ILRAD has begun a major investigation into the genetic control of trypanotolerance in the N'Dama by means of a cross-breeding and gene-mapping programme. This work is being conducted in close collaboration with a number of groups around the world, notably with Professor Soller's group at The Hebrew University of Jerusalem which is expected to play an important part in the experimental design and data analysis. The genemapping component of this work is being conducted in cooperation with the growing international group of laboratories which are actively involved in the construction of a bovine linkage map.
The programme may be viewed as having two separate threads which are running in parallel and which will come together with the phenotyping and genotyping of the F2 generation of N'Dama/Boran crossbred animals.
N'Dama/Boran cross-breeding and challenge.
The cross-breeding component began with a small group of N'Dama (Bos taurus) which was brought to ILRAD as frozen embryos from Gambia in 1983. These animals were shown to be highly trypanotolerant by comparison with Boran (Bos indicus) cattle. Multiple ovulation and embryo transfer techniques are being used to generate four large full sibling families of N'Dama/Boran crosses. The sires are four of the N'Dama bulls from Gambia and the dams are four Boran females. This programme is well under way and two of the families are complete with over 30 full sibling cross-bred calves in each.
These families are an important resources for linkage mapping and will contribute to the reference, material in use by ILRAD and other groups working on the bovine gene map.
The F1 crosses are also being phenotyped for trypanotolerance. This will provide the information needed to optimally construct the critical next generation.
Calves are born at the ILRAD ranch which is free of trypanosomiasis. After weaning at 8 months of age and a 2 month post-weaning period on the ranch, they are moved to the Laboratory. After a further 2 month period of acclimatisation during which baseline physiological data and data on their immune status is collected, they are challenged at 1 year of age with Trypanosoma congolense IL1180 by the bites of 5 infected tsetse files (Glossina morsitans centralis).
Cattle are monitored for up to 100 days post-challenge and data are collected on a regular basis on:
packed cell volume (PCV) percent and blood haemoglobin concentration
parasite counts in peripheral blood
parasite antigen levels in peripheral blood
total and differential leukocyte counts in peripheral blood
serological response to invariant trypanosome antigens
Animals in which the PCV falls to or below 15 percent are treated and effectively removed from the experiment. Animals remaining under observation 100 days after challenge are treated to effect a cure.
Plasma and sera are stored against future needs to examine other parameters of response as they may be identified.
Initial indications are that the F1's trypanotolerance lies between that of either parent. PCV falls on challenge but not to the levels of the pure boran controls.
Work on the F2 generation will begin during 1991. At the present time we plan to produce and phenotype approximately 150 F2 animals. DNA is collected and stored from all animals.
The objective of the gene mapping component of the project is to produce large numbers of highly polymorphic genetic markers. These will be applied to the F2 cross-bred animals, their parents and their grandparents. This will allow the origin of the DNA surrounding a marker to be ascribed to either N'Dama or Boran with a confidence which depends on its closeness to the marker. It is hoped to have available approximately 150 such markers by the time the F2 challenge is begun. The possible approaches to utilizing this phenotyping and genotyping information to narrow-down the location of the gene or genes responsible for the components of trypanotolerance were well described by Soller at this meeting (p. 44).
The ILRAD gene mapping programme is presently developing polymorphic markers based principally on microsatellites. Microsatellites are regions of simple nucleotide repeats, the most common being the dinucleotide motif CA. In the majority of microsatellites the repeat number is highly polymorphic and since there are known to be thousands of these regions in the bovine genome, they represent a rich source of genetic markers.
Microsatellites are exploited by means of the polymerase chain reaction (PCR). Synthetic oligonucleotides of about 20 nucleotides in length are designed according to the sequence which flanks a particular microsatellite. They are then used as primers in the PCR to specifically amplify from genomic DNA the region containing that microsatellite. The polymorphism of this PCR product is then revealed by high resolution electrophoresis. The prerequisite for utilizing a microsatellite as a genetic marker is therefore information on the DNA sequence surrounding it. This sequence will uniquely identify a microsatellite and it will become a form of “sequence tagged site” (STS).
Some suitable sequences are available from the published databases. We have used GENBANK as a source of primers for three microsatellites. However, to obtain the large numbers of markers for this study requires a programme of cloning, screening and sequencing aimed at obtaining microsatellite sequences. This is a relatively slow and laborious exercise, but having obtained a sequence and used it to synthesize primers, there is a high probability that it will be useful and its exploitation is simple and rapid.
Such a programme is presently underway at ILRAD and it is beginning to yield useful genetic markers. By pooling resources with other laboratories also developing microsatellite markers, it seems likely that a 20cM bovine linkage map will be available in the next few years.
A spin-off of the gene mapping exercise is the ability to characterize cattle populations with large numbers of markers. This offers a way to investigate historical relationships between cattle groups with a high resolution and at relatively low cost. Such studies may provide insights into, for instance, the evolution of trypanotolerance.
Mitochondrial DNA (mtDNA) is also receiving attention for the purpose of breed characterization at ILRAD. Because of its relatively high rate of mutation, mitochondrial DNA is a frequently used means of measuring genetic distance. We are using PCR to amplify fragments of mt DNA from crude DNA samples and then cutting with restriction enzymes to screen for sequence differences between individuals.
It is important to note that recent advances in technology based upon the PCR and the new classes of highly informative markers have made the genetic characterization of populations significantly cheaper and easier. A single crudely preserved blood sample may now provide all the material necessary to study thousands of polymorphic systems.
The study of the genetic control of trypanotolerance at ILRAD is critically dependent upon new technology and upon close collaboration with other groups. Indeed, PCR-based approaches to mapping greatly facilitate cooperation.
To exchange a new STS merely requires the exchange of some 40 bases of sequence data - no material need ever change hands. By using this approach and by working on common reference families, the bovine gene mapping community hopes to quickly establish a unified linkage map.
Furthermore, the state of knowledge of the human and mouse genomes is increasing rapidly. The demonstrated conservation of genetic structure between mammalian species will allow us to use this knowledge to facilitate the interpretation of data relating to the inheritance of trypanotolerance.
The objective of the mapping exercise is to make possible the next important steps in the study of trypanotolerance. Homing-in on single genes rather than areas of the genome and understanding how these genes exert their effect will be a major task. The exploitation of mapping information, by marker assisted selection or by the use of transgenic animals will be another challenge for the future.