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Modern Approach in Animal Breeding by Use of Advanced Molecular Genetic Techniques

Vaishali Khare Ankur Khare
Vol 7(5), 1-22

Genetic progress for quantitative traits in livestock made by selection on phenotype or on estimated breeding value, is successful but, limitations like routinely recording of phenotypes, sacrifice of animal for meat quality traits, recording in a particular sex for sex limited traits etc constraints the amount of genetic progress made through conventional selection and breeding method. Molecular techniques are almost free from above mentioned limitations, if applied with care. Molecular techniques like detection of DNA-level polymorphism by restriction fragment length polymorphism (RFLP), AFLP, SNP and a number of molecular markers are frequently used to improve animal performance, both knock-out and over-expression of a gene provided better understanding of gene and its relevance to animal physiology, RNA interference is used to “silent” undesired disease specific genes in domestic animals and avian species, Mitochondrial DNA markers are useful for studying evolutionary relationships among taxa, DNA bar-coding emerged as a powerful strategy for species identification.

Keywords : Estimated Breeding Value RFLP AFLP SNP DNA bar-coding Conventional Breeding


Genetic improvement in domesticated animal populations through conventional livestock improvement programmes mainly involves selection of males and females that, when mated, are expected to produce progeny that perform better than the average of the current generation. Performance usually includes a combination of multiple characteristics, or traits, most of which are quantitative in nature. Quantitative traits that are controlled by multiple to many genes (>100 to perhaps thousands), affected by environment, in livestock include traits such as milk yield, fat yield, protein yield in dairy animals and growth rate, fatness and feed intake in meat producing animals. The main criteria that are used to identify individuals to be used for breeding are estimates of their breeding values for the traits of interest. The breeding value of an individual is defined as the sum of the additive effects of all loci that contribute to the trait (quantitative trait loci or QTL), deviated from the population mean (Falconer, 1996). To date, extensive data bases of recorded phenotypes for traits of interest, or for traits that are genetically correlated to traits of interest, have been used as the main source of information to estimate the breeding value of selection candidates. To this end, sophisticated statistical methods based on best linear unbiased prediction (BLUP), mixed linear model methodology (Lynch, 1998) has been implemented. These methods capitalize on information contained in the recorded phenotypes of not only the individual itself but also that of its relatives, in order to maximize the accuracy of the resulting estimated breeding value (EBV). Although selection programs based on EBV estimated from phenotype have been very successful, they also face a number of limitations. These primarily relate to the ability to routinely record phenotypes on selection candidates and/or their close relatives in a timely manner, such that accurate selection decisions can be made at an early age to reduce generation intervals. Cost of phenotype recording also plays an important role here. Unfortunately, some traits of interest are only recorded late in life (e.g. longevity), only on one sex (e.g. milk yield in dairy cattle), require animals to be sacrificed (e.g. meat quality), or require animals to be exposed to conditions that would hamper the ability to market or export their germplasm (e.g. disease resistance) (Dekkers et al., 2012). These phenotyping constraints limit the amount of genetic progress that can be made through conventional selection and breeding method.

Molecular genetics is the study of the genetic makeup of individuals at the DNA level. It is the identification and mapping of genes and genetic polymorphisms. By the use of molecular genetics techniques it is possible to identify genes that are involved in variety of traits. Armed with this information it would be possible to select improved livestock on the basis of their genetic makeup. If applied with care, the use of molecular information in selection programmes has the potential to increase productivity, enhance environmental adaptation and maintain genetic diversity. The use of molecular genetics technologies potentially offer a way to select breeding animal at an early age (even embryos); to select for a wide range of traits and to enhance reliability in predicting the mature phenotype of the individual.

Beginning of Use of Molecular Genetic Techniques in Animal Breeding

Starting in the 1970’s, the advent of the era of molecular genetics provided new opportunities to improve breeding programs in livestock by allowing the use of DNA markers to identify genes or genomic regions that control traits of interest. For quantitative traits, these advances promised the identification of QTL and the development of DNA tests that could be use to select animals at an early age to help selection decision through marker-assisted selection (MAS), i.e. selection on a combination of information derived from genetic markers associated with QTL and the traditional phenotypic information (Soller, 1978; Smith et al., 1986; Lande et al., 1998). To this end, large numbers of candidate gene and QTL mapping studies were conducted (Andersson, 2001; Dekkers and Hospital, 2002). This resulted in the discovery of substantial numbers of QTL and marker-phenotype associations and some causative mutations (Dekkers, 2004).

Advantages of Molecular Approach over Traditional Approach

  1. Assuming no genotyping errors, molecular genetic information is not affected by environmental effects and therefore has heritability equal to 1.
  2. Molecular genetic information can be available at an early age, in principle at the embryo stage, thereby allowing early selection and reduction of generation intervals.
  3. Molecular genetic information can be obtained on all selection candidates, which is especially beneficial for sex-limited traits, traits that are expensive or difficult to record, or traits that require slaughter of the animal (carcass traits).
  4. Molecular approach helps us to select for a wide range of traits which in turn saves time and efforts.
  5. Molecular genetic information enhances reliability in predicting the mature phenotype of the individual.

Application of Advanced Molecular Genetics Technique in Animal Breeding and Genetics

Use of Genetic Markers in MAS (Marker Assisted Selection) as a Tool in Breeding Programs

The fast development of molecular techniques has opened up foundation of genes to animal breeding that were not available before through conventional breeding, creating a lot of interest about MAS (Marker Assisted Selection). The advance use of molecular genetic technologies prospectively presents the way to select the breeding animals at an early stage (even embryo); to select for a superior variety of traits (Naqvi, 2007). Animal researchers are currently formulating their trust on genetic markers. To discover the effect of the genes on the phenotype of animals, we can follow the inheritance of these markers in families of animals and see whether inheritance any of these is associate with the develop performance. If they are, then we presume that one or more genes in this region of marker are associated with beneficial effects. Then we use the information on the genetic markers to make future selection decisions, so the any animal that inherits the marker will also inherit the valuable effect correlated with it (Naqvi, 2007), this is known as marker assisted selection (MAS). In genome wide association studies (GWAS), both the candidate gene and QTL (quantitative trait loci) mapping strategies have been widely utilized in domestic animals for the finding of genetic markers appropriate for MAS (Fan et al., 2010).

Assisted Reproductive Technology (ART) in Animal Breeding

Advances in assisted reproductive technologies (ART) like artificial insemination, In vitro production, superovolution, embryo transfer, trangenesis and cloning have become significant in livestock breeding. These techniques have been introduced initially to overcome reproductive problems but have significant impact on livestock breeding (Vikrama and Balaji, 2002). All these technologies able to speed up genetic changes due to shorter generation interval and improving accuracy in selection program (Anonymous, 1992). Artificial insemination (AI) and embryo transfer (ET) are probably the most well known methods that have been adopted in developed and developing livestock production (Kahil and Rewe, 2008). The recent advances in biotechnology technologies in reproduction included production of transgenic animals and cloning (Smidt and Niemann, 1999). RT has prolonged effects on animal breeding in the future, as it increases the rate of reproduction and decrease the generation time (Abu et al., 2008). The most successful reproductive technologies like AI and ET necessitated applying on large extent, some emerging biotechnologies such as Multiple Ovulation and Embryo Transfer (MOET), In Vitro Fertilization (IVF) and cloning provides prevailing tool for rapidly changing the animal populations genetically (Wajid et al., 2013).

Use of Transgenic Technology in Animal Breeding

Transgenic animal technology is in the progress of revolutionizing the manner we domesticate the livestock. The transgenesis means transfer of foreign gene (gene of interest) into the genome of other species in a way that it stably passed from generation to generation. It has been a potential way in accelerating and facilitating genetic improvement in livestock. The process to produce transgenic animals initiated with the purpose of producing better breed lines, which are strong, more carcass, high growth rate and increase milk production (Venkatesh, 2008). In breeding, transgenic animals are created to improve qualitative and quantitative traits in livestock and to reduce susceptibility to diseases (Duszewska1 et al., 2010). This technology uses the transgene encoding a particular trait is clone into a vector which may be synthetic, virus or plasmid DNA, and hybrid vector is inserted into the genome of the host organism. A variety of methods have been developed to produce transgenic animals, some have had much success and others are being further researched. There are several methods have been introduced to create transgenic animals, in these the most common method is the microinjection of a transgene into pronucleus of a newly fertilized egg, the introduction of desired gene into embryonic stem cells and the transgenic somatic cell nuclear transfer (TSCNT) which is the variant of SCNT (Wajid et al., 2013).

Pronuclear Microinjection

The microinjection into pronucleous is the most common method known in microinjection of exogenous DNA into the pronucleus of a newly fertilized egg (zygote). This technique is used to produce transgenic sheep and pigs (Hammer et al., 1985) and also transgenic cattle (Krimpenfort et al. 1991).

Sperm Mediated Gene Transfer (SMGT)

Sperm mediated gene transfer (SMGT) is an alternative technique using natural ability of spermatozoa as a vector to transfer exogenous DNA into the egg at fertilization (Bacci 2007; Lavitrano et al., 2002 and Zani et al., 1995).

DNA Recombination in Embryonic Stem Cells

ES cells are derived from inner cell masses (ICM) of embryo at blastocysts stage. This type of embryo manipulation is used when inserting a transgene into a specific location in the genome (Bradely and Brosius, 2006). Two complementary strategies have been considered for the insertion of transgene in ES cell- homologous recombination and integrase mechanisms (Norman and MacInnes, 2002). With the introduction of homologous recombination the scientists and researchers are able to restore gene function (knock-in animals), take out gene function (knock-out animals), inactivated, or introduce any alteration in gene of interest. In vitro the gene of interest is inserted into ES cells by microinjection, viruses, electroporation or chemicals.

Cloning Technology in Animal Breeding

Cloning is an asexual reproduction of genetically identical organism can be achieved by nuclear transfer (NT) or by embryo splitting (Abu et al., 2008). Cloning is a significantly useful breeding tool, considering a perfect way to improve the performance of farm animals. One of the principle purposes of cloning is to increase the number of species in a population with superior characteristics. Cloning technology has concerned the interest of breeders for many years. Animal cloning is the most topical development of selective assisted breeding in livestock (Wells, 2003). Cloning has been used to replicate elite breeding animals (Plume, 2009). As Dolly the sheep was the first animal to be cloned in 1996 (Wilmut et al,. 1997) by somatic cell nuclear transfer (SCNT). Since that time many other species have been cloned by the same process. According to Plume K. (2009) there are around 6000 farm animals’ clones worldwide. The cloning technology has been applied in the breeding of elite cattle (Kato et al., 1998), goat (Baguisi et al., 1999), pig (Polejaeva et al., 2000), buffalo (Shi et al., 2007), camel (Wani et al., 2010), Rabbet (Chesne et al., 2002) and other pet species like dog, cat, rat, ferret, mouse (Wakayama et al., 1999; Roslin Institute Online, 2003; Lee et al., 2005; Li et al., 2006 and Shin et al., 2002). High cost of cloning is the factor that limited the use of this technology in practical animals breeding (Hugo, 2006).

DNA Microarray Technology for Assisting in Breeding Decision

A variety of techniques have been available to identify difference in gene expression including subtractive hybridization, differential display, serial analysis of gene expression and microarray hybridization. Among these microarray technology has become one of the significant tools for scientists and animal breeders to monitor genome wide expression levels of genes in livestock. DNA microarray also known as DNA/RNA Chips, BioChips or GeneChips are a collection of DNA segments, immobilized on a solid surface (e.g. glass, plastic or silicon chip) which allow the quantitative and high throughput analysis of several genes through hybridization to a set of specific probes. Each particular hybridization reaction on an array is referred to as a spot or feature, and a characteristic array may include thousands of spots (Walsh and Henderson 2004). Microarray is novel technology that has attracted a huge deal of attention from animal geneticists and breeders. It has been used for large scale gene expression studies. The high throughput system of microarray makes genotyping efficient and low cost, particularly for single nucleotide polymorphism (SNP) and indel polymorphisms (Galbraith, 2006). DNA microarrays are widely applied in genome wide genotyping for identifying variation (Petricoin et al., 2002). Whole genome genotyping tools based on SNP markers are now available as microarray based genotyping arrays. These arrays are now allocated genotyping for the entire genome with tens of thousands of SNP markers in a single hybridization step, so it appreciably increasing the throughput and decreasing the cost of current gel based techniques for molecular mapping (Kadarmideen, 2006). These expression studies have been carried out to compare gene expression between breeds. Relatively apart from gene expression profiling to established fingerprint of an animal with desirable characteristics, direct genotyping of DNA for variants associate with genes that produce desirable traits will be of interest (Feilotter, 2004). The DNA microarray used for this purpose would be allow the large scale screening of many hundred of such markers in a sole experiment, allowing selection based on multiple traits. The newly genomic approaches such as DNA microarray, SNP discovery and genotyping are hopeful tools for improving and advancing farm animal breeding.

Sex Determination of Offspring/Embryo sexing by the Help of Molecular Markers

Molecular markers can be applied in the determination of sex of pre-implantation embryos. This can be achieved by using as probes, Y-chromosome-specific (male-specific) DNA sequence. Peura et al. (1991) reported that using the PCR-based method of sex determination has the advantage of being carried out in less than five hours with almost 100% accuracy. The sexing of pre-implantation embryos can serve as an important tool for improving a herd for a desired purpose (Machaty et al., 1993).Although embryo sexing may not have dramatic effects on rates of genetic gain it can have considerable increases in efficiency. It is concluded from a study that an all-female heifer system using ET was 50% more efficient than the highest achievable in a traditional system. It has been suggested that, if multiple sexed-embryo transfer became a routine operation such as AI, beef operations based on this system could become competitive with pig and poultry production in terms of efficiency of food utilization.

Use of High-Density SNP Genotyping for Whole-Genome Selection

For most livestock species, commercial platforms are currently available that allow the genotyping of an individual for tens of thousands of SNP across the genome at a reasonable cost (<$150 per individual, depending on volume). The first such high-density SNP genotyping platform available in livestock was the 50k bovine illumina SNP panel (Matukumalli et al., 2009). To date, tens of thousands of dairy and beef bulls and cows have been genotyped using this platform. Similar SNP panels of 40 to 65 thousand SNP are now available for other livestock species, including pigs, poultry, sheep, and horse. Recently, panels with over 700k SNP have become available in cattle and such higher density panels are also under development in other species. In dairy cattle, the main use of high-density SNP genotyping has been to implement genomic or whole-genome selection (Meuwissen et al., 2001). Genomic selection involves estimation of the effect of each SNP on the high-density panel using models that fit all SNP simultaneously, with their effects treated as random variables. Once estimates of the effect of each SNP are obtained, they can be used to estimate the breeding value of selection candidates based on their SNP genotypes across the genome.

Alternatively, the high-density SNP genotypes can be used to construct a so-called genomic relationship matrix among all individuals in the population and use it instead of the traditional pedigree-based relationship matrix in the BLUP mixed model procedures that are routinely used to estimate breeding values in livestock (Henderson, 1984). This procedure, known as GBLUP, has been shown to be equivalent to the Bayesian SNP effect estimation method in which the prior distribution of SNP effects assumes that the genetic variation for the trait is equally distributed across all SNP on the panel, similar to the infinitesimal model of quantitative genetics (Stranden et al., 2009). Thus, in contrast to the phenotype-based models for prediction of breeding values, methods that utilize genomic data do depend on having some knowledge of the genetic architecture of traits (Coster et al., 2010; Verbyla et al., 2010). Alternatively, non- or semi-parametric methods have been advocated for use in genomic selection (Gonzalez et al., 2008; Bennewitz et al., 2009). Methods to combine data on genotyped individuals with phenotypic data on individuals that have not been genotyped have been developed also (Legarra et al., 2009).

Estimation of breeding values using high-density SNP data has been implemented in dairy cattle breeding programs in several countries and research to implement genomic selection in other livestock species is underway (Dekkers, 2010). In dairy cattle, this has resulted a substantial increase in the accuracy of EBV at a young age (increases of 35% on average and up to 50%, depending on the trait and size of the data set used for training) (VanRaden et al., 2009). The availability of genome-enhanced breeding values (GEBV) at a young age is having a major impact on breeding programs in dairy cattle, in particular by allowing young bulls to be selected for breeding prior to the availability of extensive progeny data. This is expected to substantially increase (up to double) the rate of genetic improvement by reducing generation intervals (Schaeffer et al., 2006) and by enhancing opportunities to select for traits with low heritability, e.g. fertility. Reduction or removal of the need for progeny testing also has the potential to substantially reduce the cost of breeding programs in dairy cattle (Konig et al., 2009).

In the last decade there have been massive advances in genotyping technology, but due to technological limitation, approximately all genotyping is partial, through these technologies only a small fraction of an individual genotype is determined rather than the whole genome’ genotype. In the near future the new innovative e.g. Illumina’s Human-1 Bead Chip or mass sequencing technologies have promise the whole genome’s genotyping. Multicolor fluorescence detection ability of CE (capillary electrophoresis) instrument such as ABI PRISM genetic analyzer have a leading role in STR genotyping, the effort is going to develop microchip platform (Liu, 2007) to perform high resolution DNA genotyping. In addition mass spectrometries (MS) with matrix assisted laser desorption/ionization (MALDI) and elctrospary ionization (ESI) techniques have been used for STR typing without allelic ladder (Butler, 1998). There are a number of genotyping techniques available, capillary electrophoresis based genotyping techniques includes AFLP®, ISSR (inter simple sequence repeat) analysis, Relative Fluorescence Quantification, Resequencing Heterozygote Detection, Single Sequence Conformation Polymorphisms (SSCP), Polymerase Chain Reaction, Confronting two pair primers (PCR-CTPP), Melting Curve Analysis of SNPs (McSNP®) and Copy Number Analysis (CNA) have been play their role (Wajid et al., 2013).

Use of High-Density SNP Genotyping for Genome-Wide Association Studies

The large amounts of high-density SNP data that are being generated for implementation of whole-genome selection can also be used for genome-wide association studies (GWAS) to identify genetic markers or genomic regions associated with traits based on population-wide linkage disequilibrium (LD). Several studies have capitalized on this to analyze the genetic architecture of traits of interest in animal agriculture (Goddard et al., 2009). For GWAS, several alternate statistical methods have been used. Most studies have used single SNP models in which each SNP is fitted separately as a fixed effect, ideally in a BLUP animal model to properly account for the family structure of the data by fitting a polygenic effect with pedigree based relationships (Kennedy et al., 1992; Mai et al., 2010; Cole et al., 2011). A problem of single SNP models is that they rely on the pair wise LD of a QTL with individual SNP. Single SNP models can also lead to excessive false positives if population structure is not properly accounted for (Yu et al., 2005). Hayes et al. (2010) used mixed linear model methodology to estimate the proportion of genetic variance associated with each genomic region of 50 SNP from the Bovine 50k Illumina SNP chip for three quantitative traits in dairy cattle. Fitting each region separately, their model simultaneously used two genomic relationship matrices, one based on 50 SNP in the region and one based on the rest of the genome, to separate genetic variance contributed by the region from variance contributed by the rest of the genome (Dekkers, 2012).

The Bayesian methods that have been developed for genomic selection have also been used for GWAS. In particular the Bayesian variable selection methods have been shown to be effective for GWAS in simulated (Sahana et al., 2010; Sun et al., 2011) and real data (Fan et al., 2011; Onteru et al., 2011). Several criteria have been used to identify important SNP or genomic regions using these methods, including the proportion of iterations of the MCMC chain that a given SNP or a set of SNP in a genomic region were given non-zero effects, or the proportion of variance that is explained by a given SNP or by a region of the genome (Sun et al., 2011; Fan et al., 2011, Onteru et al., 2011). An advantage of the genomic selection methods over the single SNP models is that all SNP are fitted simultaneously. This allows capture of all information if multiple SNP are in LD with a QTL and also implicitly accounts for any population structure that is present in the data, reducing false positives. In addition, by fitting SNP effects as random rather than fixed, estimates are shrunk towards zero depending on the amount of information that is contained in the data and the priors that are specified(Dekkers, 2012).

In July 2010, Illumina released two new genotyping SNP chips including a low-density chip (Bovine3K) having 2,900 SNP (Illumina, 2010c) and a high density chip (Bovine HD) with 777,962 SNP (Illumina, 2010a) and in January 2011 affymetrix released a high density chip with 648,855 SNP (Wiggans, 2011). Although such chips can provide genotypes that enhance the precision of genomic evaluation by better tracking of the loci responsible for genetic difference (VanRaden and Tooker, 2010). SNP chips are currently available for human, ovine, bovine, canine, porcine and equine species (Ajmone-Marsan, 2011).

Molecular Markers – A Tool for Exploring Genetic Diversity

Diversity among organisms is a result of variations in DNA sequences and of environmental effects. Genetic variation is substantial, and each individual of a species, with the exception of monozygotic twins, possesses a unique DNA sequence. DNA variations are mutations resulting from substitution of single nucleotides (single nucleotide polymorphisms – SNPs), insertion or deletion of DNA fragments of various lengths (from a single to several thousand nucleotides), or duplication or inversion of DNA fragments. DNA variations are classified as “neutral” when they cause no change in metabolic or phenotypic traits, and hence are not subjected to positive, negative, or balancing selection; otherwise, they are referred to as “functional”. Mutations in key nucleotides of a coding sequence may change the amino acid composition of a protein, and lead to new functional variants. Such variants may have an increased or decreased metabolic efficiency compared to the original “wild type”, may lose their functionality completely, or even gain a novel function. Mutations in regulatory regions may affect levels and patterns of gene expression; for example, turning genes on/off or under/over expressing proteins in specific tissues at different development or physiological stages. In the absence of reliable phenotype and QTN data, or to complement the existing data, the most rapid and cost-effective measures of genetic diversity are obtained from the assay of polymorphisms using anonymous molecular genetic markers. Anonymous markers are likely to provide indirect information on functional genes for important traits, assuming that unique populations that have had a particular evolutionary history at the neutral markers (e.g. because of ancient isolation or independent domestication) are likely to carry unique variants of functional variations. Molecular techniques have also proved useful in the investigation of the origin and domestication of livestock species, and their subsequent migrations, as well as providing information on evolutionary relationships (phylogenetic trees), and identifying geographical areas of admixture among populations of different genetic origins(Hood et al., 2004).

Using Markers to Estimate Effective Population Size

Hill (1981) suggested estimation of effective population size (Ne) using gametic phase disequilibrium of DNA polymorphisms. This estimation can be based on genotypes for linked markers (microsatellites or SNPs). The expected correlation of allele frequencies at linked loci is a function of Ne and the recombination rate. Ne can, therefore, be estimated from the observed disequilibrium. Hayes et al. (2003) suggested a similar approach based on chromosome segment homozygosity, which, in addition, has the potential to estimate Ne for earlier generations, and therefore allows a judgment of whether an existing population was of increasing or decreasing size in the past. The study demonstrated, with example data sets, that the Holstein-Friesian cattle breed underwent a substantial reduction of Ne in the past, while the effective population size of the human population is increasing, which is in agreement with both census and pedigree studies.

Use of Mitochondrial DNA based Diversity and Phylogeographical Analysis in Livestock Breeding

Mammalian mitochondria contain a small circular DNA plasmid of 16.5 kb which codes for 37 genes required to be expressed within the inner mitochondrial membrane. The mitochondrial genome evolves 17 times faster than nuclear DNA; probably due to lack of DNA repair mechanisms. As a result, the mitochondrial DNA sequence can be used to monitor evolution on a shorter time scale than is possible with chromosomal DNA. Mitochondrial genome polymorphisms are therefore frequently used to analyse population structure and demographic history. Mitochondrial DNA is haploid, and so each individual has a single haplotype. It is maternally inherited as a result of the limited contribution to the zygote of mitochondria from sperm. This limits its use in relation to domestic species where gene flow through the male line forms an important determinant of evolution and population structure, for instance through artificial insemination, but provides the advantage that it allows introgression through the female line to be distinguished from that through the male(Flint and Woolliams, 2008).

The value of mitochondrial DNA for animal breeding is that it allows an understanding of population history and structure in time and space. For instance, a low level of mitochondrial DNA polymorphism within a species suggests it has survived a reduction in population size or bottleneck, whereas a high level of variation is characteristic of a large and well-established population. Mitochondrial DNA is extremely valuable in resolving important taxonomic questions when distinguishing subspecies and in identifying evolutionary significant units. Mitochondrial DNA bar coding has been suggested as an aid to assessment of biodiversity (Hebert et al., 2003), but this has been questioned, particularly in species subject to parasite infestation or with a high incidence of symbiont infection (e.g. arthropods; Hurst & Jiggins, 2005). The general conclusion from large-scale analyses of livestock populations, which are perhaps the most informative for this purpose, suggests that variation in maternal lineages explain at most a small fraction of the variation in traits of commercial interest (e.g. Roughsedge et al., 2000 a, b).

Use of mtDNA has broadened the perspective on the origin and evolution of domesticated cattle (Maji et al., 2009). Further, one of the persistent challenges in the analysis of population genetic data is to account for the spatial arrangement (nonrandom distribution of genetic variation among individuals within populations) of samples and populations. mtDNA data have been extensively used to understand the spatial distribution of genetic lineages within species allowing the historical factor with the highest effect on the lineages spatial patterns. mtDNA has been used for the identification of maternal and paternal lineages (Erhardt and Weimann, 2007) as well as test hypothesis related to past genetic history and evolution of different species. The recognition of mitochondrial DNA molecule as a genetic marker in population and evolutionary biology derives in part from the relative ease with which clearly homologous sequences can be isolated and compared. Simple sequence organization, maternal inheritance and absence of recombination make mtDNA an ideal marker for tracing maternal genealogies (Sodhi et al., 2014).

Use of Cytogenetic and Molecular Methods for Screening of Genetic Defects in Livestock

Genetic diseases occur due to abnormalities in genetic material, at any nucleotide to chromosome level, of any kind of individual. Mostly genetic defects are rare in nature because of continuous natural selection against them. Although, all of the genetic defects do not culminate into a disease condition, but sometime, individual looking normal may have genetic defect in heterozygous condition and can act as carrier during inheritance. During recent time, we are now able to diagnose the genetic defect in the individuals. Now, biotechnology offers to diagnose genotypes, such as normal, carrier, or affected individuals. Understanding the molecular basis of a defect, the direct detection of the heterozygous carriers is thus possible even during embryonic stage. In livestock, genetic screening has become much essential in view of intensive selection in dairy and meat industry, which has predisposing only few of the high valued males. In such circumstance, any individual with genetic defect may inherit the defective gene or chromosome to a larger number of progenies; thereby have more economic concern in livestock industry. Because most genetic diseases are inherited from the carriers, which generally produce no noticeable indications, the undesirable trait can proliferate extensively in absence of screening of genetic defects. Now a day, cytogenetic and molecular screening of all breeding males has been made essential in the new National Programme on Cattle and Buffalo breeding (NPCBB) to keep our farm animals free from genetic defects aroused by any chromosomal abnormalities or nucleotide mutations. Multiple congenital malformations are seen with many types of chromosomal abnormalities, particularly deletions and aneuploidy. Animals with a balanced set of chromosomes will generally be normal phenotypically. If an individual does not have a balanced set of chromosomes, this will normally be visible through more or less deviation of phenotype from normality. Animals with a non-balanced set of chromosomes will most often be sterile and have low vitality. Chromosome deviations, in animals with a normal phenotype, are normally detected due to low fertility or complete sterility. The subfertility is caused by problems in chromosome pairing and segregation during meiosis. In general, however, it shows a substantial, often greater than 50% reduction in fertility. Chromosomal fusion in heterozygote form causes a slightly lower fertility. The karyotype of a bull with low fertility has shown having a 1/8 translocation. In twinning of foetus with different sex, a mixture of stem cells is established for the white and the red blood cells by mixing the blood in the early foetal stage. If the mixing is too extensive the heifer in a mixed twin pair gets abnormal sexual organs and is infertile and called Freemartins. The bull birthed from such twining generally has normal fertility, however, might show the genotype of the other twin. Cytogenetic screening i.e. by studying the chromosomes, we generally study the inheritance pattern from one generation to another. It also gives an opportunity to locate the genes and their arrangement on the chromosomes, which become important for the linked loci. Now a day’s advanced molecular cytogenetics like Fluorescent In Situ Hybridization (FISH) and Comparative Genomic Hybridization (CGH) has also been come out, which is analyzing the chromosome with more refinement, however, limited use is there in routine due to high cost (Niranjan et al., 2014). Kingsbury (1990) reported that a particular RFLP in the Prion protein gene was responsible for the variation in host’s response to the causative agent, and the incubation time of bovine spongiform encephalopathy (BSE). The PCR-RFLP assay has been used to identify carrier animals possessing the defective recessive allele in bovine leucocyte adhesion deficiency in cattle (Shuster et al., 1992), hyperkalemic periodic analysis in horses and malignant hyperthermia in pigs (Fujii et al., 1991). Georges et al. (1993) identified carrier animals of weaver disease in cattle using microsatellite (TGLA 116) marker.

Table 1: Important genetic diseases in cattle

Disease Gene Mutation Reference
Bovine leukocyte adhesion deficiency (BLAD) Integrin beta 2 (ITGB2) Shuster et al., 1992
A383G leading to D128G
Beta mannosidosis Mannosidase beta A, Lysosomal (MANBA) Leipprandt et al., 1999
G2574A leads to premature stop codon(Trp858Stop)
Citrullinemia Argininosuccinate Synthetase (Ass) C→T in exon 5 Dennis et al., 1989
Arginine 86 (CGA) to nonsense codon (TCA)
Deficiency of uridine monophosphate synthase Uridine monophosphate Synthetase (UMPS) Schwenger et al., 1993
C→T, Arginine 405 (CGA) to stop codon (TGA),
Complex vertebral malformation UDP-Nacetylglucosamine Kanae et al., 2005
(SLC35A3) 559 G→T, homozygosity for V180F
Sex reversal XY female Sex determining region Y (SRY) Kawakura et al., 1996
Possible deletion of SRY gene
Factor XI deficiency Blood coagulation factor XI Marron et al., 2004
(F11) 76-bp insertion in exon 12
Renaltubular dysplasia Claudin 16 (CLDN-16) Hirano et al., 2002
Deletion of 37-kb region including exons 1 to 4 (type 1 mutation)
Congenital myasthemic syndrome Cholinergic receptor, nicotinic, epsilon polypeptide 470del20 in exon 5 leads to frame shift followed by premature stop codon Thompson et al., 2007

Use of Molecular Genetic Tools for Livestock Germplasm Conservation

The genetic resources of farm animals in India are represented by a broad spectrum of native breeds of cattle, buffaloes, goat, sheep, swine, equines, camels and poultry. The genetic biodiversity among this livestock has developed and stabilized over millions of years of evolution and endowed the indigenous breeds with capabilities to withstand hostile climate, epidemic pests and diseases, and to survive on inadequate quantities of feed, fodder and water. However, over the years due to many reasons the population size of many breeds is declining. As genetic diversity equips farmers and breeders to utilize a wide range of production environments and develop diverse products to meet the needs of local communities, the unavailability of such diversity in future may hamper sustainable development. Hence the need for conservation of animal genetic resources has been accepted in India as well as globally. Conservation methods can be broadly categorized as In situ and Ex situ. In situ conservation means that animals are kept within their production system, in the area where the breed developed its characteristics. Ex-situ conservation applies to situation where animals are kept outside their area of origin (herds kept in experimental farms, farm parks, within protected areas or in zoos) or more often, when genetic material is conserved and stored in gene banks in the form of semen, ova, embryo or DNA. methods that include organized flocks/herds needs lots of space and infrastructure however ex situ conservation methods that include cryopreservation of embryos, somatic cell banking, epididymal sperms banking, cryopreservation of embryonic stem cell lines, cryopreservation of spermatogonial stem cell lines, storage of DNA, frozen semen are the advanced techniques which can be used for long time conservation of number of species and breeds in a small area (Rajeev Aggarwal, 2014).

Determination of Parentage by the Use of Molecular Techniques

The identification of parentage 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. Highly polymorphic DNA fingerprinting markers have been reported to be very useful in parentage testing (Mitra et al., 1999). Molecular markers can be employed for sire identification in Artificial Insemination programmes.

Use of DNA Barcoding Markers for Species Identification

A DNA barcode is a short DNA sequence from a standardized region of the genome used for identifying species. The essential aim of DNA barcoding is to use a large-scale screening of one or more reference genes in order to assign unknown individuals to species, and to enhance discovery of new species (Hebert et al., 2003). Biological taxonomists apply this principle to species classification. The first application of using the DNA sequences in systematic biological taxonomy (also called DNA taxonomy) was conducted by Tautz et al., 2002 and then , Hebert et al., 2003 proposed the concept of DNA barcoding and suggested its use for a single mtDNA gene, mitochondrial cytochrome c oxidase I (COI), as a common sequence in animal DNA barcoding studies. DNA barcoding has a high accuracy of 97.9% (Goodfellow, 1992), and provides a new, quick, and convenient identification strategy for animal genetic diversity (Morin et al., 2004).

Hurdles in the Application of These Molecular Genetics Techniques

Economic Factors

According to Dekkers and Hospital (2002), “economics is the key determinant for the application of molecular genetics in genetic improvement programmes. The use of markers in selection incurs the costs that are inherent to molecular techniques.” Developing costs (e.g. identifying molecular markers on the genome, detecting association between markers and the traits of interest) and running costs (e.g. typing individuals appropriate in the selection programme) are quite expensive. If the Developing and running costs of the technology is much high it may certainly out-weigh whatever benefits that could be derived from it for this the accessibility and affordability of the technology needs to be increased.

Need for Global Data Infrastructure

A global molecular data infrastructure needs to be built to share and access molecular data, in combination with performance traits between countries. This requires a re-think of how to redefine intellectual property rights.

Lab to Land Transfer of Techniques

“Dissemination Scenarios” must be developed on how to extend the genetic progress from lab to land. One could imagine that genomics selection is practised in a local nucleus herd from which selected sires (and surplus dams) are sold / distributed to farmers. Given the unique local environmental challenges, it is important to maintain a local level of input into the selection programme.

Lack of Funds/Grants to Researchers

The researches involving molecular technologies are being hampered in developing nations due to the inability of researchers to access grants and funds. Many times researchers are denied opportunity to secure research grants because their institutions or their basic affiliations could not provide the basic equipment/facilities required to effectively carry out some researches. Sometimes when research grants are provided, the amount is hardly sufficient to procure all the necessary reagents and other consumables. For all this to happen, considerable funds need to be allocated both at national and international levels.

Lack of Adequately Trained Personnel

The application of molecular markers to the improvement of animal species is also being hampered by the non-availability of enough number of adequately trained personnel with the requisite practical experience in the universities. It is therefore advocated that training and re-training of personnel be carried out to forestall the problem of inadequate human resources.


Numbers of molecular techniques together with conventional breeding methods are frequently making their impact in animal improvement in present era. Molecular techniques like detection of DNA-level polymorphism by restriction fragment length polymorphism (RFLP), AFLP, SNP and a number of molecular markers are in frequent use to improve animal performance from one generation to next, both knock-out and/or over-expression of a gene have provided us a better understanding of a given gene and its relevance with respect to overall animal physiology, RNA interference (RNA I ) has been extensively leveraged to “silent” undesired disease specific genes in domestic animals and avian species, Mitochondrial DNA markers are particularly useful for studying evolutionary relationships among various taxa, DNA bar- coding based on mitochondrial genes has emerged as a powerful strategy for species identification. The origin and refinement of genetic markers clearly led to the development of innovative strategies against conventional breeding methods, which are slowly becoming obsolete. Standardized protocols are now followed to define, develop and bring into practice a successful method, which then becomes routine among global animal breeders. SNPs are beginning to influence and refocus our strategies, and certainly along with next-generation sequencing technologies, have major role to play in the near future. These later tools have given the animal geneticists a formidable opportunity to incorporate “desirable traits” at will, while simultaneously getting rid of undesirable elements in given animal populations.

These high-tech DNA techniques, such as PCR, DNA sequencing, SSR, SNP, etc., have revolutionised all aspects of molecular biology, particularly its use in animal breeding strategies and in the conservation of genetic diversity. 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. It is obvious that molecular markers will continue to serve as a potential tool to geneticists and breeders to evaluate the existing genetic potential, and to manipulate it, to create animals as desired and needed by the farmers. It is not likely that these advanced molecular technology, will replace ‘conventional’ methods for genetic improvement. Instead, they probably will begin to be gradually incorporated into current genetic improvement programs that use efficiently classical improvement methods to achieve particular objectives.

In summary, for molecular genetic tools to make a positive contribution to sustainable livestock production we need a joined-up strategy addressing genetic progress as well as conservation overcoming all the hurdles in the application of molecular genetic techniques in routine use for animal improvement programmes.


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