models were used, such as *Drosophila melanongaster* Meigen (Misquitta & Paterson, 1999; Dzitoyeva et al., 2001; Schmid et al., 2002), *Tribolium castaneum* (Herbst) (Bucher et al., 2002; Tomoyasu & Denell, 2004; Tomayasu et al., 2008), honeybees (Amdam et al., 2003), *Acyrthosiphon pisum* (Harris) (Mutti et al., 2006; Jaubert-Possamai et al., 2007), the green peach aphid *Myzus persicae* Sulzer (Pitino et al., 2011), the western corn rootworm *Diabrotica virginifera virginifera* (LeCompte) (Baum et al., 2007; Alves et al., 2010). In almost all the experiments carried out, dsRNA was injected directly in the organism, but this kind of dsRNA delivery is not feasible in the protection of crops against phytophagous insects.

The big obstacles to make RNAi approach extensively feasible for crop protection is related to the selection of target sequences, to the production of big quantities of dsRNA to be

Concerning the selection of target sequences, up to now RNAi application tooks advantages from already known genes while cDNA libraries allowed to identify novel genes (Turner et

The recent availability for many organisms of the full genome sequences makes possible, at least theoretically, the silencing of every single gene to evaluate how the organism reacts. A great improvement in the selection of target genes can be given by the next-generation sequencing (NGS) technologies, with the direct sequence of the cDNA generated from messenger RNA (RNA-seq) (Wang et al., 2009; Haas & Zody, 2010). This new method, called also "Whole Transcriptome Shotgun Sequencing", uses recently developed high-throughput sequencing technologies to convert long RNAs into a cDNA fragments with adaptors attached to one or both ends. Then these fragments are sequenced in a high-throughput manner to obtain short sequences. The resulting reads are aligned to a reference genome or a reference transcript, or assembled ex-novo without the genomic sequence to produce a genome-scale transcription map (Wang et al., 2009). This new method results very useful in the case of non-model organisms for which any genome or transcriptome sequence information is available. Wang et al. (2011) used Illumina's RNA-seq and digital gene expression tag profile (DGE-tag) technologies to screen optimal RNAi targets from *Ostrinia furnalalis* (Guenée) demonstrating that the combination of these two technologies is a rapid, high-throughput, cost less and easy way to select the candidate target genes for RNAi.

One of the biggest limitations to a cost-effective commercial use of this technology is the production of mass quantities of dsRNAs. Tenllado et al. (2004) proved the efficacy of crude preparations of dsRNA in silencing genes of plant viruses using bacterial crude preparations obtained by lysing cell pellets with a French Press avoiding the use of time-consuming protocols to purify nucleic acids. So they anticipated that inducible expression of dsRNA in bacteria could easily be scaled-up as a rapid and cost effective expression system for a large variety of viral dsRNAs. They suggested the use of large fermenters normally used for the commercial production of entomopathogenic nematodes for the mass production of interfering products in agriculture. Moreover they stated that bacterial cell cultures provide a practicable and reproducible approach for mass production of dsRNA because they are easily pelleted, stored and distributed, without losing their interfering properties with plant

Concerning the delivery system of dsRNA, the feeding approach instead of the haemocoel injection is more attractive because more feasible in crop protection, that is the target

released, and to the delivery systems of dsRNAs.

al., 2006; Baum et al., 2007).

virus infection.

organism should be able to take up the dsRNA autonomously, especially in the case of insects , e.g. by feeding and digestion (Huvenne & Smagghe, 2010). Several studies have proved the possibility of introducing dsRNA in insect through feeding as resumed by Huvenne & Smagghe (2010) and recently confirmed by Pitino *et al.* (2011) who demonstrated that siRNAs can travel from the plant phloem through the aphid *M. persicae* stylet and reach the intestinal tissues triggering the silencing of aphid target genes. Thus the solution for a practical crop protection application is the introduction of dsRNA in the plants that are ingested by insect herbivores. That is possible by *Agrobacterium* infection, by particle bombardment or by virus infection. The first method refers to transgenic RNAi plants that are created, as other genetically modified plants, by means of isolation, amplification and cloning into a plasmid of the target gene. Then the plasmid is allowed to undergo recombination into an *A. tumefaciens* binary vector. Then plants are transformed with *A. tumefaciens* and the vectors replicate to produce ds-RNA for RNAi induction (Johansen & Carrington, 2001). Alternatively bombarding cells with dsRNA can produce transient silencing. On the contrary with Virus Induced Gene Silencing (VIGS) RNA is introduced in the plants by means of modified viruses as RNA silencing trigger (Watson et al., 2005; Wani et al., 2010).

Tenllado et al. (2004) found that a spray made of crude dsRNA was efficient at gene silencing of plant viruses. But even if the dsRNAs were able to stable remain on the leaves for several days conferring resistance to viral infection, this method was recognized not to be cost-effective. Likewise a crude extract of *E. coli* HT115 containing large amounts of dsRNA and applied to maize plants as a spray was able to inhibit Sugarcane mosaic virus (SCMV) infection (Gan et al., 2010). More recently Wang et al. (2011) demonstrated that a direct spray of dsRNA can produce a down-regulation of gene and finally a development delay and death in the Asian corn borer *O. furnalalis* larvae by penetration through the exoskeleton before reaching the haemolimph and producing RNAi effects. This finding opens new perspectives in dsRNA delivery and provides new insight that RNAi targets can be selected from the whole insect instead of gut-specific target after feeding with dsRNA.

Concerning the future of RNAi in crop protection there are good chances for its application in the field as well as interesting commercial implications in particular against viruses and insects. Probably the future of crop protection against insect pests by means of RNAi technology is more related to sap-sucking pests such as aphids, whiteflies, psyllids and leafhoppers. In fact the current expression of Bt insecticidal proteins in plant offers a better degree of protection in field condition against Lepidoptera and Coleoptera, while none of Cry toxins of *B. thuringiensis* is effective on sup-sucking pests (Virla et al., 2010). The good new results obtained with the whitefly *Bemisia tabaci* (Gennadius) (Upadhyay et al., 2011), the aphid *M. persicae* (Pitino et al., 2011) and the brown planthopper *Nilaparvata lugens* Stål (Zha et al., 2011) are promising. Anyhow, recent reports of resistance to Bt toxins expressed in the plants (Tabashnik et al., 2008) will provide more interest for alternative strategies also for Lepidoptera and Coleoptera.

There is also another application of RNAi that could open new opportunities in crop protection. Allowing the specific down-regulation of genes, this technology will permit the identification of new targets for crop protection compounds such as fungicides and insecticides (Busch et al., 2005) for an ever more specific action. With this approach vulnerable targets for new pest-specific insecticides with novel mode of action can be assessed.

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Anyhow for a wide practical application of RNAi in crop protection some challenges remain. For instance the long-term effectiveness of the approach will need to be established, in particular it is necessary to investigate how the internal defense mechanism of the insects will respond to long-term exposure to dsRNA.

### **6. Conclusion**

The general trend in crop protection is the progressive reduction of chemicals because of a more consciousness towards human health and environment. Nowadays biotechnology and genomics offer new important possibilities for this purpose. The possibility to transfer genetic information between different organisms allowed to create transgenic plants resistant to different pathogens and pests. The application of this technology to the most economically important crops and the dissemination throughout the world provided a considerable decrease in pesticide use. In parallel genomics help us to understand the precise role of simple sequences and the proteins they encode within the organism opening new perspectives for the production of pesticides with innovative mode of action and for the development of new specific control strategies against plant pathogens and pests such as the RNAi approach. The improvement of the sequencing technology and in particular the advent of the NGS together with the evolution of bioinformatics enable large-scale sequencing projects permitting a new comparative genomics approach and thus it will facilitate the identification of candidate genes to be used in crop protection. The possibility to down-regulate or over express genes in plants or in plant pathogenic organisms is a very powerful tool, within pest management and agriculture in general, to meet farmer and consumer demands.

#### **7. References**

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**5**

*India*

**DNA Sequencing**

G. Darshan Raj

**Improvement of Farm Animal Breeding by**

*Department of Animal Husbandry and Veterinary Services, Government of Karnataka,*

Animal breeds with specific qualities like adaptability to local climatic conditions, farming situations , resistance to diseases and those meet the requirements of local needs and perform under available feeds and fodder are valuable and present requirement for the world. Main farm animals cattle, sheep, goat and pigs were domesticated 9,000–11,000 years ago, whereas the dog was domesticated earlier, ~14,000 years ago, and the chicken ~4,500 years ago. Most domestic animals have a much broader genetic basis. Molecular studies have shown that the two main forms of domestic cattle, European–African (*Bos Taurus taurus*) and Asian (*Bos taurus indicus*), originate from two different subspecies of the wild ancestor (Loftus *et al*., 1994). There are thirty well recognized cattle breeds in India which are highly adoptive to local conditions and each breed has distinctive characters. The world population of buffalo is about 158M in comparison with around 1.3B cattle, 1B sheep and 500-600M goats. There are two types of domestic water buffalo, the River Buffalo (*Bubalus bubalis*) which are more widely spread globally, and are the predominant type found in the west from India to Europe. The second type, the Swamp Buffalo (*Bubalus carabanesis*), is found more frequently in the east from India to the Philippines. The two major types of buffalo can be distinguished by their different karyotypes, the Swamp Buffalo has 48 chromosomes, whereas the River buffalo has 50. However, the two types crossbreed to produce fertile hybrid progeny and so cannot be

In India and Pakistan there are well defined breeds, each with their own individual qualities. In South Asia 18 River buffalo breeds are recognized, which can be placed into 5 major groups designated as the Murrah, Gujarat, Uttar Pradesh, Central Indian and South Indian breeds. The Mediterranean Buffalo is of the River type and has been strongly selected for milk production, particularly in Italy. It has been genetically isolated for a long time and as a result has become genetically distinct from other breeding populations. The high level of milk production of the Mediterranean Buffalo has resulted in it been exported to many

Sheeps belong to the Bovidae family and a ruminant animal. It has 54 chromosomes and average life span 10-15 years. Sheeps are excellent weed destroyer. Sheep Breeds of India are based on the breed distribution in different regions 18 well recognized breeds were

**1. Introduction**

considered as truly different species.

countries worldwide.

## **Improvement of Farm Animal Breeding by DNA Sequencing**

G. Darshan Raj

*Department of Animal Husbandry and Veterinary Services, Government of Karnataka, India*

#### **1. Introduction**

84 DNA Sequencing – Methods and Applications

Wolfe, K.H., Li, W.H. & Sharp, P.M. (1987). Rates of nucleotide substitution vary greatly

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among plant mitochondrial, chloroplast and nuclear DNAs. *Proceedings of the National Academy of Sciences USA,* Vol. 84, No. 24, pp. 9054–9058, ISSN 0027-8424. Zha, W., Peng, X., Chen, R., Du, B., Zhu, L. & He, G. (2011). Knockdown of midgut genes by

dsRNA-transgenic plant-mediated RNA interference in the Hemipteran insect

RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. *Phytopathology*, Vol. 101,

plants expressing two *Bacillus thuringiensis* toxins delay insect resistance evolution.

Animal breeds with specific qualities like adaptability to local climatic conditions, farming situations , resistance to diseases and those meet the requirements of local needs and perform under available feeds and fodder are valuable and present requirement for the world. Main farm animals cattle, sheep, goat and pigs were domesticated 9,000–11,000 years ago, whereas the dog was domesticated earlier, ~14,000 years ago, and the chicken ~4,500 years ago. Most domestic animals have a much broader genetic basis. Molecular studies have shown that the two main forms of domestic cattle, European–African (*Bos Taurus taurus*) and Asian (*Bos taurus indicus*), originate from two different subspecies of the wild ancestor (Loftus *et al*., 1994). There are thirty well recognized cattle breeds in India which are highly adoptive to local conditions and each breed has distinctive characters. The world population of buffalo is about 158M in comparison with around 1.3B cattle, 1B sheep and 500-600M goats. There are two types of domestic water buffalo, the River Buffalo (*Bubalus bubalis*) which are more widely spread globally, and are the predominant type found in the west from India to Europe. The second type, the Swamp Buffalo (*Bubalus carabanesis*), is found more frequently in the east from India to the Philippines. The two major types of buffalo can be distinguished by their different karyotypes, the Swamp Buffalo has 48 chromosomes, whereas the River buffalo has 50. However, the two types crossbreed to produce fertile hybrid progeny and so cannot be considered as truly different species.

In India and Pakistan there are well defined breeds, each with their own individual qualities. In South Asia 18 River buffalo breeds are recognized, which can be placed into 5 major groups designated as the Murrah, Gujarat, Uttar Pradesh, Central Indian and South Indian breeds. The Mediterranean Buffalo is of the River type and has been strongly selected for milk production, particularly in Italy. It has been genetically isolated for a long time and as a result has become genetically distinct from other breeding populations. The high level of milk production of the Mediterranean Buffalo has resulted in it been exported to many countries worldwide.

Sheeps belong to the Bovidae family and a ruminant animal. It has 54 chromosomes and average life span 10-15 years. Sheeps are excellent weed destroyer. Sheep Breeds of India are based on the breed distribution in different regions 18 well recognized breeds were

Improvement of Farm Animal Breeding by DNA Sequencing 87

markers (generally micro satellites) and the trait being analyzed. Markers that tend to cosegregate with the analyzed trait provide approximate chromosomal location of the underlying gene (or genes) involved in part of the trait variability determinism (Yahyaoui,

The candidate gene approach studies the relationship between the traits and known genes that may be associated with the physiological pathways underlying the trait (Liu *et al*., 2008). In other words, this approach assumes that a gene involved in the physiology of the trait could harbor a mutation causing variation in the trait. The gene or part of gene, are sequenced in a number of different animals, and any variation found in the DNA sequences, is tested for association with variation in the phenotypic trait (Koopaei and Koshkoiyeh, 2011). This approach has had some success. For example a mutation was discovered in the estrogen receptor locus (ESR) which results increased litter size in pig. There are 2 problems with the candidate gene approach. Firstly, there are usually a large number of candidate genes affecting the trait, so many genes must be sequenced in several animals and many association studies carried out in a large sample of animals. Secondly, the causative mutation may lie in a gene that would not have been regarded prior as an obvious candidate for this particular trait. Candidate gene approach is performed in 5 steps: 1) collection of resource population. 2) Phenotyping of the traits. 3) Selection of gene or functional polymorphism that potentially could affect the traits. 4) Genotyping of the resource population for genes or functional polymorphism. Lastly, one is statistical analysis of phenotypic and genotypic data (Da, 2003). This is an effective way to find the genes associated with the trait. So far a number of genes have been investigated. Candidate gene approach has been ubiquitously applied for gene disease research, genetic association studies, biomarker and drug target selection in many organisms from animals (Tabor *et al*., 2002). The traditional candidate gene approach is largely limited by its reliance on existing knowledge about the known or presumed biology of the phenotype under investigation, unfortunately the detailed molecular anatomy of most biological traits remain unknown

A candidate gene approach has been already successfully applied to identify several DNA markers associated with production traits in livestock The principle is based on the fact that variability within genes coding for protein products involved in key physiological mechanisms and metabolic pathways directly or indirectly involved in determining an economic trait (e.g. feed efficiency, muscle mass accretion, reproduction efficiency, disease resistance, etc.) might probably explain a fraction of the genetic variability for the production trait itself. The first step is the identification of mutations in candidate genes that can be analyzed in association studies in specific designed experiments. For the roles they play, growth hormone (*GH*) and myostatin (*MSTN*) genes can be considered candidate genes for meat production traits and casein genes in milk production traits (Rothschild and

A typical mammalian genome including most of the farm animal species, contain s around 3x109 base pairs contained in a specific number of chromosomes typical of each species. The

**<IP_ADDRESS> Candidate gene polymorphism and DNA sequencing technique**

2003).

**2.1.2 Candidate gene approach**

(Zhu and Zhao, 2007).

Soller, 1997).

identified in India. There are nearly 102 breeds of goats in the world, of which 20 breeds are in India. Goats are reared for two purposes i.e. meat & milk. Meat and Milk production is the main objective. The domestic goat (*Capra aegagrus hircus*) is a subspecies of goat domesticated from the wild goat of southwest Asia and Eastern Europe. The goat is a member of the Bovidae family and is closely related to the sheep as both are in the goatantelope subfamily Caprinae. Goats are one of the oldest domesticated species. Goats have been used for their milk, meat, hair, and skins over much of the world. In the twentieth century they also gained in popularity as pets.

Farm animal populations harbor rich collections of mutations with phenotypic effects that have been purposefully enriched by breeding. Farm animals are of particular interest for identifying genes that control growth, energy metabolism, development, appetite, reproduction and behavior, as well as other traits that have been manipulated by breeding. Genome research in farm animals will add to our basic understanding of the genetic control of these traits and the results will be applied in breeding programmes to reduce the incidence of disease and to improve product quality and production efficiency (Leif Andersson, 2001).

### **2. Evaluation of animal traits**

Many of the traits of interest in animal production are quantitative traits*.* Evaluation of genetic merit of animals is still essentially based on the application of the theory of quantitative genetics. Traditionally, the genetic improvement of livestock breeds has been based on phenotypic selection. The past century was characterized by the development of quantitative theory and methodology towards the accurate selection and prediction of genetic response *(*Walsh*, 2000)*.This resulted in the selection of a number of economically important genetic traits in cattle, sheep, pigs and poultry. The conceptual basis of this theory is the polygenic model, which assumes that quantitative traits result from the action (and interaction) of a large number of minor genes, each with small effect. The resulting effects are then predicted using powerful statistical methods (animal model), based on pedigree and performance recording of traits from the individual animal and its relatives (Yahyaoui, 2003). Most of morphological markers are sex limited, age dependent, and are significantly influenced by the environment. Biochemical markers show low degree of polymorphism. The various genotypic classes are indistinguishable at the phenotypic level because of the dominance effect of the marker and low genome coverage *(*Montaldo and Meza-Herrera, *1998)*.

#### **2.1 Genome mapping**

The advances in molecular genetics technology in the past two decades, particularly Nucleic acid-based markers, has had a great impact on gene mapping, allowing identification of the underlying genes that control part of the variability of these multigenic traits. Broadly, two experimental strategies have been developed for this purpose: linkage studies and candidate gene approach (Jeffreys *et al*., 1985).

#### **2.1.1 Linkage studies**

Linkage studies rely on the knowledge of the genetic map and search for quantitative trait loci (QTL) by using pedigree materials and comparing segregation patterns of genetic markers (generally micro satellites) and the trait being analyzed. Markers that tend to cosegregate with the analyzed trait provide approximate chromosomal location of the underlying gene (or genes) involved in part of the trait variability determinism (Yahyaoui, 2003).

#### **2.1.2 Candidate gene approach**

86 DNA Sequencing – Methods and Applications

identified in India. There are nearly 102 breeds of goats in the world, of which 20 breeds are in India. Goats are reared for two purposes i.e. meat & milk. Meat and Milk production is the main objective. The domestic goat (*Capra aegagrus hircus*) is a subspecies of goat domesticated from the wild goat of southwest Asia and Eastern Europe. The goat is a member of the Bovidae family and is closely related to the sheep as both are in the goatantelope subfamily Caprinae. Goats are one of the oldest domesticated species. Goats have been used for their milk, meat, hair, and skins over much of the world. In the twentieth

Farm animal populations harbor rich collections of mutations with phenotypic effects that have been purposefully enriched by breeding. Farm animals are of particular interest for identifying genes that control growth, energy metabolism, development, appetite, reproduction and behavior, as well as other traits that have been manipulated by breeding. Genome research in farm animals will add to our basic understanding of the genetic control of these traits and the results will be applied in breeding programmes to reduce the incidence of disease and to improve product quality and production efficiency (Leif

Many of the traits of interest in animal production are quantitative traits*.* Evaluation of genetic merit of animals is still essentially based on the application of the theory of quantitative genetics. Traditionally, the genetic improvement of livestock breeds has been based on phenotypic selection. The past century was characterized by the development of quantitative theory and methodology towards the accurate selection and prediction of genetic response *(*Walsh*, 2000)*.This resulted in the selection of a number of economically important genetic traits in cattle, sheep, pigs and poultry. The conceptual basis of this theory is the polygenic model, which assumes that quantitative traits result from the action (and interaction) of a large number of minor genes, each with small effect. The resulting effects are then predicted using powerful statistical methods (animal model), based on pedigree and performance recording of traits from the individual animal and its relatives (Yahyaoui, 2003). Most of morphological markers are sex limited, age dependent, and are significantly influenced by the environment. Biochemical markers show low degree of polymorphism. The various genotypic classes are indistinguishable at the phenotypic level because of the dominance effect of the marker and

The advances in molecular genetics technology in the past two decades, particularly Nucleic acid-based markers, has had a great impact on gene mapping, allowing identification of the underlying genes that control part of the variability of these multigenic traits. Broadly, two experimental strategies have been developed for this purpose: linkage studies and candidate

Linkage studies rely on the knowledge of the genetic map and search for quantitative trait loci (QTL) by using pedigree materials and comparing segregation patterns of genetic

century they also gained in popularity as pets.

Andersson, 2001).

**2.1 Genome mapping**

**2.1.1 Linkage studies**

gene approach (Jeffreys *et al*., 1985).

**2. Evaluation of animal traits**

low genome coverage *(*Montaldo and Meza-Herrera, *1998)*.

The candidate gene approach studies the relationship between the traits and known genes that may be associated with the physiological pathways underlying the trait (Liu *et al*., 2008). In other words, this approach assumes that a gene involved in the physiology of the trait could harbor a mutation causing variation in the trait. The gene or part of gene, are sequenced in a number of different animals, and any variation found in the DNA sequences, is tested for association with variation in the phenotypic trait (Koopaei and Koshkoiyeh, 2011). This approach has had some success. For example a mutation was discovered in the estrogen receptor locus (ESR) which results increased litter size in pig. There are 2 problems with the candidate gene approach. Firstly, there are usually a large number of candidate genes affecting the trait, so many genes must be sequenced in several animals and many association studies carried out in a large sample of animals. Secondly, the causative mutation may lie in a gene that would not have been regarded prior as an obvious candidate for this particular trait. Candidate gene approach is performed in 5 steps: 1) collection of resource population. 2) Phenotyping of the traits. 3) Selection of gene or functional polymorphism that potentially could affect the traits. 4) Genotyping of the resource population for genes or functional polymorphism. Lastly, one is statistical analysis of phenotypic and genotypic data (Da, 2003). This is an effective way to find the genes associated with the trait. So far a number of genes have been investigated. Candidate gene approach has been ubiquitously applied for gene disease research, genetic association studies, biomarker and drug target selection in many organisms from animals (Tabor *et al*., 2002). The traditional candidate gene approach is largely limited by its reliance on existing knowledge about the known or presumed biology of the phenotype under investigation, unfortunately the detailed molecular anatomy of most biological traits remain unknown (Zhu and Zhao, 2007).

A candidate gene approach has been already successfully applied to identify several DNA markers associated with production traits in livestock The principle is based on the fact that variability within genes coding for protein products involved in key physiological mechanisms and metabolic pathways directly or indirectly involved in determining an economic trait (e.g. feed efficiency, muscle mass accretion, reproduction efficiency, disease resistance, etc.) might probably explain a fraction of the genetic variability for the production trait itself. The first step is the identification of mutations in candidate genes that can be analyzed in association studies in specific designed experiments. For the roles they play, growth hormone (*GH*) and myostatin (*MSTN*) genes can be considered candidate genes for meat production traits and casein genes in milk production traits (Rothschild and Soller, 1997).

#### **<IP_ADDRESS> Candidate gene polymorphism and DNA sequencing technique**

A typical mammalian genome including most of the farm animal species, contain s around 3x109 base pairs contained in a specific number of chromosomes typical of each species. The

Improvement of Farm Animal Breeding by DNA Sequencing 89

suggest that an animal is heterozygous. However animal may be misclassified as homozygote if there is polymorphism in the primer sequence, which prevents the allele from being amplified and therefore not being detected on the electrophoresis gel referred to as null allele. A null allele will often be detected when misparentage routinely found for a marker system. An animal might also be misclassified, if another, non allelic form was amplified with the PCR primers and digestion of the PCR product results in a different sized fragments. A Non allelic form is revealed by sequencing the fragments contained within the electrophoresis bands, which is recommended step when establishing marker system (Jiang

However new technologies have significantly advanced our ability to identify SNP's and then explore multiple candidate genes at one time at a much lower cost/polymorphism than the PCR-RFLP method. The identification SNP's within a gene or genetic region is now relatively easy To do this , genomic DNA of key animals within a population are sequenced using high throughput automatic sequencing and then compared with the other sequences

This approach is already being employed with regard to bovine leukocyte adhesion deficiency (BLAD; Shuster *et al*., 1992) and genes with major effects, such as the halothane locus in swine (Rempel *et al*., 1993) and alpha S1-casein in goat (Manfredi *et al.,* 1995).Currently, direct sequencing is one of the high throughput methods for mutation detection, and is the most accurate method to determine the exact nature of a polymorphism. Sanger dideoxy-sequencing can detect any type of unknown polymorphism and its position, when the majority of DNA contains that polymorphism. Fluorescent sequencing can have variable sensitivity and specificity in detecting heterozygotes because of the inconsistency of base-calling of these sites (Yan *et al.,* 2000). Thus, it has only limited utility when the polymorphism is present in a minor fraction of the total DNA (for example in pooled samples of DNA or in solid tumors) due to low sensitivity. DNA sequencing is usually used as a second step to confirm and identify the exact base altered in the target

The revolution in molecular genetics, and particularly genome sequencing, has already provided benefits for animal breeding, but in comparison with what the future holds, our present tools will undoubtedly be seen as primitive. Complete genome sequences are available for a number of species, genome sequences for the chicken, cow, horse, mouse and chimpanzee are either completed or nearing completion, and single nucleotide polymorphism (SNP) libraries for these species are growing rapidly. This information will underpin most of the developments in livestock breeding and breed management during the coming two decades A quite recent source of obtaining affordable sequence data are high throughput next-generation sequencing (NGS) technologies (Illumina GA/Solexa, RocheGS/454, Solid, Helicos). The massively parallel approaches in these technologies allows much faster and more cost-effective sequence 16 determination than the traditional dideoxy chain terminator method described by Sanger in 1977. The throughput for these new sequencing approaches is measured in billions of base pairs per run, thousand times more than the daily output of an automated 96 capillary DNA sequencer using dyeterminator sequencing technology. High throughput is achieved by immobilization of 400

within the population or to sequences which are publically available databases .

region previously identified as polymorphic by using scanning methods.

*<IP_ADDRESS>.1 Different methods of DNA sequencing*

and Ott, 2010).

number of human genes was initially estimated at around 50,000-1, 00,000. However, based on the recent complete sequencing of the human genome in the 2002 year , this number has been revised to 25000-30000 only. A similar estimate has been reported for most of the farm animal species. These genes prescribe the development of the living species and contribute to the biological biodiversity of our planet. A feature of the genome that has confused geneticists for years is that the coding sequences (genes) only represent some 3to 5 percent on the genome. Whereas the rest is sequences (genes) unknown functions including different types of repetitive DNA, introns, 5' and 3' untranslated regions and pseudogenes. The sizes of genes vary from less than 100 bp to over 2000kb (Fields et *al*., 1994).

Recent developments in DNA technologies have made it possible to uncover a large number of genetic polymorphisms at the DNA sequence level and to use them as markers for evaluation of the genetic basis for observed phenotypic variability. These markers posses unique genetic properties and methodological advantages that make them more usefull and amendable for genetic analysis and other genetic markers. The possible applications of molecular markers in livestock industry are by conventional breeding programme to transgenic breeding technologies. In conventional breeding strategies molecular markers have several short range or immediate applications viz., parentage determination , genetic distance estimation , determination of twin zygosity and free martinism, sexing preimplation embryo and identification of genetic disease carrier and long range applications viz ., Genome mapping and marker assisted selection. In transgenic breeding molecular markers can be used as reference points for identification of the animals carrying the trasgenes. The progress in the genetic markers suggests their potential use for genetic improvement in the livestock species.

The progress in the DNA recombinant technology , gene cloning and High throughput Sequencing technique during last two decades have brought revolutionary changes in the field of basic as well as applied genetics which provide several new approaches for genome analysis with greater genetic resolution . it is now possible to uncover a large number of genetic polymorphisms at DNA sequence level and to use them as markers for evaluation of the genetic basis for observed phenotypic variability. The first demonstration of DNA level polymorphism, known as restriction fragment polymorphism (RFLP) an almost unlimited number of molecular markers have accumulated. Currently, more powerful and less laborious techniques to uncover new types of DNA makers are steadily being introduced. The introduction of polymerase chain reaction (PCR) in conjunction with the constantly increasing DNA sequence data also represents a mile stone in this endeavor.

In the past polymorphisms in a candidate gene were routinely detected by PCR –RFLP , which involves the amplification of the gene and digesting the amplicon with restriction enzymes and then using gel electrophoresis to separate resulting fragments. In PCR-RFLP technique the gene difference among animals are detected by whether or not restriction enzymes cuts or not resulting in different sized fragments. The genetic differences are usually due to an SNP within the restriction enzyme recognition site, although there might be genetic differences due to insertions and deletions (ins /del) in the gene, which will also results in fragment size differences , although there is no variation in the restriction enzyme recognition site. Animals are expected to have two alleles for every gene except for X and Y chromosomes in males, so that one fragment on the electrophoresis gel would indicate homozygous for the PCR-RFLP allele, where as two different sized fragments would

number of human genes was initially estimated at around 50,000-1, 00,000. However, based on the recent complete sequencing of the human genome in the 2002 year , this number has been revised to 25000-30000 only. A similar estimate has been reported for most of the farm animal species. These genes prescribe the development of the living species and contribute to the biological biodiversity of our planet. A feature of the genome that has confused geneticists for years is that the coding sequences (genes) only represent some 3to 5 percent on the genome. Whereas the rest is sequences (genes) unknown functions including different types of repetitive DNA, introns, 5' and 3' untranslated regions and pseudogenes.

Recent developments in DNA technologies have made it possible to uncover a large number of genetic polymorphisms at the DNA sequence level and to use them as markers for evaluation of the genetic basis for observed phenotypic variability. These markers posses unique genetic properties and methodological advantages that make them more usefull and amendable for genetic analysis and other genetic markers. The possible applications of molecular markers in livestock industry are by conventional breeding programme to transgenic breeding technologies. In conventional breeding strategies molecular markers have several short range or immediate applications viz., parentage determination , genetic distance estimation , determination of twin zygosity and free martinism, sexing preimplation embryo and identification of genetic disease carrier and long range applications viz ., Genome mapping and marker assisted selection. In transgenic breeding molecular markers can be used as reference points for identification of the animals carrying the trasgenes. The progress in the genetic markers suggests their potential use for genetic

The progress in the DNA recombinant technology , gene cloning and High throughput Sequencing technique during last two decades have brought revolutionary changes in the field of basic as well as applied genetics which provide several new approaches for genome analysis with greater genetic resolution . it is now possible to uncover a large number of genetic polymorphisms at DNA sequence level and to use them as markers for evaluation of the genetic basis for observed phenotypic variability. The first demonstration of DNA level polymorphism, known as restriction fragment polymorphism (RFLP) an almost unlimited number of molecular markers have accumulated. Currently, more powerful and less laborious techniques to uncover new types of DNA makers are steadily being introduced. The introduction of polymerase chain reaction (PCR) in conjunction with the constantly increasing DNA sequence data also represents a mile stone in this endeavor. In the past polymorphisms in a candidate gene were routinely detected by PCR –RFLP , which involves the amplification of the gene and digesting the amplicon with restriction enzymes and then using gel electrophoresis to separate resulting fragments. In PCR-RFLP technique the gene difference among animals are detected by whether or not restriction enzymes cuts or not resulting in different sized fragments. The genetic differences are usually due to an SNP within the restriction enzyme recognition site, although there might be genetic differences due to insertions and deletions (ins /del) in the gene, which will also results in fragment size differences , although there is no variation in the restriction enzyme recognition site. Animals are expected to have two alleles for every gene except for X and Y chromosomes in males, so that one fragment on the electrophoresis gel would indicate homozygous for the PCR-RFLP allele, where as two different sized fragments would

The sizes of genes vary from less than 100 bp to over 2000kb (Fields et *al*., 1994).

improvement in the livestock species.

suggest that an animal is heterozygous. However animal may be misclassified as homozygote if there is polymorphism in the primer sequence, which prevents the allele from being amplified and therefore not being detected on the electrophoresis gel referred to as null allele. A null allele will often be detected when misparentage routinely found for a marker system. An animal might also be misclassified, if another, non allelic form was amplified with the PCR primers and digestion of the PCR product results in a different sized fragments. A Non allelic form is revealed by sequencing the fragments contained within the electrophoresis bands, which is recommended step when establishing marker system (Jiang and Ott, 2010).

However new technologies have significantly advanced our ability to identify SNP's and then explore multiple candidate genes at one time at a much lower cost/polymorphism than the PCR-RFLP method. The identification SNP's within a gene or genetic region is now relatively easy To do this , genomic DNA of key animals within a population are sequenced using high throughput automatic sequencing and then compared with the other sequences within the population or to sequences which are publically available databases .

This approach is already being employed with regard to bovine leukocyte adhesion deficiency (BLAD; Shuster *et al*., 1992) and genes with major effects, such as the halothane locus in swine (Rempel *et al*., 1993) and alpha S1-casein in goat (Manfredi *et al.,* 1995).Currently, direct sequencing is one of the high throughput methods for mutation detection, and is the most accurate method to determine the exact nature of a polymorphism. Sanger dideoxy-sequencing can detect any type of unknown polymorphism and its position, when the majority of DNA contains that polymorphism. Fluorescent sequencing can have variable sensitivity and specificity in detecting heterozygotes because of the inconsistency of base-calling of these sites (Yan *et al.,* 2000). Thus, it has only limited utility when the polymorphism is present in a minor fraction of the total DNA (for example in pooled samples of DNA or in solid tumors) due to low sensitivity. DNA sequencing is usually used as a second step to confirm and identify the exact base altered in the target region previously identified as polymorphic by using scanning methods.

#### *<IP_ADDRESS>.1 Different methods of DNA sequencing*

The revolution in molecular genetics, and particularly genome sequencing, has already provided benefits for animal breeding, but in comparison with what the future holds, our present tools will undoubtedly be seen as primitive. Complete genome sequences are available for a number of species, genome sequences for the chicken, cow, horse, mouse and chimpanzee are either completed or nearing completion, and single nucleotide polymorphism (SNP) libraries for these species are growing rapidly. This information will underpin most of the developments in livestock breeding and breed management during the coming two decades A quite recent source of obtaining affordable sequence data are high throughput next-generation sequencing (NGS) technologies (Illumina GA/Solexa, RocheGS/454, Solid, Helicos). The massively parallel approaches in these technologies allows much faster and more cost-effective sequence 16 determination than the traditional dideoxy chain terminator method described by Sanger in 1977. The throughput for these new sequencing approaches is measured in billions of base pairs per run, thousand times more than the daily output of an automated 96 capillary DNA sequencer using dyeterminator sequencing technology. High throughput is achieved by immobilization of 400

Improvement of Farm Animal Breeding by DNA Sequencing 91

from 50 to 400 bp. Further, it is promised that new, third generation single molecule sequencing will soon be available and should deliver complete genome sequence that is in

NGS technologies allow cost effective sequencing of multiple individuals which is beneficial for SNP detection. SNP discovery through parallel pyrosequencing (454) of an individual human genome identified 3.32 million SNPs, with 606,797 of those as novel SNPs (Wheeler *et al* ., 2008) which is comparable with the shotgun-sequenced Venter genome (cost \$70 million) that had 3.47 million SNPs, with 647,767 of those being novel (Wheeler *et al* ., 2008) .These authors stated that at least a 20X genome coverage is required to call 99% of heterozygous bases correctly within a single individual, resulting in a sequencing cost of 2 million dollar. For the Illumina method similar results (3.07 million SNPs of which 420 thousand novel SNPs) can be obtained by sequencing an individual genome with 36X coverage for less than half a million dollars (Wang *et al* ., 2008) or ~ 4 million SNPs of which 26% are novel SNPs at 30X coverage for a quarter of a million dollar. (Bentley *et al* ., 2008). More recent developments using single molecule sequencing further reduce the sequencing costs. For example, the discovery of 2.8 million SNPs by whole genome resequencing at 28X coverage using this technology reduces the costs to less than 50 thousand dollars (Pushkarev *et al* ., 2009). These developments indicate that extremely high throughput sequencing machines that produce relatively short reads are favorable for SNP discovery in a whole genome resequencing approach. In addition, simulations suggest that 85% of 35 bp reads can be placed uniquely on the human genome whereas 95% of 35 bp paired end reads with 200 bp insert sizes have unique placement (Li *et al*. ,2008). However SNP discovery in species with limited public genomic resources benefits from longer read lengths. It has been demonstrated that reads produced with pyrosequencing technology can be assembled de novo into reasonably long contigs that subsequently can serve as a genome reference on which short reads of other individuals can be mapped to detect SNPs (Novaes *et al*., 2008). At present whole genome sequence assembly by alignment of relative short NGS fragments without the availability of a reference genome is tedious, but possible for less complex mammalian sized genomes (Li *et al* ., 2009). Besides the consistent pattern of non-uniform sequence coverage each NGS platform generates (Harismendy *et al.,*  2009), which is substantially lower in AT-rich repetitive sequences, repetitive regions are hard to reconstruct by short-read sequence assembly. Possible NGS SNP detection strategies circumventing the requirement of a sequenced reference genome are (ultra short read) sequencing of more than one genotype and alignment of that data by using: (1) genome or transcriptome sequence data from model species closely related to the species of interest, (2) whole transcriptome or reduced representative genome sequence data for the species of

time and pricing affordable for every research group (Perez-Enciso and Ferretti, 2010)

interest, based on Roche/454 sequence technology (Wiedmann *et al* ., 2008).

Farm animals are quite valuable as resources, often notable as models for pathology and physiology studies. The reproductive physiology of farm animals is more similar to humans than that of rodents because farm animals have longer gestation and pre-pubertal periods than mice. Specific farm animal physiology, such as the digestive system of the pig is similar to that of humans. These attributes of farm animals reveal that they are an unparalleled resource for research replicating human physiological function. For decades, breeders have altered the genomes of farm animals by first searching for desired phenotypic traits and then selecting for superior animals to continue their lineage into the next generation. This genomic work has already facilitated a reduction in genetic disorders in

thousand (Roche) to 40 million (Illumina) target DNAs and sequencing these fragments simultaneously. Furthermore, these new technologies allow the direct sequencing of DNA or cDNA without any cloning step and at decreasing cost per sequenced base . Although all of these new technologies produced extremely short reads when they initially entered the market, the performance of (NGS) platforms have increased substantially. As an example: the first nextgeneration sequencer released by 454 (GS20) had an average read-length of 110 bp. A second improved sequencer has since then been introduced, the GS-FLX, which is able to obtain average read lengths of 250 bases and is able to perform mate-paired reads. A more recent release is the 454 FLX Titanium which has an average read length of 400 bp

Table 1. Accuracy of different DNA sequencing methods: Kerstens, (2010).

Throughput is compared by giving the maximum number of bases per second a platform generates at it's optimal read length. Paired-end sequencing is for some platforms restricted to a read length. Sequencing cost is compared by indicating the price per mega base and only includes the costs of reagents and costs to perform one sequencing run.

The impact of massive parallel sequencing or next generation sequencing (NGS) in Biology, and hence in Animal Genetics, is difficult to overstate. It is a revolution comparable with the one that followed Sanger sequencing forty years ago. It is not just a dramatic increase in sequencing speed: it means a change in paradigm that obliges researchers, institutions and funding agencies alike. It is also a tremendous and passionate technological race worth millions. Although it is uncertain which, if any, of the extant technologies will prevail in the future, and whether they may be replaced by new technologies, one thing is certain: next generation sequencing is this generation sequencing. Current NGS technologies provide a throughput which is at least 100 times that of classical Sanger sequencing and the technologies are quickly improving. Compared with standard sequencing, these technologies do not need cloning, i.e. they are basically shotgun sequencing and result in shorter sequences, currently

thousand (Roche) to 40 million (Illumina) target DNAs and sequencing these fragments simultaneously. Furthermore, these new technologies allow the direct sequencing of DNA or cDNA without any cloning step and at decreasing cost per sequenced base . Although all of these new technologies produced extremely short reads when they initially entered the market, the performance of (NGS) platforms have increased substantially. As an example: the first nextgeneration sequencer released by 454 (GS20) had an average read-length of 110 bp. A second improved sequencer has since then been introduced, the GS-FLX, which is able to obtain average read lengths of 250 bases and is able to perform mate-paired reads. A more recent release is the 454 FLX Titanium which has an average read length of 400 bp

**(bps)**

dye terminators 1 Mbp/day(12) 800 99.0-

3 Gbp/5days(6.9 K)

3Gbp/5days

7.5 Gbp/4days

Throughput is compared by giving the maximum number of bases per second a platform generates at it's optimal read length. Paired-end sequencing is for some platforms restricted to a read length. Sequencing cost is compared by indicating the price per mega base and

The impact of massive parallel sequencing or next generation sequencing (NGS) in Biology, and hence in Animal Genetics, is difficult to overstate. It is a revolution comparable with the one that followed Sanger sequencing forty years ago. It is not just a dramatic increase in sequencing speed: it means a change in paradigm that obliges researchers, institutions and funding agencies alike. It is also a tremendous and passionate technological race worth millions. Although it is uncertain which, if any, of the extant technologies will prevail in the future, and whether they may be replaced by new technologies, one thing is certain: next generation sequencing is this generation sequencing. Current NGS technologies provide a throughput which is at least 100 times that of classical Sanger sequencing and the technologies are quickly improving. Compared with standard sequencing, these technologies do not need cloning, i.e. they are basically shotgun sequencing and result in shorter sequences, currently

Table 1. Accuracy of different DNA sequencing methods: Kerstens, (2010).

only includes the costs of reagents and costs to perform one sequencing run.

1. Applied Biosystems http://www.appliedbyosystems.com, 2. Roche Applied Science http://www.roche-applied-science.com,

4. Applied Biosystems http://www.appliedbyosystems.com 5. Helicosbio http://www.helicosbio.com (Kerstens, 2010)

**Read length**  **%**

(3.7K) 250 96.0-99.5 2x110 <sup>30</sup>

(6.9K) 35 99.0>99.94 2x25 1.30

(22K) 25 93->99.0 no n/a

>99,999 no

36 96.2-99.7 2x36 2.10

**Accuracy Paired end \$/Mbp**

1000

**Technology Approach Maxthrougput**

Synthesis with

FLX2 Pyrosequencing 100Mb/7.5hr

Sequencing by synthesis

ligation

sequencing

3. Illumina, Inc. http://www.illumina.com

ABI/SOLiD4 sequencing by

Helicos5 single-molecule

Sanger ABI3730xl1

xa3

454/Roche

Illumina/Sole

from 50 to 400 bp. Further, it is promised that new, third generation single molecule sequencing will soon be available and should deliver complete genome sequence that is in time and pricing affordable for every research group (Perez-Enciso and Ferretti, 2010)

NGS technologies allow cost effective sequencing of multiple individuals which is beneficial for SNP detection. SNP discovery through parallel pyrosequencing (454) of an individual human genome identified 3.32 million SNPs, with 606,797 of those as novel SNPs (Wheeler *et al* ., 2008) which is comparable with the shotgun-sequenced Venter genome (cost \$70 million) that had 3.47 million SNPs, with 647,767 of those being novel (Wheeler *et al* ., 2008) .These authors stated that at least a 20X genome coverage is required to call 99% of heterozygous bases correctly within a single individual, resulting in a sequencing cost of 2 million dollar. For the Illumina method similar results (3.07 million SNPs of which 420 thousand novel SNPs) can be obtained by sequencing an individual genome with 36X coverage for less than half a million dollars (Wang *et al* ., 2008) or ~ 4 million SNPs of which 26% are novel SNPs at 30X coverage for a quarter of a million dollar. (Bentley *et al* ., 2008). More recent developments using single molecule sequencing further reduce the sequencing costs. For example, the discovery of 2.8 million SNPs by whole genome resequencing at 28X coverage using this technology reduces the costs to less than 50 thousand dollars (Pushkarev *et al* ., 2009). These developments indicate that extremely high throughput sequencing machines that produce relatively short reads are favorable for SNP discovery in a whole genome resequencing approach. In addition, simulations suggest that 85% of 35 bp reads can be placed uniquely on the human genome whereas 95% of 35 bp paired end reads with 200 bp insert sizes have unique placement (Li *et al*. ,2008). However SNP discovery in species with limited public genomic resources benefits from longer read lengths. It has been demonstrated that reads produced with pyrosequencing technology can be assembled de novo into reasonably long contigs that subsequently can serve as a genome reference on which short reads of other individuals can be mapped to detect SNPs (Novaes *et al*., 2008). At present whole genome sequence assembly by alignment of relative short NGS fragments without the availability of a reference genome is tedious, but possible for less complex mammalian sized genomes (Li *et al* ., 2009). Besides the consistent pattern of non-uniform sequence coverage each NGS platform generates (Harismendy *et al.,*  2009), which is substantially lower in AT-rich repetitive sequences, repetitive regions are hard to reconstruct by short-read sequence assembly. Possible NGS SNP detection strategies circumventing the requirement of a sequenced reference genome are (ultra short read) sequencing of more than one genotype and alignment of that data by using: (1) genome or transcriptome sequence data from model species closely related to the species of interest, (2) whole transcriptome or reduced representative genome sequence data for the species of interest, based on Roche/454 sequence technology (Wiedmann *et al* ., 2008).

Farm animals are quite valuable as resources, often notable as models for pathology and physiology studies. The reproductive physiology of farm animals is more similar to humans than that of rodents because farm animals have longer gestation and pre-pubertal periods than mice. Specific farm animal physiology, such as the digestive system of the pig is similar to that of humans. These attributes of farm animals reveal that they are an unparalleled resource for research replicating human physiological function. For decades, breeders have altered the genomes of farm animals by first searching for desired phenotypic traits and then selecting for superior animals to continue their lineage into the next generation. This genomic work has already facilitated a reduction in genetic disorders in

Improvement of Farm Animal Breeding by DNA Sequencing 93

Cat Felis catus Mammal Draft Assembly WUGSC Completed

**(7X)**

Draft Assembly

familiaris Mammal Draft Assembly BI/MIT Completed

**BCM-**

(6 ,6X) WUGSC Completed

(7X) BI/MIT Completed

(~ 2X) BI/MIT Completed

(BAC to BAC) Sanger Completed

(BAC to BAC) In process

**HGSC Completed**

HGSC In process

HGSC Completed

**Animal Publication Year Journal**  Chicken ( Gallus sonneratii) 2004 Nature Bovine (Bos taurus) 2009 Science Horse (Equus caballus) 2009 Science Cat (Felis catus) 2007 Genome research Dog (Canis familiaris) 2005 Nature Pig 2009 Nature Sheep 2010 Animal Genetics

Table 2. Important Species with known reference sequences:

Chicken Gallus gallus

Dog Canis

Horse Equus

Rabbit Oryctolagus

Buffaloe Bubalus

USA.Sangers institute, UK.

**Cow Bos taurus Mammal Draft Assembly**

Pig Sus scrofa Mammal Draft Assembly

Washington University Genomic sequencing Center USA

Table 3. Livestock Genome sequenced. Eggen, (2008).

Non mammalian vertebrate

caballas Mammal Draft Assembly

cuniculus Mammal Low coverage

bubalis Mammal Draft Assembly

BCM –HGSC - Baylor College of Medicine, Houston, Human Genome sequence center USA Wu

BI/MIT Broad institute / Massachusetts Institute of Technology center for Genome research

**2.3 Practical advantages animal breeding by DNA sequencing technology**

Advances in genomic technology in recent years were driven primarily by human DNA sequencing and genotyping projects. The human sequence was completed in 2001, the cattle sequence in 2004. Design of the cattle SNP chip required obtaining equally spaced SNP that are highly polymorphic across many dairy and beef breeds (Van Tassell *et al*., 2008) Breeders now use thousands of genetic markers to select and improve animals. Previously only phenotypes and pedigrees were used in selection, but performance and parentage information was collected, stored, and evaluated affordably and routinely for many traits

Sheep Ovis aries Mammal Draft Assembly BCM-

Goat Caprine Mammal Draft Assembly BCM-

farm animals, as many disease carriers are removed from breeding populations by purifying selection. By studying diverse phenotype over time, researchers can now monitor mutations that occur as wild species become domesticated (Fadiel *et al*., 2005).

#### **2.2 Animal genome projects**

According to Wikipedia, Genome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, anarchaean, a protist or a virus) and to annotate protein-coding genes and other important genome-encoded features. The genome sequence of an organism includes the collective DNA sequences of each chromosome in the organism. The release of the first draft of the chicken genome in March 2004 spawned the current boom in chicken genomic research (Antin and Konieczka, 2005) evolutionary standpoint; investigation of the chicken genome will provide significant information needed to understand the vertebrate genome evolution, since the chicken is between the mammal and fish on the evolutionary tree. Furthermore, the chicken remains significant as a food animal which comprises 41% of the meat produced in the world and serves as a reliable model for the study of diseases and developmental biology (Dequeant and Pourquie, 2005). With this sequenced genome, chicken breeders will have a framework for investigating polymorphisms of informative quantitative traits to continue their directed evolution of these species (Fadiel *et al*., 2005)

The sequencing of the pig genome generated an invaluable resource for advancements in enzymology, reproduction, endocrinology, nutrition and biochemistry research (Wernersson, *et al*., 2005 and Rothschild, *et al*., 2003) Since pigs are evolutionarily distinct to both humans and rodents, but have co-evolved with these species, the diversity of selected phenotypes make the pig a useful model for the study of genetic and environmental interactions with polygenic traits (Blakesley *et al*., 2004). The sequencing of the pig genome is also instrumental in the improvement of human health. Clinical studies in areas such as infectious disease, organ transplantation, physiology, metabolic disease, pharmacology, obesity and cardiovascular disease have used pig models (Rothschild, 2004). In the near future, the sequencing of the porcine genome will allow gene markers for specific diseases to be identified, assisting breeders in generating pig stocks resistant to infectious diseases (Klymiuk, and Aigner, 2005 and Fadiel *et al* ., 2005)

Cattle is of great interest since it represents a group of eutherian mammals phylogenetically distant from primates (Larkin, *et al*., 2003 and Kumar, and Hedges, 1998). Working with the cow species, B.taurus, is significant because the cow is such an economically important animal. This form of livestock makes up the beef and milk production industry. The identification of numerous single-nucleotide polymorphisms (SNPs) makes it possible for geneticists to find associations between certain genes and cow traits that will eventually lead to the production of superior-quality beef (Adam, 2002).

Bioinformatics researchers from New Zealand, US, UK and Australia have come together to work on the sheep genome map. The focal point of interest in sheep is based on the quest to maximize sheep meat and cotton wool production (Fadiel *et al*., 2005).These studies have revealed the existence of mutations that yield phenotypes unique to the sheep, emonstrating that genetic analysis of the sheep can enhance our knowledge of biological pathways in other mammalian species (Cockett *et al*., 2001 and Mouchel *et al*.,2001).

farm animals, as many disease carriers are removed from breeding populations by purifying selection. By studying diverse phenotype over time, researchers can now monitor mutations

According to Wikipedia, Genome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, anarchaean, a protist or a virus) and to annotate protein-coding genes and other important genome-encoded features. The genome sequence of an organism includes the collective DNA sequences of each chromosome in the organism. The release of the first draft of the chicken genome in March 2004 spawned the current boom in chicken genomic research (Antin and Konieczka, 2005) evolutionary standpoint; investigation of the chicken genome will provide significant information needed to understand the vertebrate genome evolution, since the chicken is between the mammal and fish on the evolutionary tree. Furthermore, the chicken remains significant as a food animal which comprises 41% of the meat produced in the world and serves as a reliable model for the study of diseases and developmental biology (Dequeant and Pourquie, 2005). With this sequenced genome, chicken breeders will have a framework for investigating polymorphisms of informative quantitative traits to continue their directed evolution of these species (Fadiel *et al*., 2005)

The sequencing of the pig genome generated an invaluable resource for advancements in enzymology, reproduction, endocrinology, nutrition and biochemistry research (Wernersson, *et al*., 2005 and Rothschild, *et al*., 2003) Since pigs are evolutionarily distinct to both humans and rodents, but have co-evolved with these species, the diversity of selected phenotypes make the pig a useful model for the study of genetic and environmental interactions with polygenic traits (Blakesley *et al*., 2004). The sequencing of the pig genome is also instrumental in the improvement of human health. Clinical studies in areas such as infectious disease, organ transplantation, physiology, metabolic disease, pharmacology, obesity and cardiovascular disease have used pig models (Rothschild, 2004). In the near future, the sequencing of the porcine genome will allow gene markers for specific diseases to be identified, assisting breeders in generating pig stocks resistant to infectious diseases

Cattle is of great interest since it represents a group of eutherian mammals phylogenetically distant from primates (Larkin, *et al*., 2003 and Kumar, and Hedges, 1998). Working with the cow species, B.taurus, is significant because the cow is such an economically important animal. This form of livestock makes up the beef and milk production industry. The identification of numerous single-nucleotide polymorphisms (SNPs) makes it possible for geneticists to find associations between certain genes and cow traits that will eventually lead

Bioinformatics researchers from New Zealand, US, UK and Australia have come together to work on the sheep genome map. The focal point of interest in sheep is based on the quest to maximize sheep meat and cotton wool production (Fadiel *et al*., 2005).These studies have revealed the existence of mutations that yield phenotypes unique to the sheep, emonstrating that genetic analysis of the sheep can enhance our knowledge of biological pathways in

that occur as wild species become domesticated (Fadiel *et al*., 2005).

(Klymiuk, and Aigner, 2005 and Fadiel *et al* ., 2005)

to the production of superior-quality beef (Adam, 2002).

other mammalian species (Cockett *et al*., 2001 and Mouchel *et al*.,2001).

**2.2 Animal genome projects**

Table 2. Important Species with known reference sequences:

BCM –HGSC - Baylor College of Medicine, Houston, Human Genome sequence center USA Wu Washington University Genomic sequencing Center USA

BI/MIT Broad institute / Massachusetts Institute of Technology center for Genome research USA.Sangers institute, UK.

Table 3. Livestock Genome sequenced. Eggen, (2008).

#### **2.3 Practical advantages animal breeding by DNA sequencing technology**

Advances in genomic technology in recent years were driven primarily by human DNA sequencing and genotyping projects. The human sequence was completed in 2001, the cattle sequence in 2004. Design of the cattle SNP chip required obtaining equally spaced SNP that are highly polymorphic across many dairy and beef breeds (Van Tassell *et al*., 2008) Breeders now use thousands of genetic markers to select and improve animals. Previously only phenotypes and pedigrees were used in selection, but performance and parentage information was collected, stored, and evaluated affordably and routinely for many traits

Improvement of Farm Animal Breeding by DNA Sequencing 95

the BovinSNP50 BeadChip (VanRaden *et al*., 2009; Cole *et al*., 2009). Genotypic and phenotypic data of these bulls were used to estimate the effects of 38,416 SNP markers (after discarding markers with low minor allele frequency and markers that were in complete linkage disequilibrium with adjacent markers) on production, type, longevity, udder health, and calving ability. Next, the estimated SNP effects were used to compute the genomic PTA of each of 2,035 young Holstein bulls born from 2000-2003 that had no progeny. Finally, the 2009 PTA of each bull in the latter group, which was based on information from its progeny, was compared with the traditional PA and the genomic PTA computed from 2004 data. The same process was repeated in the Jersey breed (1,361 older animals and 388 young bulls) and the Brown Swiss breed (512 older animals and 150 young bulls). Results in Table IV show the increase in reliability (**REL**) due to genomic information, as compared with the

**Trait Increased reliability due to inclusion Genomic data Holstein Jersey Brown Swiss**  Net merit +24 +8 +9 Milk yield +26 +6 +17 Fat yield +32 +11 +10 Protein yield +24 +2 +14 Fat percentage +50 +36 +8 Protein percentage +38 +29 +10 Productive life +32 +7 +12 Somatic cell score +23 +3 +17 Daughter pregnancy rate +28 +7 +18 Final classical score +20 +2 +5 Udder depth +37 +20 +8 Foot angel +25 +11 -1 Table 4. Reliability changes due to the inclusion of genomic data in national genetic

Recent advancements in sequencing and genotyping technologies have enabled a rapid evolution in methods for beef cattle selection. New tools will become available for beef producers to implement in the endeavor to efficiently produce high quality beef for today's consumer. Tools such as high-density genotyping assays and next generation sequencing instruments will help to shorten the generation interval, aid in the identification of causal mutations, increase the accuracy of EPDs on young sires and dams, provide information on gene expression and enhance our understanding of epigenetic and gut microbiome effects on cattle phenotypes. The ultimate objective of improving beef quality either by breeding or rearing factors. For genetic purposes, polymorphisms in some key genes have been reported for their association with beef quality traits. The sequencing of the bovine genome has dramatically increased the number of available gene polymorphisms. The association of these new polymorphisms with the variability in beef quality (e.g. tenderness, marbling) for different breeds in different rearing systems will be a very important issue. For rearing purposes, global gene expression profiling at the mRNA or protein level has already shown that previously unsuspected genes may be associated either with muscle development or

REL from parent average information only.

evaluations VanRaden *et al*.,(2009).

**<IP_ADDRESS> Beef animals**

and many millions of animals. Genetic markers had limited use during the century after Mendel's principles of genetic inheritance were rediscovered because few major QTL were identified and because marker genotypes were expensive to obtain before 2008. Genomic evaluations implemented in the last two years for dairy cattle have greatly improved reliability of selection, especially for younger animals, by using many markers to trace the inheritance of many QTL with small effects. More genetic markers can increase both reliability and cost of genomic selection. Genotypes for 50,000 markers now cost <US\$200 per animal for cattle, pigs, chickens, and sheep. Lower cost chips containing fewer (2,900) markers and higher cost chips with more (777,000) markers are already available for cattle, and additional genotyping tools will become available for cattle and other species VanRaden *et al*., (2011). Livestock selection has used estimated breeding values (EBV) based on phenotypic data and pedigree records for more than 40 years. More recently, advances in molecular genetic techniques, in particular DNA sequencing, have led to the discovery of regions of the genome that influence traits in livestock. However utilizing both sources of data in genetic evaluation schemes, such as BREEDPLAN, has been a challenge due to the heterogeneity of data sources, the multi-trait nature of the evaluations, and unknown effects of the marker information on all traits in the evaluation. The SmartGene for Beef project identified significant effects of the Catapult Genetics GeneSTAR tenderness markers on meat tenderness as recorded by the objective measure of shear force These results have been used to further develop methods for combining EBVs (i.e. phenotypic and pedigree data) and gene marker information into a single marker-assisted EBV called an EBVM. Flight time is an objective measure of an animal's temperament which has been shown to be heritable and moderately genetically correlated with SF, thus representing a potential genetic indicator trait for meat tenderness (Johnston and Graser 2009).

#### **2.3.1 Cattle**

#### **<IP_ADDRESS> Dairy animals**

An Example for milk trait, Schild and Geldermann (1996), Koczan *et al*. (1991), and Bleck *et al.* (1996) identified mutations in the 5' flanking regions of bovine casein genes. In their experiments nine of these mutations were screened by DNA sequencing to see whether they might change affinity to nuclear proteins– transcription factors affect expression of relevant genes and quantitatively influence composition of milk proteins. Eighty-one Polish Black and White (BW) and 195 Polish Red (PR) cows were screened by DNA sequencing for polymorphism. Both breeds were found to differ significantly in the distribution of various genotypes in the 5'-flanking regions of αS1- and αS2-casein genes. No polymorphism was found in the 5'-flanking region of the β-casein gene. However, preferential associations were found between individual promoter genotypes and protein variants of αS1- and β-caseins. It provided strong evidences for the existence of specific haplotype combinations, including both coding and regulatory sequences at the casein *locus* in the Polish cattle breeds. These results showed that nucleotide sequence variations in the promoter regions of bovine casein genes might change the affinity of these regions to nuclear proteins – transcription factors – and thus affect the expression of relevant genes that quantitatively influence the composition of milk proteins (Martin *et al*., 2002).

A recent project by scientists at the USDA-ARS Beltsville Agricultural Research Center, a total of 5,369 Holstein bulls and cows that were born from 1952-1999 were genotyped with

and many millions of animals. Genetic markers had limited use during the century after Mendel's principles of genetic inheritance were rediscovered because few major QTL were identified and because marker genotypes were expensive to obtain before 2008. Genomic evaluations implemented in the last two years for dairy cattle have greatly improved reliability of selection, especially for younger animals, by using many markers to trace the inheritance of many QTL with small effects. More genetic markers can increase both reliability and cost of genomic selection. Genotypes for 50,000 markers now cost <US\$200 per animal for cattle, pigs, chickens, and sheep. Lower cost chips containing fewer (2,900) markers and higher cost chips with more (777,000) markers are already available for cattle, and additional genotyping tools will become available for cattle and other species VanRaden *et al*., (2011). Livestock selection has used estimated breeding values (EBV) based on phenotypic data and pedigree records for more than 40 years. More recently, advances in molecular genetic techniques, in particular DNA sequencing, have led to the discovery of regions of the genome that influence traits in livestock. However utilizing both sources of data in genetic evaluation schemes, such as BREEDPLAN, has been a challenge due to the heterogeneity of data sources, the multi-trait nature of the evaluations, and unknown effects of the marker information on all traits in the evaluation. The SmartGene for Beef project identified significant effects of the Catapult Genetics GeneSTAR tenderness markers on meat tenderness as recorded by the objective measure of shear force These results have been used to further develop methods for combining EBVs (i.e. phenotypic and pedigree data) and gene marker information into a single marker-assisted EBV called an EBVM. Flight time is an objective measure of an animal's temperament which has been shown to be heritable and moderately genetically correlated with SF, thus representing a potential genetic

An Example for milk trait, Schild and Geldermann (1996), Koczan *et al*. (1991), and Bleck *et al.* (1996) identified mutations in the 5' flanking regions of bovine casein genes. In their experiments nine of these mutations were screened by DNA sequencing to see whether they might change affinity to nuclear proteins– transcription factors affect expression of relevant genes and quantitatively influence composition of milk proteins. Eighty-one Polish Black and White (BW) and 195 Polish Red (PR) cows were screened by DNA sequencing for polymorphism. Both breeds were found to differ significantly in the distribution of various genotypes in the 5'-flanking regions of αS1- and αS2-casein genes. No polymorphism was found in the 5'-flanking region of the β-casein gene. However, preferential associations were found between individual promoter genotypes and protein variants of αS1- and β-caseins. It provided strong evidences for the existence of specific haplotype combinations, including both coding and regulatory sequences at the casein *locus* in the Polish cattle breeds. These results showed that nucleotide sequence variations in the promoter regions of bovine casein genes might change the affinity of these regions to nuclear proteins – transcription factors – and thus affect the expression of relevant genes that quantitatively influence the

A recent project by scientists at the USDA-ARS Beltsville Agricultural Research Center, a total of 5,369 Holstein bulls and cows that were born from 1952-1999 were genotyped with

indicator trait for meat tenderness (Johnston and Graser 2009).

composition of milk proteins (Martin *et al*., 2002).

**2.3.1 Cattle**

**<IP_ADDRESS> Dairy animals**

the BovinSNP50 BeadChip (VanRaden *et al*., 2009; Cole *et al*., 2009). Genotypic and phenotypic data of these bulls were used to estimate the effects of 38,416 SNP markers (after discarding markers with low minor allele frequency and markers that were in complete linkage disequilibrium with adjacent markers) on production, type, longevity, udder health, and calving ability. Next, the estimated SNP effects were used to compute the genomic PTA of each of 2,035 young Holstein bulls born from 2000-2003 that had no progeny. Finally, the 2009 PTA of each bull in the latter group, which was based on information from its progeny, was compared with the traditional PA and the genomic PTA computed from 2004 data. The same process was repeated in the Jersey breed (1,361 older animals and 388 young bulls) and the Brown Swiss breed (512 older animals and 150 young bulls). Results in Table IV show the increase in reliability (**REL**) due to genomic information, as compared with the REL from parent average information only.

Table 4. Reliability changes due to the inclusion of genomic data in national genetic evaluations VanRaden *et al*.,(2009).

#### **<IP_ADDRESS> Beef animals**

Recent advancements in sequencing and genotyping technologies have enabled a rapid evolution in methods for beef cattle selection. New tools will become available for beef producers to implement in the endeavor to efficiently produce high quality beef for today's consumer. Tools such as high-density genotyping assays and next generation sequencing instruments will help to shorten the generation interval, aid in the identification of causal mutations, increase the accuracy of EPDs on young sires and dams, provide information on gene expression and enhance our understanding of epigenetic and gut microbiome effects on cattle phenotypes. The ultimate objective of improving beef quality either by breeding or rearing factors. For genetic purposes, polymorphisms in some key genes have been reported for their association with beef quality traits. The sequencing of the bovine genome has dramatically increased the number of available gene polymorphisms. The association of these new polymorphisms with the variability in beef quality (e.g. tenderness, marbling) for different breeds in different rearing systems will be a very important issue. For rearing purposes, global gene expression profiling at the mRNA or protein level has already shown that previously unsuspected genes may be associated either with muscle development or

Improvement of Farm Animal Breeding by DNA Sequencing 97

processes. It has the potential to increase genetic progress as well as offering an insight into pig disease and immunity traits and will assist in efforts to preserve the global heritage of rare, endangered and wild pigs. It also will be important for the study of human health because pigs are very similar to humans in their physiology, behavior and nutritional needs. To date, researchers have identified several genes or DNA regions that are associated with traits of economic importance including reproduction, growth, lean and fat quantity, meat quality traits and disease resistance. A number of gene and marker tests are now available commercially from genotyping service companies. Examples are CAST (meat quality), ESR and EPOR (litter size), FUT1 (E. coli disease resistance), HAL (halothane – meat quality, stress), IGF2 (carcase),

The equine genome map was completed in the year 2007. Its applications are comparable to the development of antibiotics. Antibiotics benefited the health of everything; this is going to benefit the health of horses to the same extent in the next 50 years because of the things we can find out .More than 45 years ago, the introduction of antibiotics for use in animals greatly improved **animal health** and productivity. Today, **genetic testing**, gene therapy and the identification of **genetic markers** for certain diseases offer an even bigger opportunity for advancement in the equine industry (Steffanus, 2010).The Equine Genetic Diversity Consortium (EGDC) represents a collaborative, international community of equine researchers who are working to build a comprehensive understanding of genetic diversity among equine populations across the world and their work in future going to stimulate new studies into the origins of breeds and breed-defining traits and guide efforts to preserve

Advances in DNA sequencing technology, the chicken genome project results, and a lot of hard work by researchers and geneticists are being combined by poultry primary breeders to change the way selection is done in pedigree flocks. Research projects involving pedigree lines from poultry primary breeders are demonstrating that genomic selection can increase the rate of progress in all traits, including economically important ones. Chickens have hundreds of thousands of SNPs in their genome. Phenotype information like growth rate, feed conversion in Broilers and egg production, egg quality and life span of the birds in layers are gathered from individuals within pedigree populations, and this is correlated with the individuals' SNPs. With the use of statistics, an individual's SNPs can be compared to information in the database and a genomic breeding value can be assigned to the animal. Analysis of an individual's SNPs can allow selection to take place earlier in the life cycle of the animal, thus genomic selection can cut

The water buffalo is vital to the lives of small farmers and to the economy of many countries worldwide. Not only are they draught animals, but water buffalo also produce meat, horns, skin and particularly the rich and precious milk that gives creams, butter, yogurt and many cheeses. Just recently, we sequenced a male water buffalo animal using the Illumina Genome

the generation time down, which speeds the rate of progress (Keefe, 2011).

MC4R (growth and fat), PRKAG3 (meat quality), RN (meat quality) (Walters, 2011).

**2.3.5 Horse**

**2.3.6 Poultry**

**2.3.7 Buffalo**

genetic diversity (Petersen *et al* .,2011).

growth, and may lead to the development of new molecular indicators of tenderness or marbling. Some of these genes are specifically regulated by genetic and nutritional factors or differ between different beef cuts (Hocquette *et al* ., 2007).

An Example for meat trait, a visibly distinct muscular hypertrophy (mh), commonly known as double muscling, occurs with high frequency in the Belgian Blue and Piedmontese cattle breeds. The autosomal recessive mh locus causing double-muscling condition in these cattle maps to bovine chromosome 2 within the same interval as myostatin, a member of the TGFβ super family of genes. Because targeted disruption of myostatin in mice results in a muscular phenotype very similar to that seen in double-muscled cattle, Kambadur *et al* ., (1997) have evaluated this gene as a candidate gene for double-muscling condition by cloning the bovine myostatin cDNA and examining the expression pattern and sequence of the gene in normal and double-muscled cattle. The analysis demonstrated that the levels and timing of expression do not appear to differ between Belgian Blue and normal animals, as both classes show expression initiating during fetal development and being maintained in adult muscle. Moreover, sequence analysis reveals mutations in heavy-muscled cattle of both breeds.

#### **2.3.2 Goat**

The International Goat Genome Consortium (IGGC) was created in March 2010 in Shenzhen and is coordinated by Professor Wen Wang, Zhang Wenguang (China) and Gwenola Tosser-Klopp (INRA). The initial activity of the consortium has focused on coordination in three areas, the results of which should benefit the planned activity in 3SR, namely: de novo goat genome assembly and resequencing; development of a Goat Radiation Hybrid Panel and Mapping; and production of a high-density SNP chip. Assembly of the goat genome is now underway in the Beijing Genome Institute (BGI). The average depth of sequencing is around 60X and optical mapping has been used to increase the scaffold size. Collection of samples for resequencing from around the world is now underway and once completed may provide information on domestication events (Zhang, 2011).

#### **2.3.3 Sheep**

The recent availability of the OvineSNP50 Beadchip (Illumina) opens promising perspectives on using molecular information in the management of breeding schemes of dairy and meat sheep populations. The large amount of information provided by this tool may have a strong impact on studies aimed at verifying the feasibility of Marker (or Gene) Assisted Selection or Genome-Wide Selection programs. The OvineSNP50 Beadchip has been recently used by the International Sheep Genomics Consortium (ISGC; *www.sheephapmap.org*) to genotype samples from 64 different sheep breeds. The sheep breed population may have great advantages by introducing molecular information in the selection scheme. Indeed the possibility to predict breeding values by genomic data might lead to reduce costs of phenotype recording and increase the number of selection objectives (Usai *et al*., 2010).

#### **2.3.4 Pig**

The draft sequence, which is about 98% complete, allows researchers to pinpoint genes that are useful to pork production or are involved in immunity or other important physiological processes. It has the potential to increase genetic progress as well as offering an insight into pig disease and immunity traits and will assist in efforts to preserve the global heritage of rare, endangered and wild pigs. It also will be important for the study of human health because pigs are very similar to humans in their physiology, behavior and nutritional needs. To date, researchers have identified several genes or DNA regions that are associated with traits of economic importance including reproduction, growth, lean and fat quantity, meat quality traits and disease resistance. A number of gene and marker tests are now available commercially from genotyping service companies. Examples are CAST (meat quality), ESR and EPOR (litter size), FUT1 (E. coli disease resistance), HAL (halothane – meat quality, stress), IGF2 (carcase), MC4R (growth and fat), PRKAG3 (meat quality), RN (meat quality) (Walters, 2011).

#### **2.3.5 Horse**

96 DNA Sequencing – Methods and Applications

growth, and may lead to the development of new molecular indicators of tenderness or marbling. Some of these genes are specifically regulated by genetic and nutritional factors or

An Example for meat trait, a visibly distinct muscular hypertrophy (mh), commonly known as double muscling, occurs with high frequency in the Belgian Blue and Piedmontese cattle breeds. The autosomal recessive mh locus causing double-muscling condition in these cattle maps to bovine chromosome 2 within the same interval as myostatin, a member of the TGFβ super family of genes. Because targeted disruption of myostatin in mice results in a muscular phenotype very similar to that seen in double-muscled cattle, Kambadur *et al* ., (1997) have evaluated this gene as a candidate gene for double-muscling condition by cloning the bovine myostatin cDNA and examining the expression pattern and sequence of the gene in normal and double-muscled cattle. The analysis demonstrated that the levels and timing of expression do not appear to differ between Belgian Blue and normal animals, as both classes show expression initiating during fetal development and being maintained in adult muscle. Moreover, sequence analysis reveals mutations in heavy-muscled cattle of

The International Goat Genome Consortium (IGGC) was created in March 2010 in Shenzhen and is coordinated by Professor Wen Wang, Zhang Wenguang (China) and Gwenola Tosser-Klopp (INRA). The initial activity of the consortium has focused on coordination in three areas, the results of which should benefit the planned activity in 3SR, namely: de novo goat genome assembly and resequencing; development of a Goat Radiation Hybrid Panel and Mapping; and production of a high-density SNP chip. Assembly of the goat genome is now underway in the Beijing Genome Institute (BGI). The average depth of sequencing is around 60X and optical mapping has been used to increase the scaffold size. Collection of samples for resequencing from around the world is now underway and once completed may provide

The recent availability of the OvineSNP50 Beadchip (Illumina) opens promising perspectives on using molecular information in the management of breeding schemes of dairy and meat sheep populations. The large amount of information provided by this tool may have a strong impact on studies aimed at verifying the feasibility of Marker (or Gene) Assisted Selection or Genome-Wide Selection programs. The OvineSNP50 Beadchip has been recently used by the International Sheep Genomics Consortium (ISGC; *www.sheephapmap.org*) to genotype samples from 64 different sheep breeds. The sheep breed population may have great advantages by introducing molecular information in the selection scheme. Indeed the possibility to predict breeding values by genomic data might lead to reduce costs of phenotype recording and

The draft sequence, which is about 98% complete, allows researchers to pinpoint genes that are useful to pork production or are involved in immunity or other important physiological

differ between different beef cuts (Hocquette *et al* ., 2007).

information on domestication events (Zhang, 2011).

increase the number of selection objectives (Usai *et al*., 2010).

both breeds.

**2.3.2 Goat**

**2.3.3 Sheep**

**2.3.4 Pig**