Recent developments in molecular biology and statistics have opened the possibility of identifying and using genomic variation and major genes for the genetic improvement of livestock. During the last five decades, the application of methods based on population genetics and statistics allowed the development of animals with a high productive efficiency.
These systems are based on simplified models of genic action that assume a large number of or genes with small individual effects in the expression of the phenotype (polygenes) and emphasizes the average genic effects (additive effects) over their interactions.
The basis is predicting the breeding values of the animals using phenotypic and genealogical information. Molecular techniques allow detecting variation or polymorphisms exists among individuals in the population for specific regions of the DNA.
These polymorphisms 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, the can prove some degree of linkage of the QTL effecting the trait and the marker. Recently, methods have been developed to detect the presence of major genes from the analysis of pedigreed data in absence of molecular information
Molecular Markers
A molecular marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci that can be observed.
A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like mini & micro satellites.
Recent years have witnessed a great interest towards molecular markers, revealing polymorphism at the DNA level, as they play an important role in animal genetics studies.
When differences in DNA occur within genes, the differences have the potential to affect the function of the gene and hence the phenotype of the individual. Genetic markers which have been used a lot in the past include blood groups and polymorphic enzymes.
We have relatively few such markers, but this has been overcome with the advent of new types of markers.
However, most molecular markers are not associated with a visible phenotype. The main types of molecular markers are VNTRs, RFLPs and RAPDs, AFLPs and SNPs.
3.1a: Variable number tandem repeat (VNTR’s) are scattered at various locations in the genome and are regions that are highly variable. These regions contain a type of DNA sequence called Variable Number Tandem Repeat which are multiple copies of a sequence of base pairs arranged in head to tail fashion.
For example, a frequently found tandem repeat is CA, and one strand containing this type of repeat reads CACACA, notated as (CA)n. The other strand would read GTGTGT.
In this example, the number of repeating base pairs is two, but it can be more. When the repeating unit is less than four, the VNTR is called a microsatellite and when the repeating unit is longer it is a mini-satellite.
Micro satellites are DNA regions with variable numbers of short tandem repeats flanked by a unique sequence. Microsatellites make good genetic markers because they each have many different ‘alleles’ – ie. There can be many different lengths of the repeat region.
An allele is defined by the number of repeats there are at the same location. With many alleles, most individuals are heterozygous, giving power to note association between marker allele and performance in progeny inheriting a favorable linked QTL allele.
Through the PCR reaction (see below), which uses the unique sequences either side of the repeat sequences as primer binding sites, microsatellite DNA can be specifically amplified.
The alleles an individual carrier at a particular microsatellite locus can then be determined by accessing the size of the amplified fragment through agarose gel electrophoresis.
3.1b:Restriction Fragment Length Polymorphisms’ (RFLP’s):here restriction enzymes enzymes cut DNA wherever they find the appropriate nucleotide sequence (e.g. Eco R1 cuts at the ‘recognition sequence’ GAATTC).
If there is a mutation at this sequence, no cut is made and the resulting DNA fragment is longer. Also mutation to give a new recognition sequence gives a pair of shorter fragments. Genetic differences (polymorphisms) of this type are known as Restriction Fragment Length Polymorphisms.
3.1c: Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments generated in PCR reactions that use a single short primer (in normal PCR a primer-pair is used). The primer must be complementary to sequences that are on opposite strands within a small number of base pairs (say 2000).
The DNA strand between these two sites is amplified in a PCR. Polymorphism is determined by individuals who have mutations at those sites, and therefore will not show a product on the gel. The advantage of RAPD‟s is that we do not need to know the DNA sequence of the species studied. A primer has a certain chance of randomly generate a PCR product.
Hence, RAPDs are cheap markers to develop. The disadvantage is that RAPDs either give or do not give a product and therefore, we cannot distinguish between homo- and heterozygotes.
3.1d: Amplified Fragment Length Polymorphism (AFLP) is based on PCR amplification of selected restriction fragments. Like RAPDs, AFLPs require no prior knowledge of DNA sequences (unlike microsatellites).
The advantage of AFLPs over RAPDs is that they are more reliable and reproducible (depend less on DNA quality and lab conditions). Also, the number of polymorphic loci (molecular markers) that can be detected is 10-100 times greater with AFLPs than with microsatellites or RAPDs
e: Single Nucleotide Polymorphisms are based on single base pair polymorphisms. A SNP is a position at which two alternate bases occur at appreciable frequency. In humans they may number greater than one in a thousand base pairs.
SNPs can be detected by a number of methods, however a relatively new technology, using DNA chips, can be used for large scale screening of numerous samples in a minimal amount of time.
Identifying Molecular Markers
Molecular techniques (such as polymerase chain reaction (PCR) or restriction enzyme digestion, followed by gel electrophoresis) can be used to identifying different alleles resulting from DNA polymorphisms.
Different alleles from a VNTR will have different size and similarly, RFLP‟s have different sizes (as defined by their name).
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Gel Electrophoresis
Gel electrophoresis separates DNA according to size. A gel is essentially a slab of gelatinous material. DNA is applied to ‘wells’ at the top of the gel (which is submerged in a tank containing some buffer), and an electrical current applied.
DNA is negatively charged and is drawn towards the positive electrode. Smaller fragments will move down the gel faster, as it is easier for them to move through the gel matrix as seen in the figure below.
Southern Blot
A southern blot involves the transfer of DNA from a gel (where it has been separated according to size) to a special type of membrane. The DNA on the membrane (which is in a denatured or single stranded state) is exposed to a probe.
A probe is a short sequence of DNA that is complimentary to, and thus binds to, a DNA sequence of interest.
Probe bound to the membrane is then visualized: this can be achieved by labeling the probe with radiation and exposing the membrane to X-ray film. A Southern Blot will usually show the alleles of VNTR‟s on all chromosomes, giving a complex pattern known as a DNA fingerprint as shown in the figure below.
Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) amplifies a specific region of DNA as defined by two primer sequences. It can thus be used to examine one particular region of the genome.
Because many copies of one specific section of the genetic material are generated, it is possible to use this technique with very, very small amounts of DNA as starting material (e.g. a single hair root or a small blood stain).PCR is a three stage process.
Firstly the DNA is denatured (made single stranded), secondly the primers bind or anneal to their complementary sequence, and thirdly the primers are extended by the addition of nucleotides complementary to that on the template sequence (this requires the action of an enzyme called DNA polymerase). This three stage process is then repeated 20-40 times as depicted in the following diagram.
Major Genes
Major geneis a gene with pronounced phenotype expression and characterizes common expression of oligogenic series, that is, a small number of genes that determine the same trait. Major genes control the discontinuous or qualitative characters in contrast of minor genes or polygene’s with individually small effects.
Major genes segregate and may be easily subject to Mendelian analysis. The gene categorization into major and minor determinants is more or less arbitrary. Both of the two types are in all probability only end points in a more or less continuous series of gene action and gene interactions.
Recent developments in molecular biology and statistics have opened the possibility of identifying and using genomic variation and major genes for the genetic improvement of livestock.
The detection of major genes using mixture models with segregation analysis can direct the work of identification of DNA marker genotypes towards populations and characteristics with greater probability of detecting a QTL.
The present trend indicates that molecular, pedigree and phenotypic information will be integrated in the future through mixture models of segregation analysis that might contain QTL effects through the markers, polygenic inheritance and uses powerful and flexible methods of estimation such as Gibbs Sampling.
Recently, methods have been developed to detect the presence of major genes from the analysis of pedigreed data in absence of molecular information.
These methods, based on mixture models and segregation analysis, allow to direct the potentially expensive and time consuming genotyping activities towards populations and characteristics with a greater probability of being controlled by a QTL and to optimize the collection of molecular data
Detection and Use of Major Genes
In the last ten years statistical methodologies of detection of major genes based on pedigree and phenotypic information on populations have been developed for animal populations.
These methods are based on the use of mixed models and segregation analysis to fit the data to a mixture genetic model that includes in addition to the polygenic effects, those of a biallelic major gene.
Calculation is performed in two stages; firstly genotype probabilities are obtained, then major gene, fixed effects and polygenic effects are fitted and used to recalculate new parameters by regressing phenotypes on estimated probabilities.
Calculation is iterated upon convergence. Segregation analysis allows inferring the unknown genotypes from the probabilities of transmission of the gene given the phenotype of the individual and their relatives.
In mixture models, regression and Gibbs sampling estimation approaches have been implemented to obtain estimates of the major gene effects and allelic frequency.
Meuwissen and Goddard (1997) evaluated the effect of including different proportions of individuals genotyped for a QTL in a mixture model that is based on the analysis of segregation of Kerr and Kinghorn (1996) and a regression approach which uses the estimated genotype probabilities as weights in the estimation process.
Unbiased estimates of QTL effect and frequency were obtained in absence of information on the genotype of the QTL, but some improvements in the precision of the estimates were observed as the proportion of genotyped individuals increased.
The main limitation of this method is that the genetic hypothesis is generally limited (one biallelic locus), thus, the presence of more alleles could not be detected. Also, the location of the locus in the genome, in absence of markers, remains unknown.
Mixture models can be modified to include markers associated with the QTL, instead of the direct effect of the QTL in addition to the information of the pedigree and the phenotype.
This is achieved by modifying the additive numerator relationship matrix (A), according to the conditional probabilities of transmission of the given QTL the information of the markers.
These developments can make possible to evaluate the likelihood of the model or another fitting criterion, to prove the relation between the markers and the QTL in population animals with outbreed mating structures.
They also may increase the possibilities of making MAS in animal populations when incomplete information exists on the genotypes of the animals for the QTL or markers so that the use of the genomic information is optimized.
Scientist has developed a method to evaluate the amount of genomic information that it allows maximizing a function of economic utility for the analysis of QTL with mixture models.
Major genes have been detected using these methods for carcass characteristics in pigs based on a mixture model of inheritance and Gibbs Sampling. Also, important effects of major genes have been detected using Findgene software for several carcass characteristics in cattle and for parasite resistance in sheep.
This methods that make use of information currently available in many animal populations, are an option for a preliminary screening for major genes that can contribute to rationalize the use of expensive QTL-marker linkage estimation experiments.
Incorporating Genetic Markers and Major Genes in Animal Breeding Programmes
Marker Assisted Selection (MAS)
The addition of genomic information to phenotypic information to increase the selection response to the traditional method is known as Marker-Assisted Selection (MAS).
The concept of Marker Assisted Selection (MAS) utilizing the information of polymorphic loci as an aid to selection was introduced as early as in 1900.
The method where marker genes used to indicate the presence of desirable genes is called as marker assisted selection.
Marker assisted selection (MAS) is indirect selection process where a trait of interest is selected not based on the trait itself but on a marker linked to it. The purpose is to combine all genetic information at markers and QTL with the phenotypic information to improve genetic evaluation and selection.
The advantage of using MAS is that the effect of genes on production is directly measured on the genetic makeup of the animal and not estimated from the phenotype. The integration of two selection methods, i.e., traditional or conventional selection methods with molecular genetics methods beneficial to the selection response.
Multiple estimated QTL effects and multiple trait selection could help to make better decisions regarding the use of MAS in animal improvement. Combined with traditional selection techniques, MAS has become a valuable tool in selecting organisms for desirable traits.
MAS is expected to increase genetic gain compared to traditional breeding programs and reduce the cost of progeny testing by early selection of the potential young bulls.
The application of MAS in breeding programmes depends on the knowledge of breeders about variable marker information from animal to animal and the different effects on multiple traits and his ability to spend in genotypic information that helps in improve their commercial breeding activities.
MAS also provide an apparently possible approach to selection for genetic disease resistance animals. In the future to make MAS effective in large breeding populations, the availability of large-scale genotyping methods and infrastructure that allows the generation of hundreds of thousands of molecular data at a reasonable cost will be necessary.
3.2.2a: Marker assisted introgression: An application that has been mentioned in the literature is the introgression a major gene in another population by means of backcrosses assisted by molecular markers.
In this case, it does not seem to exist advantage in using single genetic marker information, in comparison with the use of only phenotypic information when the characteristic is continuous and the considered genetic effects are additives.
Nevertheless, it seems feasible that using a dense map that involves many chromosomal regions and with more than one allele of interest, the time for fixation of the major genes can be reduced.
An example of introgression in pigs breeding is the introduction of litter size genes from the Meishan breed into Western pig breeds. The possible gains from such strategies depend heavily on the gene effect and the frequency in the commercial lines.
Introgression is expensive, as it involves several generations of backcrossing to the desired genotype, while keeping a desired haplotype from the introgressed QTL.
At the same time markers can be used to select against haplotypes for background genes from the imported line. This generally speeds up the introgression process and reduces the number of generations needed to arrive at the desired genotype (possibly in two generations).
Marker assisted selection can also be used in crosses of lines of about equal economic value. In that case, population wide linkage disequilibrium can be exploited, giving potentially large increases in response (Lande and Thompson, 1990).
Genetic evaluation models can have a significant effect on the achieved genetic response, models with random marker (haplotype) effects being superior, because the approach takes better account of the uncertainty of certain haplotype effects.
In summary, a rational use of the molecular methodologies requires the simultaneous optimization of selection on all the genes affecting important traits in the population.
The maximum benefit can be obtained when these techniques are used in conjunction with reproductive technologies like the artificial insemination, and collection and production in vitro of embryos to accelerate the genetic change.
There is a danger associated with a potentially inadequate use of QTL information, giving an excessively high emphasis to simple molecular information in detriment of the overall economic gain through all traits and their polygenic effects in the population.
Dissemination of the information to the industry is therefore a complex issue concerning QTL effects and molecular markers
The characteristics on which the application of the MAS can be effective are those that are expressed late in the life of the animal, or those that are controlled by a few pairs of alleles
Because of its high cost, the use of MAS could be justified, in animal nuclei that allow dilution of the costs when germplasm is extensively used towards the commercial population. Also in those characteristics in which the procedures of conventional selection have reached their limits in efficiency or the results have been not satisfactory
Before the molecular information on the QTL which control the characteristics of economic interest is generated, the detection of major genes using segregation analysis could direct the work of identification of genotypes towards populations and characteristics with greater probability of detecting a QTL using molecular markers
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