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WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: [email protected] Journal of Food, Agriculture & Environment Vol.12 (2): 752-761. 2014 www.world-food.net Review of modern strategies to enhance livestock genetic performance: From molecular markers to next-generation sequencing technologies in goats Muhammad Iqbal Qureshi 1, Jamal Sabir 1, Mohamed Mutwakil 1, Amr Abd Mooti El Hanafy Hassan El Ashmaoui 1, 3, Hassan Ramadan 1, 3, Yasir Anwar 1, Mahmoud Abdel Sadek 1, Mohamed Abou-Alsoud 1, Kulvinder Singh Saini 1 and Mohamed Morsi Ahmed 1, 2 1, 2 *, Department of Biological Sciences, Faculty of Science, PO Box 80203, King Abdulaziz University, Jeddah, 21589, KSA. Nucleic Acids Research Dept., Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, Borg El-Arab, PO Box 21934, Alexandria, Egypt. 3 Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Tahrir St., Dokki-Cairo 12311, Egypt. *e-mail: [email protected] 1 2 Received 11 February 2014, accepted 9 April 2014. Abstract Domestic goats (Capra hircus) breeding and husbandry practices, once a major revenue generator for the farmer in the developing world, now stands neglected primarily due to economics coupled with the loss of grazing land to agriculture. The putative loss of diverse genetic variability in these animals has necessitated a proper cataloguing to analyse and conserve their genetic pool. While genetic markers for QTL that are linked to the “desired” gene(s) could be further leveraged for selective breeding programmes, the most effective markers would be the functional mutations within these physiologically important genes. Recently, the emergence of next generation sequencing (NGS) technology allowed de novo sequencing of the goat genome, which in turn revived the opportunity of establishing the International Goat Genome Consortium (IGGC), whose objectives were to integrate research efforts at the international level. The collective strategy of utilising whole genome NGS and genome mapping, complemented with Illumina design tools proved to be efficient in designing the GoatSNP50 chip. This review evaluates different aspects of molecular biology from conventional platforms to emerging state of the art NGS and chip technologies with an aim to enhance livestock genetic performace. All these research endeavours and technological advancements are potentially expected to accelerate genomic research in goats. Key words: Domestic goats, functional polymorphisms, molecular markers, next generation sequencing, GoatSNP50. Introduction Domestic goats (Capra hircus) are extremely diverse species and principal animal genetic resources of the developing world. According to the FAO, the world goat population has been estimated to be around 921 million animals, with an increase of more than 20% during the last ten years (http://www.fao.org/cor/ statistic/en). Goats provide a persistent supply of meat, milk, fibre, and skin and are adapted to a wide range of grazing environments. To date, however, they lack genomic research tools available in cattle and sheep 1, 2. Due to their overall adaptive capabilities, goats are considered as essential for the sustainability and development of the ever-demanding meat and milk industry. Various breeds are largely specified based on their geographical location, morphological features and prolific nature. Classifications based on productivity, i.e. milk and meat production and dual type is also known. Differences have also been reported among populations in terms of size, coat colour, ear, and horn pattern. Classical Mendelian genetics has been employed in the past to select desirable traits. However, these breeding protocols do not allow for optimal control over precise phenotypic traits. Due to consistently volatile nature of agricultural practices, limited breeds have been selected for short-term economic growth. Thus, natively tailored breeds have been ignored or displaced without knowing their genetic significance 3. Currently, the focus of attention has 752 been shifted to DNA marker technology and breeding through marker assisted selection (MAS) programs. This field is strongly focusing on gene loci and polymorphisms revealed to be exclusively linked with desirable traits 4. Polymorphisms at nucleotide/DNA level assist in understanding the overall genetic portrayal of the livestock population. This contributes to the recognition of hybridization events as well as past evolutionary tendencies. Variations within the exonic regions of a gene introduce changes in the amino acid sequence which in turn results in an altered structure of the translated protein. Intronic variations, although, do not change the overall amino acid sequence, play an important role during splicing or binding of the regulatory proteins during transcription. Comparison studies involving these genomic alterations across species are ways forward to recognize functionally important genetic mutations and will assist in the identification of regulatory elements in non-coding regions. In farm animals, such variations can be linked to economic traits governed by either an individual gene or in case of a cumulative effect of many genes (polygenes) “Quantitative trait loci (QTLs)” 5, 6. An evaluation of the genetic variability in domestic goats provides an opportunity to conserve genetic resources and achieving improved productivity. Attaining targeted improvement, genes encoding desired traits must be characterised which to Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 date has not been significantly accomplished in goats 7. Techniques associated with the identification of gene markers (DNA markers) based on molecular data and the fabrication of genetic maps as selection criteria will possibly aid in calculating genetic distances and constructing trees, particularly in cases where the pedigree information is inaccessible or the targeted traits are of low heritability 8. Additionally, to attain targeted genetic improvement, genes encoding desired traits must be molecularly characterised by employing DNA markers and associated techniques. Current research endeavours by the animal biotechnologists striving to analyse single-nucleotide polymorphisms “SNPs” among genes and DNA markers are also helping to improve breeding strategies. Recently, the advent of next generation sequencing (NGS) technology allowed de novo sequencing of the goat genome, which in turn revived the opportunity of establishing the International Goat Genome Consortium (IGGC, www.goatgenome.org) 2 in 2010, whose objectives were to integrate research efforts at the international level. The collective strategy of utilising whole genome NGS and genome mapping, combined with Illumina design tools proved to be efficient in designing the GoatSNP50 chip. The aim of this review is to assess different aspects of molecular biology from conventional platforms to emerging state of the art NGS and chip technologies with an aim to enhance livestock genetic performance. All these research endeavours and technological advancements are potentially expected to accelerate genomic research in goats. Current Status of Molecular Markers Contemporary genetics especially in the livestock sector highlights the importance of characterising novel polymorphisms associated with economic traits. Detailed studies have been done and are still underway to re-design genomic maps, understanding the influence of allelic variants on quantitative phenotypes and performing linkage analysis to accelerate genetic improvement via marker assisted breeding programs. Utilising prolific varieties to categorise and map genes has been considered a principle approach by geneticists 9. Genes isolated from hyperprolific Chinese pigs have long been reported to enhance the number of pigs weaned per litter and with enhanced disease resistance 10. Similarly, the identification of the famous Booroola FecB gene accountable for high fertility rates in the Australian Merino sheep 11. Technologies like cloning, transgenics, and molecular markers to manoeuvre genotypes have been actively employed in recent years. However, researchers should critically analyse the viability of these techniques, with the intention, that the technical elegance might not lose sight of the practical 10. Livestock selection based on phenotypic data has remained a key modality in animal breeding. This artificial selection resulted in the development of animals with distinctive phenotypes that can be classified as individual breeds. Recent years have witnessed quantitative and molecular genetics equally dominating the theoretical and practical aspects of animal breeding. Genotypic assessment generally starts by scrutinizing phenotypic data to categorize genetic controls, while molecular genetics commences via known alleles or genes followed by examining their influences on phenotypes. Eukaryotic genomes reveal substantial amount of DNA polymorphisms between species alongside individual differences within a species 12, 13. The appraisal of genetic variability is important within and among populations, especially Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 in highly specialised livestock breeds. This may contribute to the selection and preservation of genetic resources since assisted reproductive techniques, such as artificial insemination (AI) and embryo transfer restrict chances of genetic variability within population 14, 15. Precise genotyping for specific genetic loci serves as a prerequisite as far as the genetic development of livestock is concerned. In line with this assumption, three kinds of recognisable polymorphic loci can be defined; (I) Direct markers: genetic loci encoding functional polymorphisms; (II) LD markers: loci that are in population-wide linkage disequilibrium with the functional mutation; (III) LE markers: loci that are in population-wide linkage equilibrium with the functional mutation in outbred populations 16. Mostly, direct markers are considered more beneficial as they control variation within the trait. Once functional mutations are precisely understood, possibilities to envisage the effects of specific alleles in all animals within a population become quite imminent 12. Restriction fragment length polymorphism (RFLP) was the earliest DNA marker used to construct first true genomic map. Although a widely utilised procedure, its gel based approach remains inappropriate for advanced throughput screening. Variants include PCR-RFLP and Amplified Fragment Length Polymorphism (AFLP). PCR-RFLP is frequently employed in diagnostic testing to determine the genotype at a known genetic mutation. AFLP is taken as a gold standard in molecular epidemiological studies involving pathogenic microorganisms 12, 17-19 . Other techniques like Randomly Amplified Polymorphic DNA (RAPD) and Single-Strand Conformation Polymorphism (SSCP) are also conventionally used to investigate polymorphisms at the DNA level 20, 21. SSCP, comparatively more efficient, is used to acquire information about levels of polymorphism within anonymous nuclear loci. Most researchers rely on it to reduce the bulk of sequencing necessary to distinguish novel alleles at relevant loci or to carefully assess allelic frequencies of populations. Additionally, it is utilised to screen genes intended to be sequenced for phylogenetic analysis. This allows researchers to determine (I) if the gene in question contains sufficient polymorphism, (II) which specific portion of the gene is highly polymorphic, (III) the level of intra-specific variation, and (IV) whether, there is a polymorphism among multi-copy genes within individuals (e.g. rDNA) 14, 22. Single nucleotide polymorphisms (SNPs) entail single nucleotide substitutions, additions or deletions and are found both in the coding and non-coding regions of the genome. SNPs are normally biallelic in nature. Hence, information content per SNP marker is lower than multiallelic microsatellite markers 23. However, these have become the most preferred tools in studying human genetic disorders and are being searched for in various livestock species, as scientists direct their attention towards functional genomics 19. Most SNPs, approximately two out of every three, involve substitution of cytosine (C) with thymine (T) with no critical effects on cellular functions. However, it is assumed that they can predispose an individual to different diseases or influence his/her reaction to a particular drug. In recent years, DNA sequencing has contributed a lot in determining SNPs. These symbolise one of the most fascinating approaches in genotype characterisation, since being profuse throughout the genome, genetically even and acquiescent to high throughput programmed investigations 24-26. Currently, an integrated approach utilising whole genome NGS 753 and genome mapping, complemented with Illumina design tools proved to be efficient in designing the GoatSNP50 chip. SNP panels allow screening of the genetic variability and thus open the way towards their use for genomic selection 27. The SNP chip technology has transformed the science the molecular markers and laid down the foundations for future triumphs in livestock development programmes. Micro- and mini-satellites cover large portions of the genome and represent potent means of mapping genes controlling economic traits. These are highly polymorphic and abundant and can easily be amplified by PCR, rendering them highly versatile for molecular fingerprinting 28. Consisting of a stretch of DNA, a few nucleotides long (roughly 2 to 6 base pairs), repeated several times in tandem (CACACACACACACACA), micro-satellites can also be termed as simple sequence repeats (SSR’s), short tandem repeats (STR’s), simple sequence tandem repeats (SSTR), variable number tandem repeats (VNTR), simple sequence length polymorphisms (SSLP) and sequence tagged micro-satellites (STMS) 29. Although both micro- and mini-satellites occur throughout the eukaryotic genome, mini-satellites tend to be more concentrated in the telomeric regions and sites associated with high frequency of recombination. The development of microand mini-satellite markers retains the potential of generating marker-saturated genetic maps and implementation of QTLs characterisation, therefore designing marker-assisted breeding programs 28, 30. Functional Polymorphisms and Their Association with Prolific Traits While genetic markers for QTL that are linked to the trait gene could be used to choose animals for selective breeding programmes, the most effective markers are the functional mutations within the trait genes 12, 31. In livestock species, existing information about genes implicated for high prolificacy highlights three major classes; (I) known mutated genes with genotyping available; (II) genes where patterns of inheritance are illustrated but with no recorded alterations; (III) putative genes with reasonable indication of segregation though due to inadequate data, the mode of inheritance is difficult to ascertain 31. The FEC genes: Detailed findings have suggested that litter size and ovulation rate can be genetically influenced by the action of single gene(s) called Fec genes. Classification and utilisation of these genes via fixation in goat population can fulfil the gap in ever soaring global meat and milk demand. Three of these Fec genes identified in sheep are bone morphogenetic protein receptor type IB (BMPRIB) or Activin Like Kinase 6, known as FecB located on chromosome 6 32-35, growth differentiation factor (GDF9), called as FecG situated on chromosome 5 and bone morphogenetic protein 15 (BMP15), termed as FecX positioned on the X chromosome 36, 37. The bone morphogenetic protein 15 (BMP15) gene is X linked and expressed in oocytes 38. The FecG (one mutated allele of gene GDF9) and FecX (four different mutated alleles of gene BMP15) mutations led to increased ovulation rates in heterozygous animals and sterility in the homozygotes. The FecB mutation (a point mutation in the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the TGF-β receptor family) led to high prolificacy in the famous Booroola sheep variety. TGF-β protein families have been described as 754 significant factors in the ovary for growth and differentiation of premature ovarian follicles. Three associated oocyte-derived members of this superfamily, i.e. GDF9, BMP15 and BMPR-IB are reportedly vital for follicular growth and ovulation 32. Careful regulation of the number of eggs shed and resulting litter size is fundamental to a successful reproduction in all species. The progression of ovarian folliculogenesis follows a complex route whereby proliferation and differentiation of the component cells take place in embryonic follicles 39. Quantity of the mature oocytes discharged in a single reproductive cycle and oestrum is gauged via an intricate transportation of endocrine signals between the pituitary gland and the ovary. There are reports suggesting the involvement of paracrine and possibly autocrine signals within ovarian follicles concerning oocyte and flanking somatic cells 37, 40-42. Various mammals including primates, goats, cattle, deer and possums in general bear an ovulation rate of 1 or occasionally 2 while there are mammalian species like rats, mice, hamsters, cats, dogs and pigs with ovulation rates between 4 and 15. In goats, limited data is available regarding different local factors governing this mechanism 43. In recent years, many aspects of the FecB gene, including reproductive endocrinology, ovary development, litter size, organ development and body mass have been studied. This gene has an additive effect on litter size and ovulation. A study in Egypt conducted to investigate FecB allelic variants in native sheep varieties via forced PCR-RFLP. Digestion of FecB amplicons (190 bp) with Ava II resulted in a non-carrier 190 bp band (wild type) revealing absence of this restriction site in all the studied animals 44. Detailed investigations are warranted to explicate these complex mechanisms together with assessing associated genetic controls. Studies should concentrate on vital functional polymorphisms linked with prolific traits in goats. Insulin-like growth factor binding protein-3 (IGFBP3): During late 80s and 90s, association of ovarian folliculogenesis with growth hormones (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) has been comprehensively examined. In vitro studies and knockout experiments involving various models have established an essential role for GH in preantral follicle development and differentiation through their binding with GH receptors situated both in oocyte and follicular somatic tissues 45. IGFs are considered as main mammalian polypeptides owing to their alleged role in controlling growth, development and metabolism 46. These also support primary enlargement and development of mammary glands and share a galactopoietic affect 47. In the bloodstream, about 75% of the IGFs circulate as a 150 KDa complex that consists of IGF-1 and -2 together with six IGFBPs of which IGFBP3 is the key binding partner. Sites for possible interference in the IGF/IGFBP pathway to enhace animal production are illustrated in Fig. 1 46. Synthesized in multiple tissues besides liver, IGFBP3 is available in the extracellular fluid manipulating actions of IGFs. The half-lives of IGFs are prolonged once incorporated with IGFBP3, influencing most of their metabolic actions. It is a growth hormone-dependent binding protein and the bloodstream constitutes approximately 40 times more concentrated IGFBP3 than IGFBP1, with higher affinity to IGF-I. IGFBP3 serum levels are supposedly 10 times higher than in lymph. Additionally, it is also presented in the cerebrospinal fluid, in human and rat lymph, in porcine and rat colostrum and milk, in Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 literature regarding functional polymorphisms in goats. On account of its key role in galactopoiesis and mammary gland development, the IGFBP3 gene is considered as a candidate marker gene for meat and milk production traits 57. Alpha-lactalbumin (α -LA): Alpha-lactalbumin (αLA) is among the most distinctive whey proteins, after beta-lactoglobulin (β-LG), found in bovine milk and other mammalian species. The concentration is relatively high, around 1.1 - 1.5 g/ L comprising approximately 3.4% of the total protein contents or 20% of the whey proteins 58, 59. In humans, α-LA, coded by the human LALBA gene, is considered as the fundamental whey protein, levels increasing from 21 to 34% between day 1 Figure 1. Sites for possible interference in the IGF/IGFBP pathway to augment animal and 14 of lactation 59. It is a small (MW 14,186), production. acidic protein (pl 4.8) composed of 123 amino acid (I) IGFs in the bloodstream are found as a [IGF]-[IGFBP3]-[ALS] ternary complex. Trials, utilising diverse models, are in progress to manipulate these complexes to regulate growth and metabolic activities. (II) Post residues 60. At the amino acid level, the similarity circulation, IGFs form binary complexes [IGF]-[IGFBP], thus, creating possibilities of influencing their actions levels between human and bovine α-LA stands at via tissue-specific uptake. (III) IGFs reportedly leave the circulation as independent entities. However, further investigations are warranted to establish regulatory switches to control “free IGFs” and further manipulate their 76% fully conserved residues (93 out of 123 amino metabolic functions. (IV) Reports have also indicated the involvement of a complex combination of autocrine and acids) and 88% homology has been revealed, while paracrine signalling mechanisms in the secretion of IGFs and IGFBPs. Opportunities regarding over-expression considering conservation of strong and weak of IGF/IGFBP activity to regulate growth are still to be determined 47. groups 61. Glycosylation of the protein in different human and porcine follicular fluid, in human seminal plasma and species provides certain levels of heterogeneity 60, 62, 63. A human last tri-mester amniotic fluid 48, 49. Comparison studies about IGF- α-LA isoform with a novel SNP has been disclosed but with major 1 and IGFBP3 concentrations at different stages of life in porcine biological influences still unclear. Fig. 2 illustrated the structure of serum have revealed lower levels of IGF-1 and IGFBP3 during the bovine α-LA gene 64. fetal phase than in later stages of life 50. Investigations have also α-LA is a globular protein rich in tryptophan (4 residues per reported declining trends in the concentrations of IGFBP3 during molecule) and in other essential amino acids (Leu, Lys) making it lactation. On the other hand, during the involution period fundamental for neonatal nutrition 65. Main functional priorities in lactoferrin is critically involved in the regulation of the IGF system the lactating mammary glands include lactose biosynthesis, where because lactoferrin has the capacity to compete with IGF binding α-LA participates as regulatory component of the lactose synthase to IGFBP-3 51. Recently, it has been acknowledged as an alleged complex 58. This makes it a potential quantitative trait locus (QTL) death-promoting factor, a utility that, in certain instances, seems for dairy cattle 66. Comprehensive research programs are in to be autonomous of its IGF-binding potentials 52. Detailed progress highlighting polymorphisms linked to milk trait and body investigations have recognized potential diagnostic and size traits in farm animals. The association between genetic therapeutic aspects associated with serum IGFBP3 levels especially variations governing milk composition and its production are of in growth disorders, where serum IGFBP3 is considered as a highly great significance to the dairy industry 67. Genetic alterations of specific screening tool for GH deficiency. Also in different the α-LA gene have been reported in cattle, goats and humans 68. malignancies like breast cancer, it acts as a growth modulator for Earlier, variants have been reported due to changes in both the cancer cells in an IGF-independent manner 53. The results of coding and non-coding regions of the genome. The 5' flanking present-day research speaks not only about the understanding region of the α-LA gene is a control region where both RNA of physiology of growth factors and their binding proteins but polymerase and transcription factors binds. Thus any modification also about their possible utilisation in medical practices, in here can potentially transform the α-LA gene expression 67. diagnostic determinations and therapeutic interventions against According to Bleck and Bremel, a SNP in the 5' flanking region of various diseases especially those associated with human the Holstein α-LA gene is associated with increased milk yield 69. endocrinology 49. Although this novel mutation is unlikely to affect the biological IGFBP3 is a structural gene whose association with animal functions of the α-LA gene, it highlights the need for further growth and development has been widely established. The bovine research of the polymorphisms among dairy goat milk proteins. IGFBP3 is located on chromosome 4 with full length as 8.9 kb Polymorphisms of the α-LA gene in dairy goats have received (mRNA 1.65 kb) and encoding five exons. Nucleotide sequences far less attention, probably because of the limited economic and of the IGFBP3 have been determined in cattle, buffaloes, sheep industrial interests. Goat’s α-LA contains 123 amino acids. The and goats, and genotype association with production traits has most fascinating aspect of goat’s milk is the homology it shares to been confirmed in cattle 54, 55. Evaluation of the IGFBP3 polymorphic that of humans’. Researchers have identified different forms of αalleles in local Egyptian sheep revealed no allelic variants in any LA gene and analysed the relationship between SNPs and their of the studied breeds 56. influence on milk and body size traits in different regional breeds 66. There are few reports suggesting polymorphic/non-polymorphic SNPs can reportedly affect the amino acid sequence or nature of IGFBP3 gene in domestic animals with limited exclusive posttranslational modifications of the milk proteins and their Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 755 Figure 2. The structure of the bovine α-LA gene 64. bioactivity in humans 70. However, there is a need of systematically designed studies aiming not only at exploring genetic variants but also to enhance our understanding about the functional significance of these DNA polymorphisms. Data indicates that new variants of the α-LA gene have extended the spectrum of genetic variation and possibly contributing to the dairy goat breeding. The impact of SNPs on goat milk protein variability represents a vast area for further research 66. Beta-lactoglobulin (β-LG): Beta-lactoglobulin (β-LG) is a major whey protein in milk of bovine and other mammalian species, a notable exception is humans. It is coded by the LGB gene located on chromosome 11 of the bovine genome. β-LG is a single stranded protein of 18 kDa comprising 162 amino acid residues with five disulphide bonds providing the necessary stability. It accounts for approximately 65% of total whey proteins of bovine milk 71, 72. β-LG is a member of the lipocalin protein superfamily and functions as a transporter protein for hydrophobic molecules 73. Its ability to bind hydrophobic bioactive substances and amphiphilic molecules has been confirmed, ranging from hexane to palmitic acid to retinol to vitamin D. Recent studies have revealed β-LGpectin complexes as molecular nano-vehicles for delivering hydrophobic nutraceuticals such as ω-3 polyunsaturated fatty acids and vitamin D in clear beverages 74-78. Other biological activities of β-LG include enzyme regulation, the neonatal acquisition of passive immunity, source of bioactive peptides and antimicrobial activity against mastitis-causing bacteria 76, 77. β-LG has actively been employed in food industry for numerous characteristics. It shows exceptional heat-set gelation capabilities 79. Supplements containing this protein are utilised in areas where water binding and texturisation properties are needed, like processed meat, fish products and minor food items 80. With brilliant whippability, foam overrun capacity and heat stability, βLG offers an ideal and economical substitute to egg albumin (egg white) in some food applications like meringues and similar products 81. Recently in the Arabian peninsula, fingerprinting of gene markers associated with productive traits has received increasing attention. In this context, a study was conducted involving two Egyptian goat breeds and their crossbreds. Results revealed that frequency of the AA genotype was higher in Damascus breed than in Barki and Damascus × Barki crossbreds. According to the reference data, milk production was significantly higher in the homozygous AA genotype compared to the heterozygous animals 82. Milk proteins show genetic polymorphism due to nucleotide sequence substitution or deletion, various degrees of glycosylation and phosphorylation. Polymorphism of the LGB gene was discovered in 1957. To date, 14 β-LG variants in Bos genus (B. taurus, B. javanicus and B. grunniens) have been 756 identified at the protein and DNA levels, including A, B, C, D, E, F, G, H, I, J, W and three nomenclature non-unified variants (X14712, EU883598 and M19088) 76, 83. The A and B variants occur at high frequency in most cow breeds. The occurrence of these variants is based on nucleotide exchanges located in exons II (C, D, F, W), III (A, H, X14712, EU883598 and M19088), IV (A, G, H, I, X14712, EU883598 and M19088), V (F and J), and VI (E, F and G) of the LGB gene. These variants have been associated with differences in protein yield, milk composition (fat, protein, casein and total solid content), technological properties of milk, and antimicrobial activity 76, 84, 85. Different investigations have revealed the polymorphic nature of β-LG with three genetic polymorphisms (A, B, and C) in sheep. Allelic variants A and B, present in all breeds, differ by a Tyr/His substitution in position 20, where β-LG A has Tyr and β-LG B has His, corresponding to a single nucleotide substitution in the βLG gene. The rare variant β-LG C is a subtype of ovine β-LG A with a single exchange of Arg-Glu at position 148. A single basepair substitution (T → C) in the β-LG gene disrupts a Rsa 1 site, permitting genotyping of animals for A and B. It is the most extensively studied polymorphism of β-LG with PCR-RFLP technique. Possible relationships between β-LG polymorphism and yield, composition, and cheese-making ability of milk have been widely studied in different sheep varieties 86. Despite available literature on β-LG polymorphisms in different mammalian species, no comprehensive reports are yet accessible at the protein or DNA level in goats. There exist preliminary studies suggesting novel genetic variants of the β-LG gene in goats 87. However, more detailed investigations are required to establish novel genetic alterations together with understanding their association with prolific traits like milk yield. Achieving High Prolificacy via Marker- and GenomeAssisted Selection Traditionally, selective breeding programmes have remained successful in improving the quantity and quality of the agricultural output. However, significant advances in molecular genetics have led to the identification of multiple genes or genetic markers associated with genes that affect traits of interest in livestock, including genes for single-gene traits and QTL or genomic regions that affect quantitative traits. This has enabled opportunities to enhance genetic improvement programs in livestock by direct selection on genes or genomic regions that affect economic traits through marker-assisted selection 12, 16. MAS can generally be divided into two classes. The first one categorises a recognised mutation within a gene or regulatory elements. Polymorphisms like this have positive impacts especially in a monogenic trait but with reported abnormalities like the Booroola gene in sheep that while enhancing the number of lambs per ewes makes the sheep Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 susceptible to numerous recessive abnormalities. The second type of MAS directly employs SNPs in LD with QTLs. First the estimation analysis is completed to calculate the effect of individual alleles. Breeding values are then approximated for selection candidates by coalescing pedigree, marker and phenotype data. This type of selection criterion has been employed to manipulate reproduction rate, nutritional intake, body composition, meat quality, muscle development and milk yield in livestock species. The primary challenge is limited capacity to properly estimate breeding values 88, 89. Meuwissen et al. 90 came up with a different proposition termed as “genomic selection”. The principle was to utilize a genomewide panel of dense markers rather than concentrating on a small number of QTLs with known associations. Thus all QTLs are in LD with at least one marker 90. This type of selection has two advantages. First, nearly all the genetic mutations linked to a specific trait can be tracked via a marker panel. Secondly, population-wise estimation can be done rather than taking individual families to gauge the effect of the marker alleles, as both the markers and the QTLs are in LD. For genomic selection, the prerequisite is a reference population evaluated for the markers and recorded for the trait. This sample of animals is then scrutinized to develop a prediction equation foreseeing a genomic breeding value in a manner that the effect of individual marker is calculated in conjunction with other markers. This results in predicting the breeding value of selection candidates with marker genotypes but no trait record. Genomic selection is quite helpful for traits that are difficult to measure at a young age as it reduces the generation interval and hence speed up the rate of genetic enhancement 89. Establishing futuristic trends in sheep and goat productivity through within-breed selection represent a gradual procedure. Conversely, incorporating prolific genes into a flock using ‘Marker Assisted Selection’ allows sustained selection pressure on other traits resulting in increased genetic gain. MAS procedure enjoys the advantage of introducing novel traits within any system while retaining the new breed’s fundamental characteristics. Prospects are there to utilize this substantial information on the organisation and functioning of the genome, however, their successful execution necessitates the implementation of a comprehensive integrated strategy closely aligned with business goals. The current attitude toward MAS is therefore one of cautious optimism 16. An integrated approach to comprehensively utilisie genetic information in breeding programs for MAS has been demonstrated in Fig. 3. NGS Technology and the Sequencing of Goat Genome With consistent technological milestones achieved in Next Generation Sequencing (NGS), the tangible beneficiary would be the animal breeding industry. Already the expenditure of sequencing human genome has moved down from millions to ~$5000/genome with eventual goal of achieving $1000/genome. Recently, a comparative study of different qualitative & quantitative parameters from the three NGS platforms, viz., Ion Torrent, Pacific Biosciences and Illumina MiSeq, have generated data sets of DNA sequencing operational efficiency and cost analyses 91. As these technologies grow and advance, prospects are to implement their applications all across the animal kingdom especially domestic animals. Advance third-generation sequencing Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 Figure 3. Components of an integrated system to utilise molecular genetic information in breeding programs for MAS 16. (TGS) technologies like the Single-Molecule Real-Time (SMRT™) Sequencer, Heliscope Single Molecule Sequencer, and the Ion Personal Genome Machine™ guarantee more complex sequence reads in a limited span of time, thus will be reasonably inexpensive in near future 92. Apart from utilising these NGS technologies for de novo sequencing, SNPs detection, epigenetic modifications, whole genome and transcriptome analyses, their usage across a broad spectrum of other areas is also emerging. These include evolutionary relationships among ancient genomes, elucidation of the roles of non-coding RNAs in health and disease etc. An outstanding review delineating template preparation, sequencing, imaging, genome alignment and assembly approaches in conjunction with a comprehensive analysis of NGS platforms has recently been published in Nature Reviews-Genetics 93. An automated whole-genome mapping of the goat genome has been accomplished by Dong et al. 94. With this achievement, goat joins the pig, cow and chicken as main domestic species to have been sequenced. The genome has been assembled de novo through small sequencing reads taken from a female Yunnan black goat. Whole-genome mapping is an advanced high-throughput optical mapping technology compared to the traditional optical mapping techniques that share complexities and low throughput for mapping large genomes. To obtain a whole-genome restriction map of the goat, an automated, high-throughput whole-genome mapping instrument plus recently developed data processing software were installed. The instrument utilizes a chip-like channel formation device (CFD) to stretch and immobilize single DNA molecules onto a positively charged glass surface within a disposable cartridge. This combined with programmed imaging and data processing tackles many of the inabilities that have restricted the application of optical mapping to large genomes. The device automatically produced 100,000 single-molecule restriction maps in 3 h, providing 12X physical coverage of the goat genome. A hybrid assembly approach was subsequently utilized to create super-long scaffolds (super-scaffolds) by joining practically computed single-molecule maps with in silico restriction maps measured from scaffolds assembled from Illumina sequencing data. The long super-scaffolds facilitated the scaffolds anchoring onto chromosomes. The goat genome is the first large genome sequenced and assembled de novo through whole-genome mapping technology, signifying its procedural elegance to be used for achieving a highly 757 contiguous genomic assembly without the assistance of traditional genetic maps. It offered an opportunity to establish the International Goat Genome Consortium (IGGC, www.goatgenome.org) 2 in 2010, whose aims were to consolidate research efforts at the international level. The goat genome sequence will be constructive for mapping reads obtained by re-sequencing additional goat breeds, which will facilitate the identification of SNP markers for genome-assisted breeding. Genomic differences between ruminants and nonruminant species have become more understandable via the goat genome. It will also aid in analysing more about the usage of goat as a potent biomedical model and bioreactor. Furthermore, established genes in association with cashmere fibre production can be utilized as markers for breeding improved cashmere goats, or might become potential targets for genetic or non-genetic manoeuvring 94, 95. For now, these high throughput technologies can be utilised in combination with marker techniques to formulate animal breeding strategies. Once SNP data from different animal genomes becomes accessible, we will be capable of drawing an enhanced version of associations among various breeds 96. This will go a long way in establishing breeding programs for conserving valuable genetic resources together with improving the enviable features among a variety of animal species. It is crucially important that while state of the art NGS technologies become more accessible, their applications across functional genomics, strain improvement and domestic animal breeding regime will become a routine practise. The Advent of Goat SNP50 Chip The triumph of Genome Wide Association Studies in identifying sequence variation associated with complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Principal aims are QTL detection and genomic selection. The Goat SNP50 chip is designed under the patronage of a cohesive strategy that utilizes NGS and whole genome mapping combined with Illumina design tools. 50 - 60K SNP chips are primarily used for linkage analysis to uncover association between markers and phenotypes. Three SNP discovery projects collaborated under the umbrella of the International Goat Genome Consortium identified approximately twelve million high quality SNP variants in the goat genome and stored in a database alongside their biological and technical characteristics. The 60,000 selected SNPs, uniformly distributed on the goat genome, were submitted for oligo manufacturing (Illumina, Inc.) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly 2. SNP panels permit screening of the genetic variability of a species and hence open the way towards their use for genomic selection 27. Chips have already been produced for numerous livestock species 97 and cattle are the best funded with a clear use for genomic selection. Tools for cattle include low (3K, 7K) 98, moderate (50K) 99 and High density SNP chips (628K & 777K). 50 - 60K SNP chips have also been developed for sheep, pigs 100 and chickens 101 and a 700 - 800K chip is under development for sheep (James Kijas, personal communication). 758 Conclusive Remarks Improvements in livestock breeding along with the knowledge of “desirable genes” among various breeds have ushered a new era in goat genomics. Researchers have traditionally relied on phenotypic expression and correlated these characteristics with the expression of individual genes. Detailed investigations have enhanced our understanding of how these gene(s) function in overall animal physiology. The sequencing of goat genome has provided unprecedented impetus to these efforts and we are one step ahead to exploit this technological expertise for the improvement of desirable traits among different goat breeds. The most important research issues now to be dealt with are, (I) integration of the knowledge gained from molecular marker studies in goats with NGS to quickly map SNPs among functionally important genes, and (II) precise selection of prolific breeds for complete genome analyses. In Asian countries, where goats remain to be the key providers of meat and milk, the government institutions need to pool their resources in expediting practical applications of the goat genome sequence. International consortiums are need of the hour and a centralized data base can potentially go a long way in improving our understanding of goat genetics. 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