Genetic Characterization of Black Bengal Goat Breed of Three Different Regions of Bangladesh
1.1 Goat: the Heritage and Pride of Bangladesh
Goat (Capra hircus) is found almost in all parts of Bangladesh. They are reared for the production of milk, meat, wool and leather particularly in arid, semitropical or mountainous countries (Morand, 2004). Goats can supply a family with several quarts of milk each day and thus providing important nutrition for undernourished children. Throughout the history of mankind, the role and contribution of goats in the sub-tropical developing countries has been prominent. Goat was the first ruminant animal that was domesticated around 8000-11000BC (Luikart et al., 2006 and Tapio et al., 2006).The importance of goat rearing in providing nutritional and financial sustenance to the economically weaker sections of the society is well recognized in developing countries. Goats have the ability to thrive under diverse climatic conditions and withstand extreme vagaries of nature. They are known for their versatility in adapting to arid, humid, tropical, cold, desert and mountain conditions and in the process providing people with many important livelihood products such as meat, milk, skin, draft and pack power, cashmere, mohair and enriching manure for crops and gardens.
1.2 Role of Goats in Subsistence Farming
Goats are numerically and economically very important and promising animal genetic resources in the developing countries, especially in Asia and Africa. At present, there are 767.93 million goats in the world. Of the total goat population, 92.76% of goats are found in Asia and Africa. Asia has the largest population of goats (63.66% of world population), within which the largest populations are found in China (35.36%), Pakistan (10.79%) and Bangladesh (7.05%) (FAOSTAT, 2005). These countries together possess about 71.61% of the population of goats in Asia. FAO data indicate that there exist 570 breeds, of which 146 are found in Asia. The annual growth rate of goats was 6.6% (FAOSTAT, 2005). Bangladesh possesses 34.5 million goats which is 4.49% of the total population in the world (FAO, 2003). Bangladesh has the fourth highest population of goats among the Asiatic countries.
There are 160 popular goat breeds available worldwide which are based on the size of populations, productivity and unique characteristics. Among these, Bangladesh has only one goat breed of its own, popularly known as the Bengal goat. These goats are of particular economic significance in the subsistence farming of rural community. Paradoxical to the breed name Black Bengal is not always solid black in coat color rather coat color polymorphism does exist in this breed, such as, full white or cream, bezoar, silver Bezoar, black with “Toggenburg pattern” of spotting, black with “Dutch belt” spotting (Nozawa et al., 1984; Alam, 2006 and Faruque and Khandoker, 2007). According to Nozawa et al., (1984) at least four loci (I, A, D, S) are involved in such morphological polymorphism. Amongst them Black, White (Cream) and Silver Bezoar coat color patterns are very frequent (Faruque and Khandokar, 2007). These coat color types might have some unique features and adaptive advantages to pertinent habitat. Jamuna pari (Nubian type) is another important goat breed in Bangladesh. It is estimated that more than 90% of goat population in Bangladesh comprised the Black Bengal goats, the remainder being Jamuna pari and their crosses (Husain, 1993).
The higher demand of meat and skin in the local as well as foreign markets focused the goat enterprise extremely prominent to the vulnerable group of people in the existing socio-economic condition of the country (Husain, 1993). But owing to small body size, meat produced by this breed does not fetch a high economic return. As Black Bengal goats are being reared primarily for meat production, body weight and growth rate could be considered as the most important factors. Quantity of meat produced from the goat depends on the number of kids produced from a single birth, birth weight of kids, rate of gain and weight at slaughter. For improving meat production in goats selection for 6-month body weight from twin birth type may be the most feasible way for having reasonable genetic progress in meat production (Acharya, 1992).
Body length and heart girth may be used as good reliable predictors to assess live weight. A functional and more useful criterion to characterize goat population is the body size which also indicates the performance orientation (Bhattacharya et al., 1984). There was a correlation of body weight with length, height and heart girth in Black Bengal goats. Correlation of body weight with heart girth was highest and this was followed by length and height respectively (Prasad et al., 1981). High relationship of body weight with chest girth was also observed by Singh et al. (1979) in Black Bengal and Mukherjee et al. (1979) in Grey and Brown Bengal goats.
1.3 Genetic Characterization – A measure to Conserve and Improve Goat Varieties
Conservation and improvement strategies ought to be based on proper genetic characterization in association with phenotypic characterization. Different developed and developing countries have already characterized their goats at molecular level. Genetic diversity may be measured through genetic markers. These have been used to determine evolutionary relationship within and between species, genera or higher taxonomic categories. However, breeders tend to concentrate on specific genotypes for determination of genetic diversity which combine traits of interest and may be used as progenitors in several breeding programs in order to introduce agronomical important traits. In an attempt to solve the problem of maintaining pure breeds using the observed morphological characteristics that require a lot of time and effort, the use of molecular markers in maintaining goat breeds is more suitable and less time consuming. Moreover; molecular markers are important tools in tagging desirable loci underlying the traits which have breeding importance.
Estimation of genetic variation are increasingly being based upon information at the DNA level by various molecular markers such as, Randomly amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR) or Microsatellite etc. Among them, RAPD markers, generated by the polymerase chain reaction (PCR) is widely used since 1990’s to assess infra-specific genetic variation at nuclear level. RAPD is a PCR based technique for identifying genetic variation. It involves the use of a single arbitrary primer in a PCR reaction, resulting in the amplification of many discrete DNA. The technique was developed independently by two different laboratories and called as RAPD and AP-PCR (Arbitrary Primed PCR) respectively. This procedure detects nucleotide sequence polymorphisms in a DNA amplification-based assay using only a single primer of arbitrary nucleotide sequence. The RAPD technology has provided a quick and efficient screen for DNA sequence-based polymorphisms at a very large number of loci. The major advantage is that no prior DNA sequence information is required. The vast range of potential primers that can be used gives the technique great diagnostic power. Reproducible RAPD bands can be found by a careful selection of primers, optimization of PCR conditions for the target species and replication to ensure that only the reproducible bands are scored. RAPD analysis has been used extensively for various purposes which include identification and classification of accessions, identification of breeds, genetic diversity analysis and predicting quantitative variation within germless.
1.4 Aims and Objectives of the Present Study
The farmers and GoB (Government of Bangladesh) in the recent past are showing interest to utilize this species to increase the supply of meat for human consumption and to alleviate poverty through creation of employment. The genetic improvement of goats might be one of the major options to fulfill this goal. Genetic characterization is the first and foremost priority for the genetic improvement of any species. However, there is little report on genotypes of Bangladeshi goats. Katsumata et al. (1984) firstly reported the genetic variation of different populations of Bangladeshi goats based on blood protein markers and morphological variations.
Goats are found abundantly in Kishoregonj, Savar and Narsingdi areas of Bangladesh but no genetic characterization of the goat of this area have been performed so far. In order to undertake any development work in the rural area, the goat production problems and prospects should be identified. Therefore, considering the above facts, the aim of the study is to genetically characterize Black Bengal Goat breeds of three different areas of Bangladesh by Random Amplified Polymorphic DNA (RAPD) technique.
The aims of the present study can be summarized as follows –
Determine the genetic diversity and gene frequency of Black Bengal goat from three different areas of Bangladesh
Determine the genetic distance among the Black Bengal goat population of the studied area.
2.1 Goats – One of the First Animals to be Domesticated by Humans
Goats were almost certainly the first ruminants to be domesticated (Devendra & Mcleroy, 1982) and were possibly the second species to be taken into the humanfold after the dog (Zeuner, 1963). South-West Asia (Iran & Iraq) is the most likely origin of the domesticated species of the bezoar, C. aegagrus (Payne & Wilson, 1999). Although not certain, the available evidence from comparative morphology and breeding experiments indicates that the bezoar of western Asia is the main ancestor of most domestic goats (Devendra & Mcleroy, 1982). Archaeological evidence indicates that goats, in the form of their wild progenitor the bezoar (C. aergagrus), were the first wild herbivores to be domesticated.
Some studies suggest that domestication of goat occurred approximately 10,000 years ago at the dawn of the Nelotic in the region known as the Fertile Crescent. Zeder & Hesse (2000) confirmed that the fertile crescent region of the Near East was the centre of domestication for a remarkable array of today’s primary agricultural crops and livestock. Wheat, barley, rye, lentils, sheep, goats and pigs were all originally brought under human control in the broad area that stretches from the southern Levant through south eastern Turkey and northern Syria, to the high Zagros mountain pastures and arid lowland plains of Iraq and Iran.
For more than 50 years researchers have sought to define the sequence, temporal placement, and social and environmental context of domestication. They described recent research that uses a study of modern wild goat C. hircus aegagrus to develop an unequivocal marker of early goat domestication, which we apply to assemblages that lies both within and outside the natural range of wild goats in the eastern fertile crescent region, long thought to be the initial heartland of goat domestication (MacHugh & Bradley, 2001). Luikart and colleagues add C. hircus to the growing list of domestic animals that have been widely surveyed for mtDNA sequence variation. In their survey, they demonstrate that the structure and distribution of mtDNA variation in domestic goats are qualitativelydifferent from the patterns observed in other large Eurasian herbivores domesticated for food, skins and fibre (cattle, buffalo, pigs, and sheep) (MacHugh & Bradley, 2001).
Domestication occurred gradually over a period beginning some 11000 years BC (before present) and it probably first spread to central and south eastern Asia. By 5500 BC goats had migrated into sub-Saharan Africa and a dwarf type was recorded from that period near Khartoum in the Sudan. Initially, the migration routes of the human population may have promoted the expansion of domestic goats and their establishment in several regions. Many breeds are adjusted to the climate, diseases and nutritional conditions and consequently, developed the capacity to survive and reproduce in difficult conditions. In addition they developed a great aptitude to increase production without losing local adaptation through selection programmes (Payne & Wilson, 1999).
2.2 Goat in Bangladesh
Goats have been reared in Bangladesh from the time of human settlement in this part of the earth. They stand second in number among the ruminant species. The population of goats in Bangladesh currently includes about 18 million animals, which are dispersed throughout the country. The average number of goats per household is 2.3 and they are mostly reared by landless, small and medium farmers. Goats are used primarily for meat production, but their skin is a valuable by-product (Joint FAO/IAEA Programme website).
Economically and culturally, the goat has played an important role in traditional Bengali society.Goat skins were exported to the world market on a large scale in the 18th and 19th centuries. The goat is important for its adaptability, fertility, high productivity, and skin softness. The goats’ skin is of superior quality for leather goods, and is in great demand both in domestic and foreign markets. The goat’s meat is famous for its tenderness, flavor, and leanness (Joint FAO/IAEA Programme website).
2.3 Genetic Resources of Goats in Bangladesh
The country has a native goat breed commonly known as the Black Bengal goat, exotic breeds such as the Sirohi, Beetal and Jamunapari, and crossbreds between the Black Bengal goat and exotics. Black Bengal goats are found all over the country. They can be classified into 3 sub populations; Bangladesh West (BBW), Bangladesh Central (BBC) and Bangladesh East (BBH); according to their geographical distribution. The exotic breeds and crossbreds are concentrated in the western part of the country and in some specific areas of central and eastern Bangladesh. Sirohi and Beetal bucks are imported by the farmers privately and used mainly for crossbreeding purposes. In the absence of any census, it is very difficult to know the exact population number of each population or breed. However, Black Bengal goats are clearly predominant (Joint FAO/IAEA Programme website).
2.4 Black Bengal Goats
Black Bengal goat or Bengal Black goat is the common name for a small breed of goat found in Bangladesh and northeastern India (Assam and West Bengal). In Bangladesh, they are known simply as “BBGs.” Despite the name, the coat color of Black Bengal goats is not black in all cases. At least seven recognizable coat color patterns can be found:, solid black, solid white, black with “Toggenburg pattern” of spotting, brown with “Toggenburg pattern” of spotting, black with “Dutch belt” spotting, silver bezoar, and brown bezoar wild-type pattern. Coat color is controlled by epistatic genes. The frequency of each color pattern varies depending upon the location. Different populations also differ in size, with adult animals ranging from 42 to 56 cm at the withers. The exotic breeds exhibit their breed-specific characteristics.
The Black Bengal goat hair is short, soft and lustrous. The back is straight, legs are short, and the ears are 11–14 cm in size and pointed forward. Both sexes have horns (5.8-11.5 cm), directed upward or sometimes backward. Beards are often observed in both sexes. The average height of an adult is 50 cm. Adult bucks weigh 16–18 kg and does weigh 12–14 kg.
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Figure 2.1: Different varieties of Black Bengal Goats.
2.5 Genetic Polymorphism: The Genetic Basis of variation
Genetic polymorphism arises from mutation. It refers to the difference in DNA sequence among individuals, groups, or populations, and can be caused by mutations ranging from a single nucleotide base change to variations in several hundred bases. In a formal sense, there are only two kinds of polymorphisms: those due to replacement of DNA bases and those due to insertion or deletion of base pairs. The simplest type of genetic polymorphism is the single nucleotide polymorphism (SNP). A position is referred to as an SNP when it exists in at least two variants, being the rarer allele more abundant than 1% in the general population. Other types of genetic polymorphisms result from the insertion or deletion of a section of DNA, which include repeat sequences (mini and microsatellites) and gross genetic losses and rearrangement. Gross alterations are mutations in which substantial portions of DNA sequence (>500 bp) are deleted, duplicated or rearranged. These types of genomic alterations can be detected by high resolution cytogenetics (for extremely large alterations such as chromosomal number and chromosomal translocations) and by fragment analysis of implicated chromosomal regions using southern blot, microsatellites, and fluorescent in situ hybridization (FISH). Hypervariable minisatellites are usually defined as the repetition in tandem of a short (6 to 100 bp) motif spanning 0.5 Kb to several kilobases. They are mostly located between genes, and are dispersed unevenly in the genome preferentially in telomeric locations (Lathrop et al., 1988). Because of their length polymorphism, and the ability to cross-hybridize with similar loci throughout the genome, minisatellites have been used in DNA fingerprinting and forensic for individual identification (Jeffreys et al., 1985). Microsatellites or short tandem repeats (STRs) are tandem repeats of multiple copies of the same sequence motif composed of 1-4 base pair long units. They are ubiquitous in prokaryotes and eukaryotes and present even in smallest bacterial genomes (Hancock, 1996). These polymorphisms show high levels of allelic variation in the number of repeat units, and are used extensively as markers in linkage studies. A subset of STRs, namely trinucleotide repeats, are implicated in many human neurodegenerative disorders such as fragile X syndrome and Huntington’s disease (Bates and Lehrach, 1994; Reddy and Housman, 1997) and in some human cancers (Wooster et al., 1994). This kind of STRs is often called dynamic mutations (Richards and Sutherland, 1992).
SNPs (Single Nucleotide Polymorphism) are the most common type of genetic diversity and are estimated to occur with a prevalence of one SNP per 1300 bases in the human genome (Lander et al., 2001; Venter et al., 2001). In principle, SNPs could be bi-, tri-, or tetra-allelic polymorphisms. However, tri-allelic and tetra-allelic are very rare and SNPs are sometimes simply referred to as bi-allelic markers (Brookes, 1999). SNPs can result from either the transition or transversion of nucleotide bases. Transition substitutions occur between purines (A and G) or between pyrimidines (C and T). Transversions are substitutions between a purine and a pyrimidine. Transition mutations are more common than transversions, with CpG dinucleotides showing the highest mutation rate, presumably due to 5-methylcytosine deamination (Duncan and Miller, 1980). Nucleotide substitutions occurring in protein-coding regions can be classified as synonymous and non-synonymous according to their effect on the resulting protein. A substitution is synonymous if it causes no amino acid change while a non-synonymous substitution results in alteration in the encoded amino acid. The latter type can be further classified into missense and nonsense mutations. A missense mutation results in amino acid changes due to the change of codon used while a nonsense mutation results in a termination codon. Even within a single chromosome, the SNPs are not uniformly distributed, and some genomic regions have significantly lower or higher diversity than the average. Polymorphisms in the regulatory regions of genes and sequence variants that alter amino acids in the coding regions are generally less common, reflecting a greater selection pressure reducing diversity at these DNA regions. Within coding exons, the nucleotide diversity is four- fold lower, with about half resulting in non-synonymous codon changes (Li and Sadler, 1991; Nickerson et al., 1998). In addition, there is enormous diversity in SNP frequency between genes reflecting different selective pressures on each gene as well as different mutation and recombination rates across the genome. Depending on where a SNP occurs, it might have different consequences at the phenotypic level. SNPs in the coding regions (sometimes termed as cSNPs) or in regulatory regions are more likely to cause functional differences than SNPs elsewhere. In case of the human monogenic Mendelian disorders, SNPs in the coding regions that alter the function or structure of the encoded protein could be the cause of the disease. In animal production, examples of direct impact of SNPs on phenotype include mutations in growth hormone receptor gene in dwarf chicken (Duriez et al., 1993; Huang et al., 1993), myostatin (GDF-8) gene in double- muscling cattle (Kambadur et al., 1997; Grobet et al., 1998) and as1-casein in goat (Grosclaude et al., 1994). In general, association studies have to be performed in order to statistically establish that particular alleles are associated with one or more phenotypic traits. However, most SNPs are located in non-coding regions, and have no direct effect on the phenotype. These SNPs are useful as markers in population genetics and evolutionary studies and to identify genes implicated in complex multigenic traits by using linkage disequilibrium.
2.6 Genetic Diversity and Conservation
Genetic diversity is defined as the sum of genetic differences in multiple loci among individuals in a population, and is most readily reflected in the phenotypic variation seen in many populations. Genetic diversity is a valuable asset as the adaptability of a population, that is the population’s ability to adapt to changes, depends on it (Woolliams et al. 2005). It is well known that species can face great environmental changes over time, such as in climate, pollution and in diseases, and genetic diversity is required for populations to adapt to these changes (Frankham, Ballou & Briscoe, 2002). The long term consequences of intense selection be that due to changing market demands or a drive towards increased economic returns, are of great concern for many populations, both those under selection and those that are considered unfavorable for the market (Woolliams et al., 2005). This is not least due to the fact that intense selection leads to inbreeding and inbreeding has been shown to increase the risk of extinction in captive populations (Brook, Tonkyn, O´Grady & Frankham, 2002; Frankham et al., 2002). Loss of genetic diversity is often associated with inbreeding and reduction in reproductive fitness (Frankham et al., 2002; Willi, Buskirk & Hoffmann, 2006) and although there has been some disagreement regarding the importance of genetic factors in population extinctions (Frankham et al., 2002) it has been established that most species do not become extinct before genetic factors negatively affect them (Spielman, Brook & Frankham, 2004). This has been demonstrated to apply to species (Spielman et al., 2004) and there is no reason that it could not apply to individual populations within species as well. Many plant and animal species around the world are at risk of extinction, largely due to human activities (Lande, 1998). During the past fifteen years 300 of the 6000 farm animal breeds identified by the Food and Agricultural Organization (FAO) have become extinct and 1350 breeds are at risk of extinction in the near future. During this period, fourteen European goat breeds have become extinct (Taberlet et al., 2008).
Goats are one of the worlds most adaptable and widespread livestock species, and are one of the main economic recourses in many developing countries (Luikart et al., 2001). Fortunately, the market demand, at least in some parts of the world, are changing and the demand for specialty products (niche-products) is growing. This gives breeders of original/rare breeds an opportunity to expand the stock and preserve its genetic diversity. One of the primary goals in the management of animal populations is to maintain their genetic diversity at a high level and their inbreeding at a low level (Fernández, Villanueva, Pong-Wong & Toro, 2005). To estimate the future breeding potential of a livestock breed it is necessary to characterize the genetic structure and estimate the level of genetic diversity within the breed.
2.7 Genetic Identification of Goat Breeds and Measuring Genetic Variability
Very limited information on the genetic variability measurements and genetic differences of Bangladesh goat breeds exists. It is unclear as to whether different breeds exist or whether only different ecotypes or populations can be identified according to the areas where they occur. Such information will contribute to the preservation of the local breeds as an investment guaranteeing the potential use in future breeding programs.
The term ‘breed’ is not well defined. The animal-orientated definition recognizes that breeds differ by the totality of average differences observed in many quantitative and qualitative traits. The differences may overlap but they have a genetic basis and these differences taken together provide a unique description. (Meghen et al., www.fao.org/ag/AGa/AGAP/FRG/FEEDback/War/t1300b/t1 300b0j.htm)
2.8 Advantage of Genetic Characterization of Goat
The use of a genetic study to determine the genetic make-up of the breeds or populations in Bangladesh will contribute to information and the better understanding of goat genetic resources. The genetic characterization of these breeds would be a powerful tool for breed conservation and improvement. For the effective utilization of indigenous Bangladeshi goat genetic resources, it is necessary to genetically characterize the different populations. Such characterization would provide a database with information on the genetic variation between and among the goat populations in the country. It would also provide information as to which of the populations represent homogeneous breeds and which are genetically distinct. Further information will contribute to the determination of the risk status of the populations and breeds. Ultimately, the information would contribute to the understanding of the evolutionary history of goats in Bangladesh as well as to the future conservation and management of goat genetic resources.
Local livestock breeds are products of indigenous knowledge and should be regarded as national asset. However, due to indiscriminate breeding within the native stock as well as with exotic breeds, there is a marked decline in the population of unique animals conforming to the true attributes of native breeds. Awareness of the value of genetic resource has stimulated the study of genetic diversity of native breeds. Genetic variations that can be effectively measured within and between populations make the basis of breed characterization (Hetzel and Drinkwater, 1992). Therefore the detailed knowledge on genetic variation within and among different breeds is very important for understanding and developing endogenous economic traits of breeds (Yeo et al., 2000) and for optimizing breeding strategies and regulating germplasm conservation.
Genetic markers provide useful information at different levels: population structure, gene flow, phylogenetic relationship, patterns of historical biogeography and the analysis of parentage and relatedness (Feral, 2002). The development in DNA technologies have made it possible to uncover a large number of genetic polymorphism at the DNA sequence level and to use them as markers for evaluation of the genetic basis for observed phenotypic variability. The DNA markers possess unique genetic properties and methodological advantages that make them more useful and amenable for genetic analysis compared to other genetic markers. The DNA markers provide information on every region of genome at nucleotide level regardless of the level of gene expression and also remain unaffected by environmental and developmental changes. Molecular markers have been shown to be an efficient tool in quantification of genetic diversity of various populations (Saitbekova et al., 1999; Barker et al., 2001).
2.9 Techniques for Genetic Characterization of Goat
Several techniques have been developed to estimate the genetic variation or polymorphisms in populations and, hence, the genetic relationship amongst populations. Some major techniques with practical application are discussed below:
2.9.1 Random Amplified Polymorphic DNA (RAPD)
RAPDs are known as arbitrarily primed Polymerase Chain Reaction (PCR) (AP-PCR), or as a DNA Amplification Fingerprinting technique (DAF). This technique is based on the use of short, arbitrary primers in a PCR reaction and can be used to produce relatively detailed and complex DNA profiles for detecting amplified fragments between organisms. In the simplest format, only one short oligonucleotide consisting of eight to ten nucleotides in length is used. However, multiple primers are usually applied and a range of five to 21 nucleotides has proven successful if detection is coupled with polyacrylamide gel electrophoresis. Relaxed PCR conditions allow for multiple unspecific priming sites on opposite DNA strands, even if the match is imperfect. A successfully amplified template sequence will, however, only span from a priming site sequence to a nearby complementary sequence. Depending on the primer template combination and ratios, amplified products range from less than ten to over a 100. In this way, a spectrum of products characteristic for each template and primer combination is typically obtained and these can be adequately resolved and visualized using polycrylamide gel electrophoresis and silver staining. Agarose gel electrophoresis and ethidium bromide staining can also be used to detect only the major fragments (Van Marle-Köster, 2002).
2.9.1.1 Difference between RAPD and Standard PCR
Often, PCR is used to amplify a known sequence of DNA. Thus, the scientists chooses the sequence he or she wants to amplify, then designs and makes primers which will anneal to sequences flanking the sequence of interest. Thus, PCR leads to the amplification of a particular segment of DNA (Figure 2.2).
Figure 2.2: Standard polymerase chain reaction (PCR). Only the particular segment bracketed by a specific set of forward and reverse primers are amplified.
However, in RAPD analysis, the target sequence(s) (to be amplified) is unknown. The scientist will design a primer with an arbitrary sequence. In other words, the scientist simply makes up a 10 base pair sequence (or may have a computer randomly generate a 10 bp sequence), then synthesizes the primer. The scientist then carries out a PCR reaction and runs an agarose gel to see if any DNA segments were amplified in the presence of the arbitrary primer. Two important things for or PCR to occur: the primers must anneal in a particular orientation (such that they point towards each other); the primers must anneal within a reasonable distance of one another.
Figure 2.3: Random Amplified Polymorphic DNA (RAPD) reaction 1. The unknown segments are amplified by arbitrary primers. Here, the arrows represent multiple copies of a primer (all primers have the same sequence). The direction of the arrow also indicates the direction in which DNA synthesis will occur. The numbers represent locations on the DNA template to which the primers anneal. Primers anneal to sites 1, 2, and 3 on the bottom strand of the DNA template and primers anneal to sites 4, 5, and 6 on the top strand of the DNA template. In the above example, only 2 RAPD PCR products are formed: Product A is produced by PCR amplification of the DNA sequence which lies in between the primers bound at positions 2 and 5. Product B is the produced by PCR amplification of the DNA sequence which lies in between the primers bound at positions 3 and 6. No PCR product is produced by the primers bound at positions 1 and 4 because these primers are too far apart to allow completion of the PCR reaction. No PCR products are also produced by the primers bound at positions 4 and 2 or positions 5 and 3 because these primer pairs are not oriented towards each other.
Figure 2.4: Random Amplified Polymorphic DNA (RAPD) reaction 2. If another DNA template (genome) was obtained from a different (yet related) source, there would probably be some differences in the DNA sequence of the two templates. Suppose there was a change in sequence at primer annealing site 2. As shown in this figure, the primer is no longer able to anneal to site 2, and thus the PCR product A is not produced. Only product B is produced.
Figure 2.5: Agarose gel electrophoresis of Random Amplified Polymorphic DNA (RAPD) reactions. Here, Lane 1 contains molecular weight markers; Lane 2 and 3 contain products of RAPD reaction 1 and 2 respectively.
2.9.1.2 Advantages and disadvantages of RAPD
RAPDs have the advantage that they can be obtained at a reasonable cost and will generally amplify a range of fragments of most DNA and show polymorphisms. Certain primers will produce unrelated patterns between unrelated animals and identical ones for very closely related animals. Presumably, primer sites are randomly distributed along the target genome and flank both conserved and highly variable regions. Wide variation in band intensity can be shown to be reproducible between experiments, which could be the result of multiple copies of the amplified regions in the template or the efficiency with which particular regions are amplified. The polymorphic bands obtained from RAPDs can also be cloned for further analysis.
A major disadvantage is that the RAPDs are very sensitive to PCR conditions and this may lead to poor reproducibility (Van Marle-Köster, 2003). The consistency of results is not guaranteed as minor differences in experimental conditions can produce erratic results. Even under carefully controlled conditions, there can be ambiguity in the scoring of bands separated on a gel. Full understanding of the manner in which the genetic variation observed is generated and the reconstruction of evolutionary histories is difficult. RAPDs are dominant markers and heterozygosity can be scored as homozygosity, which affects the accuracy of the information content. In comparison to other genetic profiling techniques described in this study, the reliability of RAPDs is regarded as moderate (Van Marle-Köster, 2003).
2.9.2 Restriction Fragment Length Polymorphisms (RFLPs)
This technique relies on the amplification of variable regions of the target genome, with amplicons then being digested with one or more sequence-specific restriction enzymes. The DNA fragments of different lengths are then subjected to electrophoresis and fragments migrate according to their weights, the smaller fragments faster and the large fragments more slowly. Thus, RFLP generally refers to the differences in banding patterns obtained from DNA fragments, after sequence-specific cleavage with restriction enzymes (Van Marle-Köster, 2003). This technique can be applied to nuclear DNA or to mitochondrial DNA (also to chloroplast DNA in the case of plants). It has applications in the study of genetic distances, genetic variation, gene flow, effective population size, patterns of historical biogeography and analyses of parentage and relatedness. Since mutational events are generally the product of base substitutions, however, the rate of mutation is likely to be extremely low (10-7 to 10-8 per generation), and this results in a similar problem to that of proteins, which is, a lack of resolving power when dealing with very closely related groups. This has been demonstrated by Theilmann et al., (1989), who carried out a study of nine RFLPs in six breeds of cattle where only Brahman (Bos indicus) cattle differed significantly from the Bos taurus breeds (Meghen et al., www.fao.org /ag/AGa/ AGAP/FRG/FEEDback/War/t1300b/t1300b0j.htm – 32k).
2.9.3 Mitochondrial DNA
In animal cells, DNA is also found outside the nucleus in the mitochondria. Animal mitochondrial DNA can be easily isolated. It evolves five to ten times more rapidly than nuclear DNA and, a particular region, the D-loop, evolves even faster and is maternally inherited. Thus, mitochondrial phylogeny offers a relatively clear picture of the evolutionary history of a single genetic element. This strictly maternal inheritance of mitochondrial DNA can cause misinterpretation of the data and, consequently, the misreading of resultant phylogenies. Some mitochondrial DNA studies have been performed on goats, for example the study by Sultana and Tsuji (2003) on Pakistan goats.
2.9.4 Amplified Fragment Length Polymorphism (AFLP)
Amplified Fragment Length Polymorphism is a DNA fingerprinting technique that is based on the detection of DNA fragments, subjected to restriction enzymes, followed by selective PCR amplification. The DNA is cut with two restriction enzymes and double stranded adapters are then ligated to the ends of the DNA fragments to generate the template PCR. The specific adapter, ligated to the DNA fragment, determines the distribution of DNA restriction sites throughout the genome in question by DNA amplification. AFLP procedures can be manipulated to suit specific applications through the selection of the restriction enzymes and the design of the PCR primers. Typically, a rare-cutter restriction enzyme is combined to ensure the generation of small fragments (frequent-cutter) but to limit the number of fragments (rare-cutter) at the same time. PCR primers can be designed to have no selective bases on the 3´ends if the targeted templates are simple elements such as plasmids or bacterial artificial chromosomes. As in other techniques for fingerprinting, fragments are separated and analysed using gel electrophoresis. The AFLP technique can be performed at a reasonable cost, development costs are low but running costs are higher than for RAPD analysis but have the advantage of a higher reproducibility than RAPDs (Van Marle-Köster, 2003). This technique has also found application in limited genetic diversity studies of goats (Ajmone-Marsan et al., 2001; Crepaldi et al., 2001).
2.9.5 Microsatellites
Simple Tandem Repeats (STRs), or microsatellites, are a relatively new class of genetic marker. Microsatellites consist of tandem repeats of very short nucleotide motifs from one to six base pairs long, the dinucleotide repeat CA being the most common in mammalian genomes. A typical microsatellite locus may consist of a stretch of DNA with the base sequence CA repeated 12 times, i.e. (CA) 12. When the unique sequence flanking both ends of the repeated sequence is known, the microsatellite can be preferentially amplified using PCR. Different length classes (alleles) vary in the number of repeats and can be separated using polyacrylamide gel electrophoresis (PAGE). This class of marker is highly polymorphic by displaying many different alleles for a given locus. It is not uncommon to find up to ten alleles per locus and heterozygosity values of 60% in a relatively small number of samples (Goldstein & Polack, 1997).
Microsatellites tend to mutate with mutation rates up to 10-2 per generation (Van Marle-Köster, 2003). This means that, although new length classes are generated at a rate fast enough to allow for the distinction of breeds, the rate is not so fast that relationships are obscured by homoplasy (identity of alleles as a result of separate mutation events as opposed to common ancestry). A large number of microsatellite markers have been listed for various species that include cattle, horses, swine, sheep, goats, chickens, ducks, buffaloes, donkeys and camelids (FAO publication on Secondary guidelines, 2004). These markers are well dispersed through the genome and are applied in studies on genetic variability, parentage verification and genome mapping projects (Zamorano et al., 1998; Saitbekova et al., 1999, Gustavo et al., 2000; Martinez et al., 2000; Ritz et al., 2000; Mburu et al., 2003; Li et al., 2002; Van Marle-Köster, 2003). There are public-domain databases of accumulated sequence data, such as GenBank and EMBL.
Several studies using microsatellites specifically for goats have been conducted on Spanish, Asian, French, Italian, Chinese, Pakistan, Namibian, South African, Indian and sub-Saharan goats determining the genetic relationships between and among populations, genetic variation estimates and genetic diversity between populations (Tuñón, et al., 1989; Luikart et al., 1999; Yang et al., 1999; Ajmone-Marsan et al., 2001; Watts, Saitbekova et al., 1999; Barker, et al., 2001; Chenyambuga, 2002; Kim et al., 2002; Li et al., 2002; Sultana & Tsuji 2003; Kotzé et al., 2004, Els et al., 2004; Martinez et al., 2004; Visser et al., 2004 & Tilagan et al., 2006). The United Nation’s Food and Agriculture Organization on Farm Animal Genetic Resources published a document that evaluated the current status of molecular genetics research in different domestic animals (FAO Publication, 2004). This document concluded that microsatellite loci were the preferred marker for molecular genetic studies worldwide. Microsatellite data were applied in 66% of all genetic distance studies. Biochemical markers were the second most frequently applied technique with a representation of 34% in studies.
2.9.6 Detection of Genetic Variation at Protein Level
Initial identification of polymorphism relied on the study of physiological and biochemical variation at protein level that follow indirectly from variation in DNA sequence. This technique was widely used during the 1980s – 1990’s in population genetics. Research has been carried out worldwide using blood biochemical polymorphisms in order to study the genetic relationship between populations (Kidd, 1974; Tuñón, Gonzalez &Vallejo, 1989; Casati et al., 1999; Kotzé et al., 2000). Protein polymorphisms have proved to be a cheap and fast method of analysing single locus variation in breeds (Thairu-Muigai, 2002). Protein polymorphism studies, however, are now of limited value in the assessment of genetic variation as they detect relatively low levels of polymorphism, resulting in a lower resolving power for genetic characterization studies.
The occurrence of genetic polymorphisms in milk protein was first reported by Aschaffenburg and Drewry (1955). Using paper electrophoresis, they observed the existence of two distinct bands of b-lactoglobulin from individual bovine milk samples. A subsequent analysis demonstrated that these two types were under genetic control, determined by two autosomal codominant alleles (Aschaffenburg and Drewry, 1957). The paper electrophoresis technique was applied fo r the detection of genetic variants of a-lactalbumin (Blumberg and Tombs, 1958) and b-casein (Aschaffenburg, 1961). Variants of as1-casein were detected by starch- urea electrophoresis (Thompson et al., 1962), and the use of reducing agents in the sample buffer or in the gel contributed to the characterization of two k-casein variants (Neelin, 1964; Schmidt, 1964; Woychik, 1964). With the first milk protein (a-lactalbumin) totally sequenced (Brew et al., 1970), the amino acid sequences of the detected variants were subsequently determined in the 1970s. The introduction of analytical techniques with increased resolving power and sensitivity, such as polyacrylamide gel electrophoresis, isoelectric focusing, chromatography, and more recently capillary electrophoresis, enabled detection of more genetic variants in milk of different livestock species.
The main limitation of protein polymorphism detection methods is that are restricted to the resolution of only proteins with differing net charges. However, mutations with amino acid substitution that do not lead to a change in the net charge on proteins should occur in theory three times more frequently than those resulting in an alteration in the charge (Ng-Kwai Hang and Grosclaude, 1992). Furthermore, protein electrophoretic mobility is also affected by post-transcriptional modifications such as different degrees of phosphorylation and glycosylation of the protein.
2.10 Determination of Genetic Relationships between the Breeds
2.10.1 Genetic Distances
The genetic relationship between populations can be measured by determining the genetic distance between populations. This difference measured between two populations provides a good estimate of how divergent they are genetically. When the genetic distance is large, the genetic similarity is high and the time they diverged from each other is smaller (Thairu-Muigai, 2002). One of the common measurements of genetic distance in use today is Nei`s standard genetic distance (DS) (Nei, 1972) whose value is proportional to evolutionary time when the effects of mutations and genetic drift are taken into consideration. However, Nei (1983) noted that the modified Cavalli-Sforza and Edwards’ distance measure (DA) is more efficient in determining the true topology of an evolutionary tree’s being constructed using allele frequency data, especially if the populations are closely related. DA has also been reported to increase more slowly with time and maintain a linear relationship for longer periods of time (Nei, 1983; Thairu-Muigai, 2002).
2. 10.2 Construction of Phylogenies
Phylogenetic analysis of populations has become an important tool for studying the evolutionary relationship of populations. It offers a simple graphic aid for visualizing the relationship between the populations, hence making interferences on the evolutionary histories easier (Thairu-Muigai, 2002). The phylogenetic relationship of populations that were under investigation in this study was constructed using the neighbour-joining method in DISPAN to construct a phylogenetic tree from DA and DS distance measurements (TREEVIEW) Bootstrap test with 1000 replicates (Ota, 1993).
2. 10.3 Genetic Differentiation
The understanding of genetic structuring or differentiation within a population is of interest to geneticists because it reflects the number of alleles exchanged between populations that influence the genetic composition of individuals within these populations. Gene flow between populations determines the effect of selection and genetic drift generates new polymorphisms and increases the local effective population size. The Fst and Coefficient for genetic differentiation (Gst) are very commonly used to describe population differentiation (Thairu-Muigai, 2002).
2.11 Previous studies on genotypic characterization of Goat breed of countries other than Bangladesh
Several researchers reported about the molecular characterization of different goat breeds of different part of the world. Summary of some of the previous works are given below:
Studies conducted in Sudan by Gaali and Satti (2009) reported the genetic variations among 14 individuals of goat (Capra hircus) from two domestic Sudanese goat breeds (Nilotic and Nubian) by using RAPD analysis. The test generated 59 entirely repeatable RAPD fragment bands and the statistical analysis showed 55 polymorphic bands among the 14 individuals. The genetic distances among the population were found 8 to 72%. The highest dissimilarity coefficient was between individuals within the Nilotic breeds while there was a comparatively low degree of differentiation among the Nubian population. The constructed UPGMA dendrogram of the coefficient of similarity showed that the Nubian clustered together while the individuals from the Nilotic form 4 groups. From their study this group reported that the link between the individual of the Nilotic is quite weak and some of them linked to the Nubian. The results of the study offer useful information about some Sudanese goat breeds.
Oliveira et al (2005) verified the genetic diversity between and within seven populations of Moxotó goat (n = 264) from the States of Pernambuco, Paraíba and Rio Grande do Norte, using RAPD (Random Amplified Polymorphic DNA). Moxotó, as well as other naturalized breeds, suffers genetic losses due to the indiscriminate miscegenation with breeds raised in the Northeast Region of Brazil. The analysis of molecular variance (AMOVA) showed that the greater part of total genetic variability (71.55%) was due to differences between individuals within populations, while 21.21% was among populations. The analysis of variance among the pairs of populations demonstrated that the populations located in Floresta, PE x Angicos, RN presented a smaller value of intrapopulational differentiation (8.9%), indicating low genetic variability among them. Nei’s genetic distances varied between 0.0546 and 0.1868 in the populations. The dendrogram generated showed that the Canindé breed, used as outgroup, clustered with the populations of Moxotó, indicating a possible common origin of the naturalized goat breeds.
Dixit et al (2008) reported about the goat breed named Kutchi which is an important dual-purpose (meat and milk) goat breed found in Banaskantha, Patan and Kutch districts of Gujarat, India. Genetic diversity within the breed was investigated using 25 microsatellite molecular markers. The PCR products were run on automated DNA sequencer for allelic differentiation at 25 microsatellite loci. The average number of alleles observed across the studied microsatellite loci was 12.0 ± 1.02. The average expected gene diversity within the population was 0.79 ± 0.02, whereas observed heterozygosity was 0.59 ± 0.06. Thirteen out of the total 25 studied loci showed significant deviations from Hardy–Weinberg equilibrium. The Fis (inbreeding) value was 0.23 ± 0.07. The genetic differentiation among sub-populations of this breed was low (Fst = 0.05 ± 0.01). The Sign and Wilcoxon tests detected significant departure from mutation drift equilibrium in the population at most of the studied loci. Finally, appropriate breeding strategies for its conservation and improvement of its unique attributes like adaptability and fitness under harsh climatic conditions of the arid/semiarid zone were warranted in this study.
Rekik et al (2010) reported about the genetic characterization of three ovine breeds in Tunisia using randomly amplified polymorphic DNA markers. The breeds were: Barbarine, Queue Fine de l’Ouest and D’man. The DNA samples were isolated from 160 animals from the three breeds. Twenty random primers were used for their study. Only from 9 primers they found clearly polymorphic and reproducible bands. Level of polymorphism varied from 71.42 to 88.88 per primer and from 75.43 to 83.02 per breed. Genetic variation in studied breeds was measured with three indices (Nei’s gene diversity, Shannon’s information and level of polymorphic loci) and showed that the highest diversity was found in the exotic D’man breed while the lowest diversity was obtained for both the Barbarine and Queue Fine de l’Ouest native breeds. The inter breed similarity indices and the UPGMA dendrogram, based on genetic distance clearly separated the three breeds. The closest relationship was observed between Tunisian Barbarine and Queue Fine de l’Ouest breeds. The AMOVA analysis indicated that the largest part of the genetic variability (97.86%) originated from differences among individuals within breeds.
Sztankoova et al (2007) reported about the Genetic polymorphism at the CSN1S1 gene in two Czech goat breeds. The genetic polymorphism of the CSN1S1 (casein alpha-S1) locus was investigated in two endangered Czech goat breeds (White Shorthair and Brown Shorthair). These breeds are kept mainly for their good dairy performance. Genetic characterization of the CSN1S1 locus contributes to the knowledge of the genetic structure of these two endangered breeds. The study was performed on 498 goats (333 White and 165 Brown Shorthair goats) by means of different polymerase chain reactions (PCR). They detected A* (associated with normal content of protein), E, F and 01 alleles. The analysis showed a prevalence of CSN1S1 F (0.658; 0.597) and CSN1S1 A* (0.269; 0.303) alleles. In both breeds, the frequency of occurrence of E and 01 alleles was very low: E (0.054; 0.085) and 01 (0.019; 0.015), respectively. No population followed the Hardy-Weinberg equilibrium, the value of polymorphic information content (PIC) being 0.426 in White and 0.472 in Brown Shorthair goats. Moreover, the test of population differences (P = 0.130) showed no significant differences between White and Brown Shorthair goats. This study was important for the preservation of the population of both breeds.
2.12 Previous Studies on Genotypic Characterization of Goat Breeds of Bangladesh
Polymorphism of the blood proteins polymorphism of Bangladeshi goats was studied by Kutsumata and co-workers (1984). Out of 33 blood protein and enzyme loci examined, they found polymorphism at 7 loci: Hb-II, Tf, Alp, PA-3, Amy, MDH and LDH-A. They concluded that Black Bengal and Jamnapari goats had the same evolutionary origin and classified them as Indian goats.
Rahman et al. (2006) reported about the genetic characterization by RAPD analysis of the Black Bengal and Jomuna pari goat breed of certain areas of Bangladesh. They observed the DNA extracted from 14 goat breeds by gel electrophoresis. Eight goat specific primers were synthesized by ASM-800 DNA synthesizer and screened in the study and all these primers were capable of priming polymorphic amplification pattern in both the breeds. Random amplification of polymorphic DNA–Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 14 Black Bengal goat and Jamunapari goat breeds. Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. Data were analyzed by using a computer programe POPGENE (Version 1.31).
3.1 Location of the Study Area
Three areas namely Narsingdi, Kishoregonj and Dhaka (Savar) were selected (Figure 3.1). A total number of 21 Black Bengal healthy goats 7 from each of the selected area were considered. Among the selected goats 70% were male and 30% were female.
Figure 3.1: Sites of sample collection. Arrow marks show the regions.
3.2 Phenotypic Characteristics of Black Bengal Goat
The Black Bengal goat breeds were phenotypically identified by the following points: Body length (Distance between the middle point of horn and the point hips), ear length, horn length, fore leg and hind leg length.
3.3 Collection of blood samples
Blood samples of goats were investigated to analyze genetic variations. A total of 21 blood samples were collected from three different selected goat populations. At least 5ml of fresh blood sample was collected from each of the animal aseptically by puncturing jugular vein in EDTA containing Vacutainer. The blood was gently mixed with anticoagulant and the vial was marked with number and sex and kept on ice to maintain low temperature in order to prevent cell lysis. Subsequently the blood samples were transported to the laboratory and stored at –20 °C until the isolation of genomic DNA. The research was conducted in the Biotechnology and Genetic Engineering Laboratory under Biotechnology Division of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh for a period from June 2010 to December 2010.
Equipments and Chemicals Employed:
Needle
EDTA containing vacuum vial
Tube rack
Cotton swab with Ethanol
Ice-box
Apron
Marker pen
3.4 DNA Extraction
DNA was extracted from whole blood following the protocol of Roe et al. 1996, namely Standard Saline Citrate Buffer method. The detail of the protocol is described below:
3.4.1 Materials and Machineries Employed
Micropipette 1000µl, 100µl, 2.5µl
Micro tips 1000µl, 100µl, 2.5µl
Tip boxes
Microcentrifuge tubes
Vortex mixture
Microcentrifuge machine
Tube racks
Beaker 250ml, 500ml
Incubator
Deep freezer (-20 ºC)
Water bath
3.4.2 Chemicals Needed
0.2 M Na-acetate
10% SDS
Proteinase – K
Ethanol – 100% and 70%
Phenol Chloroform Isoamyl Alcohol (PCIA) solution
2 M Na-acetate
TE buffer solution
3.4.3 Methods of DNA Extraction
Day – 1
Blood samples were taken from the refrigerator and kept at room temperature overnight.
Day – 2
The blood samples were homogenously mixed by inverting the vial several times and 400 µl blood samples was taken in a microcentrifuge tube with the help of 1500 µl micropipette.
800 µl of SSC buffer solution was added in each tube and homogenized by mixing with vortex machine.
The mixture was centrifuged at 13,000 rpm for 5 minutes and supernatant was discarded.
Further 1000 µl of SSC buffer solution was taken in each tube and homogenized by vortex machine.
The mixture was then centrifuged at 13,000 rpm for 5 minutes and supernatant was discarded.
375µl 0.2 M Sodium acetate was added in each and homogenized by vortex machine.
Then 25µl 10% SDS and 5µl Proteinase-K was added in each tube and homogenized by vortex machine.
Then the tubes were incubated at 55 °C temperature for 1 hour.
Then 20µl PCIA solution was added and mixed by vortex machine. Then centrifuged at 13,000 rpm for 5 minutes and aqueous layer was removed in new microcentrifuge tube.
1000µl 100% ethanol was added in each new microcentrifuge tube and homogenized by mixing with vortex machine.
The mixture was then centrifuged at 13,000 rpm for 4 minutes and supernatant was discarded.
180µl TE (10:1) buffer was added and homogenized by mixing with vortex machine. Then incubated at 55 °C temperature for 10 minutes.
20µl 2 M sodium acetate was added and the mixture was then centrifuged at 13,000 rpm for 4 minutes and supernatant was discarded.
Then 1000µl 70% ethanol was added and the mixture was then centrifuged at 13,000 rpm for 4 minutes and supernatant was discarded.
Then the pellet was dried in DNA concentrator for 15 minutes to remove alcohol and resuspended the pellet by adding