Download
1 / 53
E N D
BIOTECHNOLOGY IN ANIMAL BREEDINGKOLEKSI SOEDITO ADJISOEDARMO
Content • Molecular markers • Marker assisted selection • Genotype assisted selection • Marker assisted introgression • Parentage anlaysis • Analysis of genetic diversity • Transgenesis • Ethics in biotechnology
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
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
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
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
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
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
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
Typing SNPs • Numerous methods • Direct sequencing • DNA chips (potential for very high throughput) • Various other methods
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 <4 base pairs: microsatellite • if >4 base pairs: minisatellite 5’ CACACACACACA 3’ 3’ GTGTGTGTGTGT 5’ Also notated (CA)n
Microsatellite markers BL25 5’ 3’ GGCAATGGAAGTGG CACACA...CACACACACTCACCCACTAGATC CCGTTACCTTCACC GTGTGT...GTGTGTGTGAGTGGGTGATCTAG 3’ 5’ Alleles differ in length
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
Variations detected by markers From: Vignal et al. GSE 2002.
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
Properties of markers: statistical considerations • Density • SNPs (~1 every 1000 bp)>> microsatellites • Mutation rate • Microsatellites (1x10-5)> SNPs (1x10-9) • Rate and type of genotyping errors • Often lab dependant – checks need to be in place
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
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
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
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
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)
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
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
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
MAS – markers in LE Accuracy of selection Ease of industry implementation Cost to detect markers • MAS – markers in LD • GAS
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
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
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
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
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
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
Numberof 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, <1 cM required? • How many markers within each haplotype?
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?
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)
Factors affecting livestock production may look like…. Smallest gains MAS/GAS Reproductive technologies Breeding program design Evaluation & selection Management Farming systems e.g. which species Largestgains
Marker assisted introgression • Introgression: • e.g. introgression 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
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
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
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.
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
How are transgenic animals produced? • Three basic methods of producing transgenic animals: • DNA microinjection • Retrovirus-mediated gene transfer • Embryonic stem cell-mediated gene transfer
How do transgenic animals contribute to human welfare? • The benefits of these animals to human welfare can be grouped into areas: • Agriculture • Medicine • Industry
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.
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
Medical Applications • xenotransplantation • nutritional supplements and pharmaceuticals • human gene therapy
