BIOTECHNOLOGY IN ANIMAL BREEDING

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BIOTECHNOLOGY IN ANIMAL BREEDING
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Content

• Molecular markers
• Marker assisted selection
• Genotype assisted selection
• Marker assisted introgression
• Parentage anlaysis
• Analysis of genetic diversity
• Transgenesis
• Ethics in biotechnology

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Molecular markers

• Molecular markers reveal polymorphisms at the DNA
  level
• Are sites where differences in DNA sequences
  occur among members of the same species
• Markers can be located in either coding or
  non-coding regions

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Types of DNA variations

• Insertions or deletions (Indels)
• Single nucleotide polymorphisms (SNPs)
• Variable number of tandem repeats (VNTRs)
• Markers detect one or more of these variations

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Restriction fragment length polymorphism (RFLPs)

• RFLPs were the first DNA based markers to be used
  
• Involve the use of restriction enzymes to cut DNA
  into fragments
• Polymorphism based on single base substitutions
  at recognition sites of restriction enzymes

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Restriction fragment length polymorphism
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Random amplified polymorphic DNA (RAPDs)

• RAPDs are detected by doing a PCR assay with a
  single short oligonuceotide primer, of arbitrary
  sequence
• Polymorphism due to either a nucleotide base
  change that alters the ability of the primer to
  anneal, or an insertion or deletion within the
  amplified fragment
• RAPD loci are distributed randomly throughout the
  genome

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RAPDs

• Polymorphisms are visualised as the presence or
  absence of a band
• RAPDs function as dominant genes, rather than
  displaying the co-dominance of RFLPs
• Dominant markers are less informative and
  homozygotes cannot be distinguished from the
  heterozygotes

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Amplified Fragment length polymorphism (AFLP)

• AFLP are a modification of the RAPDs
• The restriction fragments are amplified by adding
  linkers that result in selective amplification
• Fragments are detected on sequencing-type
  polyacrylamide gels through radioactive or
  fluorescent labelling

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SNP markers

• Single nucleotide polymorphism single base
  change in DNA sequence, with usually two
  alternative nucleotides
• Why not 4 alternative nucleotides?
• low prob. of 2 independent base change occurring
  at any single position
• (1-5 x 10-9 / nucleotide / generation at neutral
  position)
• Bias for transitional mutations (A ? G, C? T)
  over transversions
• Least frequent allele present at 1 or greater

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Typing SNPs

• Numerous methods
• Direct sequencing
• DNA chips (potential for very high throughput)
• Various other methods

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Microsatellite markers

• Type of VNTR, which are multiple copies of a
  sequence of base pairs arranged end to end
• Length of repeating unit varies
• if lt4 base pairs microsatellite
• if gt4 base pairs minisatellite

5 CACACACACACA 3 3 GTGTGTGTGTGT 5
Also notated (CA)n
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Microsatellite markers
BL25
5
3
GGCAATGGAAGTGG CACACA...CACACA
CACTCACCCACTAGATC CCGTTACCTTCACC
GTGTGT...GTGTGT GTGAGTGGGTGATCTAG
5
3
Alleles differ in length
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Typing microsatellites

• Most commonly use PCR based methods
• Steps are
• amplify region by PCR
• primers labelled via radioactivity or
  fluorescence
• separate PCR products according to size
• polyacrylamide gel, capillary based systems
• score alleles

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Variations detected by markers
From Vignal et al. GSE 2002.
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Properties of markers
statistical considerations

• Heterozygosity
• SNPs two co-dominant alleles
• Microsatellites numerous co-dominant alleles
• Thus a single locus microsatellite is usually
  more informative than a single locus SNP (but
  multiple locus SNPs can have similar information
  content to a microsatellite)
• Note that marker heterozygosity is always
  population dependent

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Properties of markers
statistical considerations

• Density
• SNPs (1 every 1000 bp)gtgt microsatellites
• Mutation rate
• Microsatellites (1x10-5) gt SNPs (1x10-9)
• Rate and type of genotyping errors
• Often lab dependant checks need to be in place

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MARKER ASSISTED SELECTION
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Selection in quantitative traits

• Based on genetic parameters i.e heritabilities,
  genetic variances, and correlations.
• Uses statistical analysis of phenotypic data from
  pedigrees
• No knowledge of number of genes, their effects or
  location in genome is used
• Assume mean performance improved by accuracy of
  breeding values, selection intensity, generation
  interval and genetic variation

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Complexity of quantitative traits

• Several limitations due to
• Phenotype is imperfect predictor of BV eg
  measured late in life, have few recordings,
  sex-limited, sacrificial traits
• Some negative associations between genes are
  caused by linkage and epistasis
• Ideal for trait to have high heriatability and
  observed before reproductive age.
• Molecular genetics alleviates some of these
  problems

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Quantitative trait loci

• QTL refers to genes with significant effects
  (major genes) large enough to be detected and
  mapped on the genome.
• Knowledge of genes located at QTL can increase
  accuracy of estimating BV
• QTL can be targeted by use of genetic markers
• Genetic markers are landmarks at the genome
  chosen for their proximity to QTL

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MAS and GAS

• Marker assisted selection (MAS)
• select on molecular marker(s) linked to the QTL
  of interest ? indirect marker
• markers may be in
• linkage equilibrium (LE) with the QTL (phase is
  specific within families)
• linkage disequilibrium (LD) with the QTL
• Genotypic assisted selection (GAS)
• select directly on the causative mutation(s) of
  interest ?direct marker

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Linked markers
m
m
m
m
G
G
G
G
Tight linkage (m almost always inherited with G)
Loose linkage (m usually but not always inherited
with G)
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Linkage phase
m
m
m
m
In different families , a certain marker allele
may be associated with a different QTL allele
G
m
G
G
Sire 1 Inheriting M is good
Sire 2 Inheriting m is bad
Markers in LE must be used within sire families
Population wide linkage disequilibrium to be
determined for markers in LD
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Some points about MAS

• MAS is less accurate than GAS
• dependant on recombination frequency (linkage
  distance) between QTL and marker(s)
• results in probabilities of inheriting certain
  genotypes
• reduction in accuracy may be small if marker
  haplotypes are used
• MAS with markers in LE requires progeny testing
  to determine linkage phase of QTL and marker in
  each family

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Some points about GAS

• Marker is the causative mutation
• Thus certainty of inheriting a particular
  genotype
• Identifying the gene and causative mutation can
  take many years
• More difficult for quantitative rather than
  discrete traits
• Causative mutation is population wide
• Thus do not need to re-establish linkage phase in
  each family

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• MAS markers in LE

Accuracy of selection Ease of industry
implementation Cost to detect markers

• MAS markers in LD

• GAS

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Traits for gene markers

• Gene markers are most beneficial for traits are
  difficult to improve under traditional selection
• Require slaughter to measure
• Carcase traits
• e.g. meat pH, tenderness, colour
• Are measured on one sex only
• Milk Production
• Are measured late in life
• Lifetime fecundity
• Are difficult or expensive to measure
• Disease resistance

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Breeding scheme structures can also be altered to
accommodate markers

• For example, progeny testing in dairy
• Candidate young sires to progeny test
• Determine marker (and thus QTL) genotypes
• Only progeny test those that have promising
  genotypes

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Accommodating markers in breeding schemes
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Response

• Relative advantage of MAS/GAS over traditional
  selection is higher if
• trait heritability is low
• the QTL is of large effect
• the favourable allele is initially rare
• markers trace QTL inheritance with a high level
  of accuracy
• mode of gene action is non-additive

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Short and long term effects of MAS
Marker assisted selection
Normal selection
Response
Short-terms benefits 2 to 60
0 5 10 15 20
25 30
Year
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Use of markers in industry

• Industry implementation
• Very difficult for MAS with markers in LE
• Some examples for MAS with markers in LD
• Some examples for GAS
• Implementation often via breeding organisations
• No clear signals in relation to whether markers
  are meeting expectations but often used as a
  marketing tool

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Issues related to industry implementation

• How many QTL and how many markers around each
  QTL?
• How well should markers be verified?
• accuracy of effect estimate
• population wide LD
• frequency of favourable allele
• epistatic (gene interaction) effects
• How to incorporate into the selection index

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Number of markers for each QTL

• Single marker versus marker haplotype
• How much additional information does a marker
  haplotype give over a single marker in LD?
• Marker haplotypes
• Is a haplotype of a 5, 2, lt1 cM required?
• How many markers within each haplotype?

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Marker verification

• Accuracy of effect estimate
• How well should effects be known before
  implementation MAS?
• For markers in LD, accuracy of effect estimate
  relates to the number of individuals with a
  particular haplotype
• Effects may depend on genetic background
• Population wide LD
• How many populations / individuals from each
  population to test before claiming population
  wide LD?

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Incorporation into a breeding objective

• Markers provide another selection criteria
• Thus (following selection index theory)
  phenotypic and genotypic relationships to other
  traits in the selection criteria should be known
• Allele frequency will change with time thus
  need to re-evaluate (as for other genetic
  parameters)

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Examples of tests on the market
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Factors affecting livestock production may look
like.
Smallest gains
MAS/GAS
Breeding program design
Reproductive technologies
Evaluation selection
Management
Largest gains
Farming systems e.g. which species
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Marker assisted introgression

• Introgression
• e.g. introgress allele from Breed A into Breed B
• A x B ? rounds of identify animals with
  favourable allele and backcross to Breed B ? 99
  Breed B with favourable allele from Breed A
• In relation to MAI, markers can be used to
• Identify animals that have inherited the allele
  being introgressed
• quantify of original breed

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Parentage

• Parentage can be determined using a marker panel
• Typically 20-30 markers
• More markers if population is inbred / markers
  are uninformative
• Parentage analysis for a number of livestock
  species is commercially available

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Analysis of genetic diversity

• Can use markers to assess level of genetic
  variation/diversity in a population by comparing
  
• Number of alleles in a population
• Differences in allele frequencies between
  population- genetic distances
• Level of inbreeding of populations
• Kinship estimates of populations

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TRANSGENIC ANIMALS
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Introduction

• The nucleus of all cells in every living organism
  contains genes made up of DNA.
• Genes can be altered artificially, so that some
  characteristics of an animal are changed.
• For example,
• an embryo can have an extra, functioning gene
  from another source artificially introduced into
  it,
• or a gene introduced which can knock out the
  functioning of another particular gene in the
  embryo.
• Animals that have their DNA manipulated in this
  way are knows as transgenic animals.

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Why are these animals being produced

• Transgenic animals are useful as
• disease models and
• producers of substances for human welfare.
• Some transgenic animals are produced for specific
  economic traits.
• transgenic cattle that produce milk containing
  particular human proteins, which may help in the
  treatment of human diseases.
• Other transgenic animals are produced as disease
  models
• animals genetically manipulated to exhibit
  disease symptoms so that effective treatment can
  be studied

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How are transgenic animals produced?

• Three basic methods of producing transgenic
  animals
• DNA microinjection
• Retrovirus-mediated gene transfer
• Embryonic stem cell-mediated gene transfer

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How do transgenic animals contribute to human
welfare?

• The benefits of these animals to human welfare
  can be grouped into areas
• Agriculture
• Medicine
• Industry

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Agricultural Applications

• Transgenesis will allow larger herds with
  specific traits.
• breeding
• Traditional breeding is a time-consuming,
  difficult task. When technology using molecular
  biology was developed, it became possible to
  develop traits in animals in a shorter time and
  with more precision. In addition, it offers the
  farmer an easy way to increase yields.

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Agric applications.

• Scientists can improve the size of livestock
  genetically.
• quality
• transgenic cows exist that produce more milk or
  milk with less lactose or cholesterol, pigs and
  cattle that have more meat on them, and sheep
  that grow more wool. In the past, farmers used
  growth hormones to spur the development of
  animals but this technique was problematic,
  especially since residue of the hormones remained
  in the animal product.
• Disease-resistant livestock is not a reality just
  yet.
• disease resistance
• Scientists are attempting to produce
  disease-resistant animals, such as
  influenza-resistant pigs, but a very limited
  number of genes are currently known to be
  responsible for resistance to diseases in farm
  animals

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Medical Applications

• xenotransplantation
• nutritional supplements and pharmaceuticals
• human gene therapy

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Industrial Applications

• In 2001, two scientists at Nexia Biotechnologies
  in Canada spliced spider genes into the cells of
  lactating goats. The goats began to manufacture
  silk along with their milk and secrete tiny silk
  strands from their body by the bucketful. By
  extracting polymer strands from the milk and
  weaving them into thread, the scientists can
  create a light, tough, flexible material that
  could be used in such applications as military
  uniforms, medical microsutures, and tennis racket
  strings.
• Toxicity-sensitive transgenic animals have been
  produced for chemical safety testing.
  Microorganisms have been engineered to produce a
  wide variety of proteins, which in turn can
  produce enzymes that can speed up industrial
  chemical reactions.

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What are the ethical concerns surrounding
transgenesis?

• Should there be universal protocols for
  transgenesis?
• Should such protocols demand that only the most
  promising research be permitted?
• Is human welfare the only consideration? What
  about the welfare of other life forms?
• Should scientists focus on in vitro (cultured in
  a lab) transgenic methods rather than, or before,
  using live animals to alleviate animal suffering?
• Will transgenic animals radically change the
  direction of evolution, which may result in
  drastic consequences for nature and humans alike?
• Should patents be allowed on transgenic animals,
  which may hamper the free exchange of scientific
  research?

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Ethics in biotechnology in general

• Inefficiency and impact on animal welfare
• Human safety and welfare
• Environmental concerns
• Regulatory laws
• Patent and ownership