Abstract: Recent developments 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 the observed phenotypic variability. The use of DNA markers to define the genetic makeup (genotype) and predict the performance of an animal is a powerful aid to animal breeding. Molecular markers are a tool to study the diversity on the genetic level. The ultimate use of DNA markers would be to identify Quantitative Trait Loci (QTL) in order to practice genotype selection. In recent years different marker systems such as RFLP, RAPD, STS, AFLP, SSR, SNP and other have been developed and applied to livestock. This study provides a brief overview of the current application of these markers in animal breeding.
INTRODUCTION
To date, most genetic progress for quantitative traits in livestock have been made by selection on phenotype or on estimates of breeding values derived from phenotype without any knowledge of the number of genes that affect the trait or the effects of gene (Zakizadeh et al., 2007). The development of molecular techniques has been created new possibilities for the selection and genetic improvement of livestock. The discovery of the PCR had a major impact on the research of eukaryotic genomes and contributed to the development and application of various DNA markers (Gholizadeh and Mianji, 2007). Molecular techniques allow detecting variation or polymorphism exists among individuals in the population for specific regions of the DNA. These polymorphism can be used to build up genetic maps and to evaluate differences between markers in the expression of particular traits in a family that might indicate a direct effect of these differences in terms of genetic determination on the trait. More probably, they can prove some degree of linkage of the QTL effecting the trait and the marker. For genetic analysis, molecular markers offer several methodological advantages that are both attractive as well as amenable. For example: (1) the DNA samples can not only be isolated very conveniently from blood of live individuals but can also be isolated from tissues like sperm, hair follicle and even from archival preparations, (2) the DNA samples can be stored for longer periods and can readily be exchanged between the laboratories, (3) the analysis of DNA can be carried out at an early age or even at the embryonic stage, irrespective of the sex, (4) once the DNA is transferred on to a solid support, such as filter membranes, it can be repeatedly hybridized with the different probes and moreover, heterologous probe and in vitro-synthesized oligonucleotide probes can also be used and (5) the PCR-based methods can be subjected to automation (Mitra et al., 1999).
APPLICATIONS OF MOLECULAR MARKERS
Genetic Diversity
Breed characterization requires knowledge of genetic variation that can
be effectively measured within and between populations (Gholizadeh et
al., 2007). Extinction of endangered farm animal breeds leads to an
irreversible loss of genetic diversity. The need to conserve genetic diversity
is widely accepted for biological, economic and cultural reasons (Oldenbroek,
1999). A main reason is that an abundant resource of genetic diversity
within each livestock species is the prerequisite of coping with putative
future changes in livestock farming conditions (Bennewitz et al.,
2006). The maintenance of high levels of genetic variability and low levels
of inbreeding are major objectives in conservation programs. Genetic variation
is a prerequisite for populations to be able to face future environmental
changes and to ensure long-term response to selection, either natural
or artificial, for traits of economic or cultural interest (Frankham et
al., 2003). Also, inbreeding levels should be kept as low as possible
in order to avoid deleterious effects on fitness related traits which
could compromise the viability of the populations (Fernandez et al.,
2005). Three factors are considered as being largely responsible for the
declining genetic diversity of livestock:
• | Destruction of the native habitats of livestock breeds. |
• | The development of genetically uniform livestock breeds. |
• | Farmer and/or consumer preferences for certain varieties and breeds (and changes in these consumer preferences over time). |
Of these, commercial interests are considered as the most important pressure on livestock diversity. Important factors in determining the direction and nature of change include: growth performance (productivity), pest and disease resistance, ease of handling, adaptation to current levels of technology and to a relatively minor extent consumer choice. A more reliable measure of differences among breeds is genetic distance, which can be estimated from the differences in the frequencies of different genetic variants (alleles) at a number of marker loci. From the patterns of within-population genetic variation at marker loci, it is possible to deduce demographic factors important to the conservation of domestic cattle diversity (Malevieiute et al., 2002) 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 (Gwakisa, 2002). Measuring diverse attributes of a population is important to its characterization, taking into account phenotypic traits, reproduction, geographic distribution, origin and habitat (Gholizadeh et al., 2006). The genetic characterization of populations, breeds and species allows evaluation of genetic variability, a fundamental element in working out breeding strategies and genetic conservation plans. Molecular markers have been comprehensively exploited to access this variability as they contribute information on every region of the genome, regardless of the level of gene expression (Pandey et al., 2006). Lynch et al. (1995) showed that small populations might decline in fitness due to the accumulation of detrimental mutations. Hedrick et al. (1996) suggested that low genetic variation in a species might be indicative of a recent population bottleneck and such a bottleneck did potentially indicate vulnerability to extinction. In small populations, genetic drift tends to reduce genetic variation, leading eventually to homozygosity and loss of evolutionary adaptability to environmental changes (Lande, 1988). Low genetic variation in a species may be an indication of a recent population bottleneck and such a bottleneck could result in inbreeding depression. Species with low genetic variation may be more vulnerable to environment change and consequently is vulnerable to extinction (Zhang et al., 2002). 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 polymorphism of nuclear DNA and mitochondrial DNA, minisatellites, randomly amplified polymorphic DNA (RAPD), microsatellite, amplified fragment length polymorphism and sequencing of mitochondrial DNA (Gwakisa, 2002). Among these, microsatellites have quickly become the favourite agents for population genetic studies as they offer advantages which are particularly appropriate in conservation projects (Canon et al., 2001). For the analysis of genetic diversity in Lithuanian cattle breeds Malevieiute et al. (2002) chose the analysis on microsatellite markers. Microsatellite markers have also important qualities that make them very practical as molecular markers:
• | They are variable and exhibit a high level of allelic variation. |
• | They are co dominantly inherited. |
• | All co dominantly inherited alleles in an individual are visible, which is not the case for dominant markers, such as blood groups. |
• | They are very versatile in their application; they may be used to detect genetic variability and population structure differentiation among populations, phylogeny; they allow paternity testing and evaluate recent genetic and demographic history, such as population bottleneck. |
• | They are easily analyzed and occur regularly throughout the genome, making them especially suitable for genetic analysis. |
When a population goes through a bottleneck rare alleles tend to be lost and the average number of alleles per locus, allelic diversity, is reduced. Heterozygosity, however, is not reduced proportionally, because rare alleles contribute little heterozygosity. The difference between allelic diversity and heterozygosity is used as the basis for statistical tests detect presence of recent genetic bottleneck (Piry et al., 1999)
Marker Assisted Selection (MAS)
The idea behind marker assisted selection is that there may be genes with
significant effects that may be targeted specifically in selection. Some
traits are controlled by single genes but most traits of economic importance
are quantitative traits that most likely are controlled by a fairly large
number of genes. However, some of these genes might have a larger effect.
Such genes can be called major genes located at QTL. Although the term
QTL strictly applies to genes of any effect, in practice it refers only
to major genes, as only these will be large enough to be detected and
mapped on the genome. Following the pattern of inheritance at such QTL
might assist in selection (Van Der Verf, 2000). For MAS to be effective,
reliable estimates of QTL positions and effects are required. An adequate
power, precision and accuracy of QTL analyses can only be expected from
large, well suited mapping populations, using a marker set with good genome
coverage and phenotypic values based on multi-environment field trials
(Van Ooijen, 1992; Utz and Melchinger, 1994; Beavis, 1998). Close linkage
between marker loci and QTL is required not only for minimizing the bias
of estimated QTL effects but also for maximizing the frequency of the
desired QTL genotypes under MAS. The importance of close linkage is even
higher, if MAS is continued in recurrent cycles with intercrossing the
selected progenies after each cycle (Geiger and Welz, 1999). The linkage
disequilibrium, genome scan approach using anonymous molecular markers
is one of the major strategies used to identify QTL affecting economic
traits. Many studies have mapped QTL affecting several economic important
traits in farm animals and meat-type chickens (Zhou et al., 2003).
Molecular marker analysis allows to identify genome segments, so-called
QTL, contributing to the genetic variance of a trait and thus to select
superior genotypes at these loci without uncertainties due to genotype
by environment interaction and experimental error. Selecting for favorable
QTL effects based on marker data therefore has great potential for improving
quantitative traits (Geiger and Welz, 1999). The earliest form of Deoxyribonucleic
Acid (DNA) marker used to construct the first true genomic maps was the
restriction fragment length polymorphism (RFLP). The development of the
Polymerase Chain Reaction (PCR) technique has revolutionized molecular
genetics. The localisation of QTL provides markers linked to the trait
genes that could be used in breeding programs to improve the selection
for a particular trait. However, to be applied in this way it is first
necessary to determine the phase of the alleles at the markers and trait
gene (Williams, 2005). Potential benefits from marker assisted selection
are greatest for traits that:
• | Have low heritability (traits with observed or measured values that are a poor predictor of breeding value). |
• | Are difficult or expensive to measure (disease resistance). |
• | Can not be measured until after the animal has already contributed to the next generation (carcass data). |
• | Are currently not selected for as they are not routinely measured (tenderness). |
• | Are genetically correlated with a trait that you do not want to increase (most likely because associated genes affects one trait of the pair but not the other) (Van Eenennaam, 2007). One of the best examples of the application of MAS within population is the selection of young sires before their induction for actual progeny testing (Kashi et al., 1990a; Weller and Fernando, 1991). Inclusion of marker information for selection of young sires in progeny-testing programmes may lead to an increase of genetic gain by 15-30% (Piper and Bindon, 1982). |
Molecular Approaches to Disease Resistance
Genetic resistance to infectious diseases has been a subject of many
controversies. One of the tasks of genetic improvement is to select animals
resistant to infectious diseases, especially those difficult and expensive
to eradicate, in order to obtain healthy animals in which endogenous potentiality
is optimized and therapeutic events reduced (Zanotti et al., 2002).
Infectious diseases are responsible for major economic losses in livestock
production. Although control of the environment by sanitation and isolation
and massive use of vaccination and drugs, reduces the incidence of many
diseases, the problem has not been eliminated (Heller et al., 1992).
The linkage disequilibrium, genome scan approach using anonymous molecular
markers is one of the major strategies used to identify QTL affecting
economic traits. Many studies have mapped QTL affecting several economic
important traits in farm animals (Zhou et al., 2003).To achieve
sufficient power to identify linkage between marker loci and QTL with
low to moderate effects requires a large sample of animals to be genotyped
(Darvasi et al., 1993). The relatively high cost of marker genotyping
limits these applications for genetic analysis and genetic improvement.
The DNA pooling, also called bulk segregant analysis, is an efficient
method to reduce costs in marker-QTL linkage determination by pooling
DNA from selected individuals at each of the two phenotypic extremes,
which are the most informative individuals (Darvasi and Soller, 1994).
To identify a DNA marker for a disease gene animal material from a few
related families should exist, comprising around 50 offspring of which
at least 15 have the disease. When linkage has been found, it is natural
to continue using markers between the two markers providing the linkage.
The final goal will always be to identify the real disease gene. When
linkage has been found, comparative studies can also be initiated. Candidate
genes for the disease might be found by looking at the corresponding chromosome
areas in other species, which are already known. An alternative to the
classic marker analysis might be a careful study of the disease and thereby
finding a candidate gene from another species. A candidate gene is a gene
with a fair chance of causing the disease when comparing the aetiology
of the disease. If one or more candidate genes exist, the analysis starts
by typing these. If it is the right gene, complete association is found.
Parentage Determination
Breeding programs have been of considerable importance to improve
productivity in the animal industry. In dairy cattle, progeny testing
is the method of choice; however, this method presents two drawbacks limiting
its use: high cost and increased generation intervals. These obstacles
can be overcome when large numbers of progenies are obtained by artificial
insemination (Baron, 2002). Since the breeding value of an animal is generally
estimated using the information available from its relatives, the knowledge
of correct parentage is therefore a prerequisite. Parentage identification
in segregating populations generally takes place by means of the exclusion
principle. That is, presence at some genetic locus in the offspring of
an allele not found in either of the putative parents effectively excludes
the particular parental pair from biological parenthood. The effectiveness
of DNA fingerprints for parentage identification derives from the fact
that over an entire population, each minisatellite locus exhibits a wide
range of alleles, differing in their fragment lengths.As a result, over
the population as a whole, numerous bands, differing in fragment length,
can be identified, but only a few of these bands will be present in any
one individual. There is thus only a small probability that two randomly
chosen individuals will share all, or even a large proportion of the bands
in their respective DNA fingerprints (Kashi et al., 1990b). DNA
testing is the most accurate and reliable genetic analysis available for
parentage testing. However, the accuracy or specificity of the DNA testing
depends on the sample and procedure that the DNA laboratory has used.
Since we utilize the most advanced genetic testing procedure, we are able
to achieve at least specificity of 99.9%. In most cases, DNA testing will
result in specificities of 99.99% or greater. If DNA patterns between
the child and the alleged father do not match on three or more genetic
markers, then the alleged father is excluded with 100% certainty. Parentage
testing using molecular markers yields much higher exclusion probability
(> 90%) than the testing with blood groups (70-90%) or other biochemical
markers (40-60%) (Geldermann, 1990). Glowatzki-Mullis et al. (1995)
demonstrated that using two triplex microsatellites, wrong parentage can
be excluded with almost 99% accuracy.
Measuring Effective Population Size
Effective population size is one of the key parameters in population
genetics. It is analogous to different measures of genetic variation within
a population, which is a function of mutation rate, gene flow and population
size (Kimura and Ohta, 1971). Several factors affect the prediction of
effective population size, including sex ratio, mating system, selection,
pattern of inheritance, changes in the population size over generations
and population subdivision (Caballero, 1994). Intuitively one might expect
the effective population size to be close to the adult population census
size, but parameters such as reproductive failures, skewed sex ratios
and substantial reproductive skews caused by specific mating systems can
bias Ne up to several orders of magnitude below census size (Frankham,
1995a). Two advances in molecular genetics hold great promise for the
application of genetic markers to the estimation of Nc or Ne
of wildlife populations. These are: (1) the development of highly polymorphic
DNA markers and (2) the ability to amplify these markers with the Polymerase
Chain Reaction (PCR) from low-quality, low quantity DNA samples (Schwartzl
et al., 1998) The effective population size (Ne) plays a central
role in how a population evolves because Ne affects the degree to which
a population can respond to selection, as well as its sensitivity to inbreeding
effects (Crow and Kimura, 1970; Lande, 1995; Lynch et al., 1995).
Ne can be estimated from genetic data in one or more samples
(Waples, 1991). Most one-sample estimators use associations among alleles
at different loci to infer Ne (Hill, 1981; Vitalis and Couvet,
2001). Multiple-sample methods infer Ne from temporal changes
in allele frequencies or the rate of coalescence of alleles between sample
periods (Nei and Tajima, 1981; Wang, 2001; Berthier et al., 2002).
Accurate estimates of effective population size (Ne) are central to the
development of appropriate conservation strategies in any species as Ne
predicts the rate of loss of neutral, genetic variation, the fixation
rate of deleterious and favourable alleles and the rate of increase of
inbreeding experienced by a population (Frankham et al., 2002).
Importantly, the Ne of a population is often many times smaller than the
census size (N) of the population, the Ne/N ratio averaging just 0.11
in a survey of vertebrate species (Frankham, 1995b). While estimates of
Ne can be gained using direct methods based on field data (estimates of
sex ratio bias, offspring production, variation in family size etc.),
obtaining such data can be very cumbersome in many wild populations, especially
in aquatic species. Hence, indirect methods for Ne estimation based on
molecular marker data have also been developed. From a practical viewpoint,
these methods can be broken down into two categories: those that require
data from a single population sample (single generation methods: e.g.,
Hill, 1981; Pudovkin et al., 1996; Beaumont, 1999; Luikart and
Cornuet, 1999) and those requiring samples from the same population collected
at least one generation apart (temporal methods: Waples, 1989; Anderson
et al., 2000; Wang, 2001; Berthier et al., 2002). An important
recent advance has been the development of methods which take into consideration
the effects of migration on Ne estimation. The major limitation to use
of these methods is that double the sampling effort is required. The change
in allele frequencies (F) between sample periods is an inverse
function of Ne. Therefore, Ne can be derived from the amount of change
in allele frequencies (Nei and Tajima, 1981; Waples, 1991). However, this
estimator uses only the first two moments of the allele frequency distribution
to obtain Ne and a number of approximations are made in its derivation.
Several studies have noted that it is often biased high.
DISCUSSION
The development of molecular markers for genetic analysis has led to great increase in our knowledge of livestock genetics and our understanding of the structure and behaviour of animal genomes. For example, Selection of bulls for artificial inseminations is a very large contributor to decisions affecting genetic progress in current dairy cattle improvement with increasing knowledge of position and effects of major loci for quantitative variation, modification of traditional selection procedures based only on phenotypes will be needed. Molecular data will help eliminate undesirable alleles and increase favorable alleles (Rahimi et al., 2006). 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. However, utilization of marker-based information for genetic improvement depends on the choice of an appropriate marker system for a given application. For example, different types of markers have been used in paternity testing. In recent years, microsatellite markers have been used, because of their large polymorphism information content, widespread distribution in the genome, the type of samples that can be used the possibility to process several samples at the same time and the fact that the results are easy to interpret (Baron, 2002). Marker assisted selection is starting to be implemented in nucleus breeding programs. Trait heritability is the most important factor influencing the effectiveness of MAS. MAS seems to be most promising for traits with low heritability. But trait heritability is also of major importance for accuracy in the mapping of QTLs. Low heritability reduces the power of detecting QTLs, which is based on correlation between phenotype and marker genotype. This could mean that for well-mapped QTLs MAS may add little to phenotypic selection, while for traits with a very low heritability the underlying QTLs cannot be identified. It is the area in between these two extreme cases that looks most promising for application of MAS. If QTLs can be mapped for a trait having a low heritability the accuracy of the QTL position may not be very high, which is reflected in a large QTL support interval on the genetic map (Lee, 1995).