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Genetic diversity in indigenous cattle for East Africa - using RAPDs

Paul S. Gwakisa (2002)

Faculty of Veterinary Medicine, Sokoine University of Agriculture, P.O. Box 3258, Morogoro, Tanzania

Introduction

Background

The primary aim of studying genetic diversity is to understand the extent of differentiation of populations within species. Population-specific genetic markers (alleles) can be generated using a range of methods available for detection of polymorphic loci. Polymorphic genetic markers are extremely useful for a number of applications, such as measurement of the amount of genetic diversity in species, discrimination between individuals, strains or species, identification of markers linked to economically useful traits as well as analyses of animal kinship relationships, behavioural and population ecology.

In the past decade there have been tremendous efforts globally to study genetic diversity in livestock species. This, in part, reflects a measurable indicator of an output to the Food and Agriculture Organisation (FAO) global programme that addresses the needs of both development and conservation of animal genetic resources in the different parts of the world. The efforts also reflect global concern that a surprisingly narrow range of animal species is used for agriculture (sheep, goats, cattle, pigs, buffaloes, chickens and others that have local importance to certain areas) and this pool is threatened with genetic dilution. Shrinkage of the animal genetic resource pool is, to a great extent, a result of the radical shift of livestock development from subsistence to commercial farming systems. The world is witnessing increased specialisation of animal production objectives and competitive pressures arising from the specialisation. In sub-Saharan Africa, where the role of the livestock sector in gross agricultural output is increasing, specialisation has resulted in breed replacement, where indigenous zebu cattle are silently upgraded via artificial insemination with semen of Bos taurus breeds to achieve productivity gains. Although indigenous zebu cattle account for as much as 90% of the total cattle populations in African countries (Teale et al. 1993), crossbreeding with taurine breeds constitutes a significant threat to the zebu genetic resource. African zebu cattle are of particular importance because of their adaptation to a wide range of environmental conditions, such as heat, drought and tropical diseases. It is important to know the diversity and relationship between the African breeds and strains of livestock. Moreover, it is important because, quite often, the same breed may be known by different names or two breeds may be known by the same name, based on geographical locations of such populations (Gwakisa et al. 1994). The need to curb the threats of dilution and extinction of the African animal genetic resources, by strategic development and conservation, has never been more justifiable and is now, with the available technology, timely. Efficient strategies for conservation require sets of genetic markers, which characterise distinct populations (Kemp and Teale 1994).

Rationale for molecular genetic approach

Traditional methods used to study individual genetic variability of animals and populations employed polymorphism in protein markers. However, these techniques lack the power to resolve differences between closely related breeds, since a great deal of genetic variation remains undetectable by using protein markers. Moreover, the genotype frequencies estimated from protein markers may be influenced by natural selection among alleles, making it difficult to interpret interpopulation comparisons. With recent developments in molecular genetics, it has been solidly established that a measure of relative genetic diversity in animal populations can be attained through description of nucleotide sequence differences and similarities in the deoxyribonucleic acid (DNA) of animals in such populations. Analysis of DNA has several significant advantages over protein markers for the study of molecular population genetics and systematics. First, the genotype rather than the phenotype is assayed. Second, the expression of DNA markers is not influenced by development or by environmental factors. Third, one or more sequences appropriate to a problem can be selected on the basis of evolutionary rate or mode of inheritance. Fourth, the methods are, for most part, general to any type of DNA and, fifth, DNA can be prepared from small amounts of tissues and is relatively stable, even in non-cryogenetically stored tissues. The last attribute means that genetic information on rare or endangered species can be obtained without destructive sampling and that it is possible to analyse DNA from extinct populations or species. More recently, molecular data from DNA markers have received particular attention in the study of population variability because of their possible use in determining the chronology of evolutionary events. This is because DNA markers are much less subject to natural selection than are phenotypic traits.

The problem

Molecular techniques in the study of animal genetic diversity

A variety of different molecular techniques are being used in various laboratories for the study of inter- and intra-specific genetic variation at the DNA level. The most widely used techniques are restriction fragment length polymorphisms of nuclear DNA and mitochondrial DNA, minisatellites, microsatellites, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism and sequencing of mitochondrial DNA. These techniques differ in the type of data they generate, in the way that they resolve genetic variations and in the taxonomic levels at which they may be most appropriate. As a rich selection of molecular techniques is available, choice of method to use for genetic diversity is quite often dictated by the power of the method to generate reproducible polymorphism that can either be tracked in a Mendelian fashion or can segregate a phenotypic trait in a predictable manner. However, aspects such as lack of specialised equipment, high running costs and lack of technical competence continue to impede many laboratories in research institutions and universities in sub-Saharan Africa. Many researchers in Africa would benefit if laboratory technology development was paralleled by (1) simplification in technology and ultimately reduction in expense, and (2) increase in analytical power per unit effort. The discovery of the polymerase chain reaction (PCR)-based RAPD technique has made a substantial contribution as a simple technique, which is affordable to a broad community working in molecular genetics with limited resources.

Principle of RAPD analyses

The PCR-based RAPD technique is an attractive complement to conventional DNA fingerprinting. RAPD analysis is conceptually simple. Nanogram amounts of total genomic DNA are subjected to PCR using short synthetic oligonucleotides of random sequence. The amplification protocol differs from the standard PCR conditions in that only a single random oligonucleotide primer is employed and no prior knowledge of the genome subjected to analysis is required. When the primer is short (e.g. 10-mer), there is a high probability that the genome contains several priming sites close to one another that are in an inverted orientation. The technique essentially scans a genome for these small inverted repeats and amplifies intervening DNA segments of variable length. The profile of amplification products depends on the template-primer combination and is reproducible for any given combination. The amplification products are resolved on agarose gels and polymorphisms serve as dominant genetic markers, which are inherited in a Mendelian fashion (Williams et al. 1990; Rothuizen and van Wolferen 1994). Amplification of non-nuclear RAPD markers is negligible because of the relatively small non-nuclear genome sizes. Two modifications of detection of RAPD markers have been described as DNA amplification fingerprinting (DAF) and arbitrarily primed polymerase chain reaction (AP-PCR). DAF uses short random primers of 5-8 base pairs (bp) and visualises a relatively greater number of amplification products by polyacrylamide gel electrophoresis and silver staining (Caetano-Anolles et al. 1991). AP-PCR uses slightly longer primers (such as universal M13) and amplification products are radioactively labelled and are also resolved by polyacrylamide gel electrophoresis (Welsh and McClelland 1990). Standard RAPD analysis is performed according to the original method (Williams et al. 1990) using short oligonucleotide primers of random sequence which can either be locally synthesised or are commercially available (Operon Technologies Inc., Alameda, California, USA). Only high-molecular weight, i.e. non-degraded, DNA should be subjected to RAPD analyses. Amplified products can be resolved by gel electrophoresis on 1-2% agarose gels.

How to generate RAPD data

By employing different oligonucleotide primers, molecular characters that are characteristic of individuals can be generated . For any given primer, RAPD amplification products can be broadly classified into two groups: variable (polymorphic) or constant (non- polymorphic). For instance, consider a RAPD analysis of several individuals within a breed and several breeds within a given species. Constant fragments characteristic for the species may be identified, as well as fragments which are polymorphic between breeds within the species. Both types of product can be exploited for establishing relationships. In this example, constant fragments operationally identify members of a certain species exclusively, if the fragment is a unique polymorphism in a comparison of several breeds (species-specific marker). Similarly, fragments polymorphic at the breed level will operationally identify individuals of the breed if the fragment is constantly detected among the individuals (breed-specific marker). RAPD fragments, polymorphic among individuals of one breed, or belonging to one pedigree or one sex have also been obtained with different primers (Kemp and Teale 1994; Rothuizen and van Wolferen 1994; Gwakisa et al. 1994). Thus, RAPD products that serve as molecular genetic markers at different levels can be generated.

Advantages and disadvantages of RAPD markers

The advantages of RAPDs are that: there is no requirement for sequence information for the design of primers or probes; the procedure is quick, simple and can be automated; and a very small amount of DNA (e.g. 10 ng per reaction) is required. Different RAPD primers produce different banding patterns because the polymorphisms generated by any primer are due to differences in spacing between primer binding sites as well as point mutations. RAPD has been successfully used in genetic studies of many animal, plant and microorganism species (Michelmore et al. 1991; Chapco et al. 1992; Kantanen et al. 1995) including African goat populations (Chalya et al. 1997), East African zebu cattle (Gwakisa et al. 1994, 1997; Teale et al. 1995) and Tanzanian local sheep ecotypes (Stephen et al. 2000).

Disadvantages of RAPDs are many. First, resultant fingerprint patterns can be complex; thus, they require skilful scoring. Second, the method is very sensitive to reaction conditions, DNA quality and PCR temperature profiles. It is absolutely critical to maintain strictly constant PCR reaction conditions in order to achieve reproducible results. Third, the markers are dominant; thus, heterozygotes cannot be detected. Fourth, in the absence of pedigree material, the identity of individual bands in the multi-band profiles is not known and there can be uncertainty in assigning markers to specific loci. This makes RAPDs difficult to use in interpopulation or interspecific comparisons. Fifth, single bands on the gel can sometimes be comprised of several co-migrating amplification products. This makes it difficult to distinguish many of the polymorphisms apparent from PCR artifacts. Moreover, RAPD amplification occurs only at low, non-specific annealing temperatures. It is therefore likely that, depending on the surrounding DNA structures, a certain site is amplified in DNA from one animal but not from another (Rothuizen and van Wolferen 1994).

In the present case study, the potential application of RAPD fingerprinting in the study of genetic diversity in East African zebu cattle will be discussed.

Source of the material used

The results of two independent studies using RAPDs to study genetic diversity in East African zebu cattle will be described here.

Study I (Gwakisa et al. 1994):

Objective: Characterisation of zebu cattle populations in Tanzania using random amplified polymorphic DNA markers.

Representative animals of three cattle breeds in Tanzania (Mpwapwa, Tanganyika Shorthorned Zebu (TSZ) and Boran) were sampled from several herds. Random samples from 30 animals of both sexes were obtained from each breed.

Study II (Gwakisa et al. 1997).

Objective: Studies of genetic diversity in some indigenous cattle breeds of East Africa.

The cattle breeds and their respective locations in parenthesis were Boran (Kongwa, central Tanzania), Maasai Zebu (Kongwa, central Tanzania), Iringa Red (Iringa, southern Highlands of Tanzania), Dwarf Chagga Zebu (Moshi, Kilimanjaro, northern Tanzania), Tarime (also called Sukuma, Tabora and Shinyanga; western Tanzania) and Ankole (Tabora, western Tanzania). Also included were Boran and Sahiwal breeds from Kenya and -West African N'Dama breeds sampled from the International Livestock Research Institute (ILRI) in Kenya. Twenty cattle (10 of each sex) were sampled from each breed.

Sampling protocol, DNA extraction and analysis

The sampling strategy for each breed avoided selection of more than 2-3 animals from herds that grazed together or exchanged breeding bulls. Blood was collected and used to prepare DNA using published procedures. In essence, the blood cells were lysed using hypotonic solutions and non-ionic detergents and the nuclei were pelleted through centrifugation. The DNA was purified by treatment with protein denaturants and proteinases and then these were removed using organic solvents and high concentrations of sodium chloride. The DNA was concentrated by precipitating it with ethanol and then re-suspending it in water (Sambrook et al. 1989). Samples were adjusted to a concentration of 20 ng/μl before PCR amplifications which were performed on single-animal DNA and on breed-specific DNA pools. The breed-specific DNA pools were each made by mixing equal quantities of DNA from 15 individuals of the same breed at a 1:3 male: female ratio.

Random amplified polymorphic DNA - Polymerase chain reaction (RAPD-PCR)

The RAPD-PCR was performed on a Programmable Thermal Controller (MJ Research Inc., Watertown, MA, USA). Each 10μl reaction mix comprised: 20 ng template DNA, 6 pmol primer, dNTPs (deoxynucleoside triphosphates) each at 50μM, 0.5 units Taq DNA polymerase (Promega Biotec, Madison, Wl, USA), 10mM Tris pH 8.3, 50μM KCI, 0.001% gelatin, 0.025% Tween-20, 0.25% NP 40 and 1.5mM MgCl2. Temperature cycling comprised 40 cycles of 10 s at 95C, 5 s at 35C and 1 min at 72C. PCR products were electrophoresed at 150 V in 2% agarose gels containing ethidium bromide. PhiX 174 RF DNA digested with HaeIII (Gibco-Bethesda Research Laboratories Paisley, Renfrewshire, UK) was used as a molecular size marker. RAPD fingerprints were visualised by ultraviolet (UV) illumination and documented by photography.

Primers

A total of 141 arbitrary oligonucleotide primers was used singly for amplification of the breed-specific DNA pools. The primers, which discriminated between the DNA pools, were investigated further on single DNA samples to determine the proportion of animals carrying the pool-specific fingerprints. All the 141 primers were 10 bp long with a GC content of 45-80%. Sequences for the primers that amplified apparent pool-specific products are shown below.

ILO 1065: 5' CCG GTG TGG G 3'

ILO 1127: 5' CCG CGC CGG T 3'

ILO 1204: 5' GAC GGC GCA A 3'

ILO 1212: 5' GCG GCC GTA A 3'

ILO 525: 5' CGG ACG TCG C 3'

ILO 526: 5' GCC GTC CGA G 3'

Scoring of bands and statistical analysis

Only distinct, prominent bands were scored. Such bands represented RAPD fingerprints ranging from 0.31 to 1.1 kb in size. RAPD fingerprints were compared only on samples run in the same gel. Amplified DNA representing separate PCR products was scored and put in a data set for analysis of the following statistics: