During the past three decades, the development of molecular genetics, especially DNA based markers has an important role in the field of animal genetics and breeding. Instead of conventional breeding program through selection alone, utilization of molecular genetic tools can ease us in optimizing the animal breeding program. Selection according to genotype has become an important tool in the breeding of farm animals. Molecular markers, revealing polymorphisms at the DNA level are the powerful tools to study the variation. These molecular markers have wide range of applications in population genetics, conservation biology and evolutionary biology. This review gives a brief summary on the characteristics of different molecular markers including hybridization and PCR-based markers.
The main focus on the activities of animal breeding started changing from quantitative to molecular genetics in the 1990s. In the past, selective breeding is the main focus of conventional breeding system, which includes progeny testing and various selection programs. In order to optimize the animal breeding program, it is important to add molecular genetic techniques along with the conventional animal breeding methods. During last three decades, 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 observed phenotypic variability and makes a vital part in animal breeding.
A genetic marker can be defined as any stable and inherited variation which is measurable or detectable 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. This kind of variations will be seen with markers at different levels, like morphological, biochemical, chromosomal and molecular level. There are some disadvantages with genetic markers other than molecular markers because of their low degree of polymorphism and influence of environment. The markers capable of revealing the genetic variation at the DNA sequence level are referred to as molecular markers. Their unique genetic properties like numerous in nature, ubiquitous distribution in the genome, selective neutral behavior, easy and fast assay, high reproducibility and co-dominance inheritance make them useful than other genetic markers. The present review summarizes the basic characteristics of molecular markers and their applications.
Molecular markers are-
Restriction Fragment Length Polymorphisms (RFLPs)
RFLP was the first DNA hybridization based molecular marker technique which is initially used by Grodzicker et al., 1974, for the physical mapping of temperature-sensitive mutations of adenoviruses. Later in 1980, this method was established by Botstein et al. for constructing genetic linkage map in humans. This technique was based on the function of restriction endonucleases which cleaves the DNA at specific nucleotide sequences. This procedure involves the digestion of genomic DNA with known restriction endonucleases and these digested fragments are separated and visualized using gel electrophoresis. Further these fragments are subjected to southern hybridization using radioactive or chemiluminescent probes and visualized by autoradiography (Drinkwater and Hetzel, 1991; Smith and Smith, 1993; Bishop et al., 1995).
The polymorphisms are determined by the number and the varying lengths of these DNA fragments, implying that the restriction endonuclease cut the DNA at unrelated locations (Mburu and Hanotte, 2005). The molecular basis of RFLP is that nucleotide base substitutions, deletions, insertions, duplications and inversions within the whole genome can abolish or create a new restriction endonuclease recognition site.
RFLPs are co-dominant in nature and can be mapped using linkage analysis, but they have some disadvantages like time consuming, labor intensive and are inconvenient for high throughput screening (Mburu and Hanotte, 2005).
Randomly Amplified Polymorphic DNA (RAPD)
RAPD is a PCR based molecular marker developed independently by two different laboratories (Williams et al., 1990; Welsh and McClelland, 1990). This technique uses short, arbitrary primers (̴ 10 bp length) that can bind to many places in the genome hence also called as arbitrarily primed PCR (AP-PCR). Target DNA was randomly amplified by PCR using these arbitrary primers (DNA amplification fingerprinting technique-DFP) (Caetano-Annoles et al., 1991) and the amplified fragments are generally separated on polyacrylamide gel electrophoresis and visualized using silver staining. The polymorphisms are detected as the presence or absence of bands of a specific size. This technique had also used for the conservation of endangered species and genetic diversity analysis (Yu and Paul, 1992; Mackill, 1995; Cao and Oard, 1997).
No prior knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. There are some disadvantages with these markers- detection of polymorphism is limited, dominant markers (difficult to distinguish between homo and heterozygotes); very sensitive to PCR conditions (Munthali et al., 1992; Lowe et al., 1996) and this may lead to poor reproducibility.
Amplified Fragment Length Polymorphism (AFLP)
AFLP is a novel molecular fingerprinting technique with the combination of RFLP and PCR, developed by Zabeau and Vos in 1993 (Zabeau and Vos, 1993; Vos et al., 1995). The technique is based on the selective PCR amplification of restriction fragments from a total restriction enzyme digested genomic DNA. Genomic DNA was digested with restriction enzymes and the fragments were ligated to synthetic adaptors and further, amplified with specified primers (complementary to a selective sequence on the adaptors). Amplified fragments are separated by a denaturing polyacrylamide gel and visualized by autoradiography or silver staining (Blears et al., 1995).
AFLPs are considered as the “gold standard” for molecular epidemiological studies of pathogenic microorganisms and it is also widely used in forensic science (Mburu and Hanotte, 2005). It is an ideal molecular approach for population genetics and genome typing. These markers overcome the labor-intensive, time consuming limitations of RFLP method and solve the reliability problem caused by non-specific amplifications in RAPDs. Like RAPD, AFLP also does not require any prior knowledge of the sequence and it detects the greater number of loci than RAPD does. AFLPs are notable for their genetic stability, they provides an effective, rapid, reproducible and economical tool for detecting a large number of polymorphic genetic markers that can be genotyped automatically (Vos et al., 1995; Vos and Kuiper, 1997). But these are dominant bi-allelic markers (Paglia and Morgante, 1998), expensive and technically demanding.
The term microsatellite was coined by Litt and Luty (1989) to characterize the simple sequence repeats. Microsatellites [Short Tandem Repeats (STR) or Simple Sequence Repeats (SSR)] are the DNA sequences of 1 to 6 nucleotide length tandem repeats and widely distributed throughout the genome (Litt and Lutty, 1989; Tautz, 1989). The change in the number of repeats caused by unequal crossing over between homologous tandem repeats or due to gain or loss of repeat units at a particular locus represents a variety of alleles with different number of tandem repeats. Therefore, in the population these loci have variable number tandem repeats (VNTR) and hence also called as VNTR loci.
Microsatellite loci are amplified using PCR with specific primers and the different alleles observed are separated using electrophoretic analysis or genotyped on a Sequencer (Jarne and Lagoda, 1996; Goldstein et al., 1999; Goldstein and Schlotterer, 1999). In the recent years, microsatellites are the marker of choice in livestock genetic characterization studies, population genetic analysis and used in evaluation of genetic resources (Sunnucks, 2001; Civanova et al., 2006). Microsatellites are co-dominant markers with high level of polymorphism and having high reproducibility. But these markers are time consuming and expensive to develop, stutter bands may complicate accurate scoring of polymorphisms and do not provide information on functional traits biodiversity (Mburu and Hanotte, 2005).
Single Nucleotide Polymorphisms (SNPs)
SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in normal individuals in some population(s). To consider the variation the least frequent allele has an abundance of at least 1% or greater (Brookes, 1999; Mburu and Hanotte, 2005). Simply, SNP is the polymorphism occurring between DNA samples with respect to single base. They involve the substitution of one nucleotide for another or the addition or deletion of one or a few nucleotides. A SNP is found where different nucleotides occur at same position in the DNA sequence. Most SNPs, usually involve the replacement of cytosine (C) with thymine (T) and are found at every 1000bp (Landegren et al., 1998; Stoneking, 2001; Vignal et al., 2002), in the genome.
DNA sequencing has allowed the discovery of SNPs and is the most recent contribution to study DNA sequence variation. Genomic selection using the SNP markers is a powerful new tool for genetic selection, breeding (Seidel, 2009) and population studies (Syvanen, 2001; Frohlich et al., 2004).There are some reasons which create interest in the use of SNPs as marker for genetics analysis. They are prevalent, abundant in the genome (Primmer et al., 2002), stably inherited, found in both coding and non-coding region, and amenable to high-throughput analysis (Vignan et al., 2002; Werner et al., 2002). SNP located in coding regions are directly associated with the protein function and as the inheritance is more stable, they are suitable for selection over time (Beuzen et al., 2000). SNPs are generally bi-allelic type, which indicates only two alleles in a population.
In recent years, these molecular markers and especially DNA-based markers, have been extensively used in many areas such as parentage determination, individual identity, genetic distance estimation and evolution; determination of twin zygosity and free martinism; identification of disease carrier animals, gene mapping, marker assisted selection (MAS), sex determination, transgenic breeding, conservation of animal genetic resources, demographic studies, pathogen identification and disease research; genomic selection and genome wide association studies; reconstruction of phylogenetic relationships among populations (Mburu and Hanotte, 2005; Teneva, 2009).
The variation revealed in terms of polymorphism at DNA level has provided a large number of markers and revealed their utility in animal breeding. In recent years, Marker Assisted Selection (MAS) is practiced, where the trait of interest is selection based on the marker linked or associated with it. Genotyping using molecular markers is a powerful tool that can be useful in optimizing animal breeding program. The development of molecular markers will continue in the near future and provide better understanding of animal genetic resources.