised and distributed to the participants over the next few months, as the final BovMap activity.
Finally, the BovMap project has proceeded in line with the timescale presented at the beginning of the projet and has demonstrated the capacity of the European laboratories to contribute to the global mapping effort on a coordinated basis. Today, expertise in bovine mapping exists in all european countries and when chromosomal regions of interest are identified (for example regions where the polled, the mh genes or a QTL lie), new research programmes devoted to the construction of fine resolution regional maps can be elaborated with better chances of success than ever encountered before. The way to QTL mapping and cloning is now open, and the practical implementation of results in breeding strategies can be expected before the end of the century.

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