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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
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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. Food and Agricultural Organisation (FAO) may take the
lead by providing guidelines and necessary knowhow to link
various international goat breeding and genomics institutes and
to provide a central pool of data sharing.
Acknowledgements
This work was supported by the NSTIP strategic technologies
program in the Kingdom of Saudi Arabia (Project No. 11-Bio15183). The authors also, acknowledge assistance from the Science &
Technology Unit, Deanship of Scientific Research and Deanship
of Graduate Studies, King Abdulaziz University, Jeddah, KSA.
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