Abstract: In genetic analysis, various types of genetic markers such as morphological, chromosomal, biochemical and molecular markers are used in buffalo production. Mainly the molecular markers, (hybridization-based markers and PCR-based markers) have tremendous uses which are capable of pinpointing genes and traits and diversity and evolution at the genetic level. In recent years, the demonstration of genetic polymorphism at the DNA sequence level has provided a large number of marker techniques with variety of applications. Genetic marker is a potential tool to geneticists and breeders to evaluate the existing germplasm and to manipulate it to create animals as desired and needed by the society. Now, genetic marker has brought a new dimension to animal breeding, especially in direct identification of the gene. This review focuses on application of different genetic markers in buffalo production.

INTRODUCTION

In recent years, various types of genetic markers such as morphological, chromosomal and biochemical and molecular markers are used to increase the productivity of animals. Morphological (e.g., pigmentation or other features) and chromosomal (e.g., structural or numerical variations) markers usually show low degree of polymorphism and hence are not very useful for genetic markers. Biochemical markers have been tried out extensively but have not been found encouraging as they are often sex limited, age-dependent and are significantly influenced by the environment. Sometimes, the various genotypic classes are indistinguishable at the phenotypic level owing to dominance effect. Furthermore, these markers reflect variability in their coding sequences that constitute <10% of the total genome. The molecular markers, capable of detecting the genetic variation at the DNA sequence level have not only removed these limitations but also possess unique genetic properties that make them more useful than other genetic markers. Moreover, they are numerous and distributed ubiquitously throughout the genome. These follow a typical Mendelian inheritance which usually expresses in a co-dominant fashion and are often multiallelic giving mean heterozygosity of >70%. They remain unaffected by the environmental factors and generally do not have pleiotropic effects on Quantitative Trait Loci (QTL). Since, gene expression is not a prerequisite, virtually the entire genome including the noncoding regions can be visualized. Genetic markers by definition are stable inherited variations that can be measured or detected by a suitable method and can be used to evaluate the specific genotype or phenotype other than itself. These markers are numerous and distributed ubiquitously throughout the genome. In some cases, genetic markers are breed-specific but more often although, there are breed overlaps in genetic-marker existence, there are marked differences in the frequencies of the controlling genes. Genetic marker based on unique sequence DNA mutation located within protein coding regions have long been used in genetic population studies with most of the markers being biochemical polymorphisms identified on the basis of electrophoretic differences (Baker and Manwell, 1977). Recent developments in DNA technologies have made it possible to uncover a large number of genetic polymorphisms at the DNA sequence level and to use them as markers for evaluation of the genetic basis for the observed phenotypic variability. Since, the 1st demonstration of DNA-level polymorphism known as the Restriction Fragment Length Polymorphism (RFLP) (Grodzicker et al., 1974), an almost unlimited number of molecular markers have accumulated. Currently, more powerful and less laborious techniques to uncover new types of DNA markers are steadily being introduced. The introduction of Polymerase Chain Reaction (PCR) (Saiki et al., 1988) in conjunction with the constantly increasing DNA sequence data also represents a milestone in this endeavour. The present review focuses on genetic markers and their various uses in buffalo production.

Genetic marker: Genetic marker can be defined as any stable and inherited variation that can be measured or detected by a suitable method and can be used subsequently to detect the presence of a specific genotype or phenotype other than itself which otherwise is non measurable or very difficult to detect. Such variations occurring at different levels, i.e., at the morphological, chromosomal, biochemical or DNA level can serve as the genetic markers (Mitra, 1994).

Molecular markers: The markers revealing variations at DNA level are referred to as the molecular markers. Molecular markers are found at specific locations of the genome. They are used to flag, the position of a particular gene or the inheritance of a particular characteristic.

Classification of molecular marker: The markers revealing variations at the DNA level are referred to as the molecular markers and on the basis of techniques used for their detection, these have been classified into two major categories: hybridization-based markers and PCR-based markers.

The hybridization-based markers: These include the traditional Restriction Fragment Length Polymorphism (RFLP) analysis (Botstein et al., 1980) as well where in appropriately labeled probes for the genes of importance (e.g., cDNA or genomic DNA sequences) are hybridized on to filter membranes containing Restriction Enzyme (RE)-digested DNA separated by gel electrophoresis and subsequently transferred onto these filters by Southern blotting.

The polymorphisms are then visualized as hybridization bands. The individuals carrying different allelic variants for a locus will show different banding patterns. Hybridization can also be carried out with the probes (e.g., genomic or synthetic oligonucleotide) for the different families of hypervariable repetitive DNA sequences namely, minisatellite (Jeffreys et al., 1985), simple repeats (Ali et al., 1986), Variable Number of Tandem Repeats (VNTR) (Nakamura et al., 1987) and microsatellite (Litt and Luty, 1989) to reveal highly polymorphic DNA Fingerprinting Patterns (DFP).

The PCR-based markers: These have however, removed the necessity of probe-hybridization step and have led to the discovery of several useful and easy-to-screen methods. Depending on the type of primers (i.e., primers of specific sequences targeted to a particular region of a genome or primers of arbitrary sequences) used for PCR, these markers can be further sub-divided into the following 2 groups:

The sequence-targeted PCR assays: In this assay system, a particular fragment of interest is amplified using a pair of sequence-specific primers. In this category, PCR-RFLP or Cleaved Amplified Polymorphic Sequence (CAPS) analysis is a useful technique for screening of sequence variations that give rise to the polymorphic RE sites. Such analysis involves amplification of a specific region of DNA encompassing the polymorphic RE site and digestion of the amplified DNA fragment with the respective RE.

However for the screening of the sequence variations that do not lead to creation or abolition of restriction sites, other approaches namely allele specific PCR (AS-PCR) (Nichols et al., 1989), PCR Amplification of Specific Alleles (PASA) (Sarkar et al., 1990), Allele Specific Oligonucleotide (ASO) hybridization assay (Saiki et al., 1986), Amplification Refractory Mutation System (ARMS) (Newton et al., 1989) and Oligonucleotide Ligation Assay (OLA) (Landegren et al., 1988) are used. These assays are based on the principle of high specificity of PCR to selectively amplify specific alleles using primers that match the nucleotide sequence of one but mismatch the sequence of other allele. The sequence-targeted PCR approach is also employed to reveal Simple Sequence Length Polymorphism (SSLP), using a pair of primers that flank the Simple Sequence Repeat (SSR) motifs. If cloned and sequenced microsatellite loci can be subjected to PCR amplification and such microsatellite loci can be recovered by PCR such loci are termed as Sequence Tagged Microsatellite Site (STMS) (Beckmann and Soller, 1990) markers.

The arbitrary PCR assays: In this assay system, however unlike the standard PCR protocol, randomly designed single primer is used to amplify a set of anonymous polymorphic DNA fragments. It is based on the principle that when the primer is short (usually 8-10 mm) there is a high probability that priming may take place at several sites in the genome that are located within amplifiable distance and are in inverted orientation. Polymorphism detected using this method is called Randomly Amplified Polymorphic DNA (RAPD) (Williams et al., 1990). Based on this principle, several techniques which do not require any prior sequence knowledge have been developed. However, they differ in number and length of primers used, stringency of PCR conditions and the method of fragment separation and detection. In Arbitrary Primed PCR (AP-PCR) (Welsh and McClelland, 1990), slightly longer primer is used (e.g., universal M13 primer) and amplification products are detected by radiactive or nonradioactive method following polyacrylamide gel electrophoresis.

In DNA Amplification Fingerprinting (DAF) (Caetano-Anolles et al., 1991) analysis, shorter primer is used (5-8 mm) which reveals relatively greater number of amplification fragments by polyacrylamide gel electrophoresis and silver staining. All these techniques having similar features can be described by a common term Multiple Arbitrary Amplicon Profiling (MAAP) (Caetano-Anolles et al., 1992).

Besides these, a number of modifications of the basic MAAP assays (namely, template endonuclease cleavage MAAP and RAPD-RFLP) have been developed as well.

In addition to arbitrary primers, semi-arbitrary primers designed on the basis of RE sites or sequences that are interspersed in the genome such as repetitive sequence elements (Alu repeats or SINEs), microsatellites and transposable elements are also used. In the Amplified Fragment Length Polymorphism (AFLP) assay (Zabeau, 1993), template DNA is digested with two REs and the resulting restriction fragments are then ligated with adapters and subsequently, PCR amplification is carried out using specially designed primers which comprise; a unique part corresponding to selective bases and a common part corresponding to the adapters and the RE site. Microsatellite-Primed PCR (MAP-PCR) assay is carried out using microsatellite as the primer.

Different markers and their application in buffalo production: There are several genetic markers used in buffalo populations pointing genes and traits and monitoring diversity and evolution at the genetic level. These have been described in subsequent sections.

Blood protein polymorphism: Blood protein polymorphism was used extensively for Parentage test, breed identification and phylogenic study of domestic animals during the 60 and 70’s decay of last century. In buffaloes, blood protein was also used for breed identification and phylogenic studies by a number of investigators.

Giri and Pillai (1956) from India were the 1st to report blood protein polymorphisms in domestic water buffaloes. Since then, many scientists from different parts of the world especially Australia, India (Khanna, 1969, 1973; Naik et al., 1969; Basavalah, 1970), apan (Amano et al., 1980, 1981, 1986, 1992, 1998), Italy (Masina et al., 1971) and Bulgaria (Makaveev, 1968, 1970) reported about blood protein polymorphisms of domestic water buffaloes (Table 1).

Though, there is variation in methods and nomenclatures used, nevertheless most of the scientists reported about polymorphic loci for blood proteins and enzymes of domestic water buffaloes.

Table 1:	List of blood protein examined electrophoretically
*Starch Gel electrophoresis (SG); *Polyacrylamide Gel (PAG); *Isoelectric Focusing (IEF)

Chromosome polymorphism: Prakandze was the 1st to research with buffalo cytogenetics in Asia giving a brief account on the chromosome number. Later, Makino (1944) reported correctly the chromosome number of swamp buffalo of Taiwan and he mentioned it as 48. Ulbrich and Fischer (1966) were the 1st to report about the details of buffalo karyotypes.

Since, then many researchers in Japan (Amano et al., 1983, 1987), India (Gupta and Chaudhury, 1978; Chakrabarti and Benjamin, 1980; Yadav and Balakrishnan, 1982), Sri Lanka (Scheurmann et al., 1974), Malaysia (Harisah et al., 1989), China (Youjun et al., 2001) and Thiland (Chavananikul, 1989) have conducted research on cytogenetics of Asian buffaloes. From their studies, it now clear and well established that that river buffaloes and swamp buffaloes differ in their karyotypes.

The diploid chromosome number of river buffaloes is fifty. Their karyotypes consists of five pairs of submetacentric chromosomes, ninteen pairs of acrocentric chromosomes and a pair of acrocentric sex chromosomes.

On the other hand, karyotypes of swamp buffaloes consists of a single pair of metacentric chromosomes, four pairs of submetcentric chromosomes and eighteen pairs of acrocentric chromosomes and a pair of acrocentric sex chromosome. The cross between two types produces a number of chromosome polymorphisms in F₁ and F₂. The details of chromosome polymorphisms have been shown in Table 2.

Mitochondrial DNA (mtDNA) polymorphism: A survey of two types of water buffaloes revealed distinct lineages of mtDNA. The sequence divergence between swamp and river buffaloes was similar to the relationship estimated from RFLP analysis of the entire mtDNA (Tanaka et al., 1995). Genetic diversity among swamp and river buffaloes was studied by Amano et al. (1984) in Bangladesh by DNA sequences analysis of the mitochondrial gene for cytochrome b, the results showed that each of the 2 groups has mitochondrial DNA (mtDNA) with specific cytochrome b haplotype.

Table 2:	Chromosome number of the offspring produced from different types of mating between swamp and river buffaloes

The pairwise nucleotide sequence divergence was calculated to be 2.67% between swamp and river buffaloes, suggesting that they might have diverged from the ancestral populations of the Asiatic domestic water buffaloes, approximately 1 million years ago. Mitochondrial DNA is maternally inherited. Changes in the nucleotide sequence of mtDNA occur faster than nuclear DNA (Faruque, 2003).

Amano et al. (1994) reported the genetic variants of ribosomal DNA and mtDNA between river buffaloes and swamp buffaloes and concluded that only mtDNA provides adequate means for classification of water buffaloes. So, analysis of mtDNA provides useful information regarding relationships between and within populations/breeds/species (Faruque, 2003). Bhat et al. (1990) described the cleavage pattern of mtDNA of Indian Murrah buffaloes.

Out of thirteen restriction endonuclease enzymes tested only one enzyme viz., Bg L^-1 had polymorphic sites. Hu et al. (1997) described the restriction endonuclease patterns of mtDNA of three local types buffalos of Yunnan province of China. They tested 18 enzymes and identified polymorphisms for BamHI, EcoRI and ScaI sites. From their result, they concluded that Chinese water buffaloes belong to swamp type with genetic variance among different local buffalo populations.

The finding of Bhat et al. (1990) and Hu et al. (1997) agree to the findings of Tanaka et al. (1995). Tanaka et al. (1995) used fifteen enzymes for swamp buffaloes and river buffaloes. They identified five restriction cleavage sites two for river buffaloes and three for swamp buffaloes. The additional site they mentioned is PstI. The restriction endonuclease cleavage analysis of mtDNA makes it clear that genetic variability exists in different buffalo populations of Asia.

Microsatellite marker maps/Short Tandem Repeats (STR): Microsatellites, alternatively known as Simple Sequence Repeats (SSRs), Short Tandem Repeats (STRs) or Simple Sequence Length Polymorphisms (SSLPs) are tandem repeats of sequence units generally <5 bp in length, e.g. (TG)n or (AAT)n. Micro satellite are sequences characterized by the tandem repetition of short motifs from 1-4 base pair in length which is why they are also referred to as Short Tandem Repeats (STR). Bovine autosomal microsatellite markers were tested for Polymerase Chin Reaction (PCR) amplification in African buffalo for population genetic studies. Amplification was observed for 139 markers (83%) and 101 markers were studied further with 91 (90%) being polymorphic.

Considering the overall high level of polymorphism, it was concluded that most bovine microsatellite markers are applicable in African buffalo (Van Hooft et al., 1999).

RFLP studies in buffaloes: The wish to improve disease resistance has been primary impetus for Major Histocompatibility Complex (MHC) research in farm animals as numerous associations between MHC polymorphism and disease susceptibility have been found.

Buffalo Lymphocyte antigen (BuLa), the MHC of buffalo has been analyzed by RFEL technique employing Pst, Pvull, HindIII and MspI restriction enzymes and human class II a-chain gene (HLA-DRA) c DNA probe. The hybridization of buffalo genome revealed the existence of homologous sequences and corresponding region in buffaloes.

Highly polymorphic pattern was revealed by using Msp I restriction enzyme whereas with other restriction enzyme a non polymorphic pattern was observed (Kumar et al., 1993). RFEL studies on Growth Hormone (GH) and Prolactin (PRL) loci in Murrah and Nili-Ravi buffaloes indicated that both the breeds were homozygous for GH and PRL loci suggesting that monomorphism, at these 2 loci may be characteristic feature of buffaloes (Mitra et al., 1995).

Chikuni et al. (1994) amplified buffalo genomic DNA by PCR using primers based on the nucleotide sequences of k-casein gene. Sequencing of the portion of the amplified gene from exon 3-4 revealed polymorphism at nucleotide position 516, resulting in glutamine/aspertic acid in the casein precursor.

Mitra et al. (1998) also using PCR/RFEL amplified a 379 bp fragment of buffalo Casein CSN3 gene and reported one polymorphism at codon 135 (Thr-ACC-IIe ATC) in buffaloes kappa casein gene. The frequency of 135 The/IIe alleles was estimated to be 0.88 and 0.12, respectively.

The polymorphism of MHC class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene were amplified and characterized. The MHC of buffalo has been mapped to chromosome 2 p (Iannuzzi et al., 1993a). Compared to other ruminant species, buffalo MHC loci are poorly characterized. Only PCR-RFLP types have been investigated in two Indian buffalo breeds (Aravindakshanet al., 2000).

Random Amplified Polymorphic DNA (RAPD): More recently, the Random Amplified Polymorphic DNA (RAPD) technique has emerged as another means for studying genetic variation at the population and species level (Welsh and McCelland, 1990). RAPD markers are based on amplification of genomic DNA by PCR using short primers homologous to random target sites in the genome.

Polymorphisms can be simply identified as the presence or absence of an amplified product on ethidium bromide stained agarose gels. RAPD may be used to detect DNA variability at different levels, ranging from single base changes to deletions and insertions (Williams et al., 1990).

Rao et al. (1996) used this technique to reveal polymorphism in buffalo and cattle genomic DNA. They used 14 random primers to amplify DNA fragments. Whereas high level of polymorphism was observed between species within species variation was less.

Gene mapping: Molecular markers have three-fold applications in gene mapping: a marker allows the direct identification of the gene of interest instead of the gene product and consequently, it serve as a useful tool for screening somatic cell hybrids use of several DNA probes and easy-to-screen techniques, a marker also helps in physical mapping of the genes using in situ hybridization and the molecular markers provide sufficient markers for construction of genetic maps using linkage analysis.

Genetic maps are constructed on the basis of two classes of molecular markers: type I markers that represent the evolutionary conserved coding sequences (e.g., classical RFLPs and SSLPs) are useful in comparative mapping strategies where polymorphism is not an essential prerequisite. However, these are mostly Single Locus and Di-Allelic (SLDA) and thus are not useful for linkage analysis. On the other hand, the type II markers (like microsatellites markers) have higher polymorphism information content (PIC, a measure of the usefulness of a marker for linkage studies) than conventional RFLPs and can be generated very easily and rapidly. A beginning in gene mapping in buffaloes has already been made through the pioneering work of Iannuzi and his associates in Italy. A few genes have been mapped recently on specific chromosomes of buffaloes using the techniques like somatic cell hybridization and In situ Hybridization (ISH). Present status of gene mapping in buffaloes has been shown in Table 3.

Table 3:	Status of physical gene map in buffaloes

CONCLUSION

The genetic improvement of animals is a continuous and complex process. Ever since, the domestication of animals by man, he has always remained busy in improving his animals. In this pursuit, many methods have been developed and tested. In recent years, the demonstration of genetic polymorphism at the DNA sequence level has provided a large number of marker techniques with variety of applications. This has in turn prompted further consideration for the potential utility of these markers in animal breeding. However, utilization of marker-based information for genetic improvement depends on the choice of an appropriate marker system for a given application. Selection of markers for different applications are influenced by several factors, viz., the degree of polymorphism skill or expertise available, possibility of automation, radioisotopes used reproducibility of the technique and finally the cost involved. Presently, the pace of development of molecular markers is tremendous and the trend suggests that explosion in marker development will continue in the near future in buffalo population.

NOMENCLATURE