Genetics Block 2
What are the SNPs and their differences with respect to abundance, utility for mapping and functional changes that may impact gene expression and/or phenotype? Disease?
1/200 bp average Identified by sequence alignment where color of chromatogram allows to determine position and each peak has different height and different colors indicating different alleles - indicates "heterozygous" Holstein - leukocyte adhesion deficiency - radioactivity can read - SNPs - from artificial insemination Main Coon cat - cardiac myosin - SNP - hypertrophic cardiomyopathy
What is known about the creation of some of our primary domesticated species?
3 main pathways of domestication Commensal - pig, dog, cat, rat mouse, guinea pig... Prey - pig?, sheep, goat, llama, alpaca, cattle... Directed - horse, donkey, camel Population size is likely to shrink dramatically and this it turns means drift will be powerful in the subsequent small population
What are components to consider in regards to genomic testing in cattle?
- more SNPs increase accuracy - degree of increased accuracy doesnt necessitate use of higher SNP panels
How are purebred animals are artificial populations man has created? And what do they require?
Active population-level management
What are benefits of genomics?
Genetic patterns are often breed specific
What are the genomic approaches toward cellular and organismal function?
Genome - each individual carries a unique genome consisting of his or her genome Whole-genome approaches - study how the entire complement of genes in a cell work together Transcriptome - example of a genomic approach towards understanding cellular function Proteomics - genome-wide study of how all proteins function within a cell Ex tumor cell expression comparison
What is the difference between germ cells and somatic cells?
Germ cells - the eggs and sperm and precursors. Haploid cells that combine and give rise to all other cells and must contain the transgene for it to be transferred to the new generation Somatic cells - all mature cells that when modified don't transfer to future generations
How do selection and drift act to change allele frequency in a population?
Selection is a non-random change in frequency of a genetic variant due to differential fitness Selection dominates drift only when s and Ne (effective population size - stable, fixed and is breeding - Ne < N (census population)) are large enough S x Ne < 1 = mutation nearly neutral and thus it is largely governed by genetic drift Strongly selected mutations ( s x Ne >>1) are likely lost by random chance 30% of new mutations lost by chance Beneficial fixed faster (2hxs) - if get to high enough frequency become under positive selection pressure
What are the implications of epigenetic reprogramming, XCl, and imprinting for cloning and stem cell therapy?
Somatic cell nuclear transfer INCOMPLETE EPIGENETIC REPROGRAMMING, AS EVIDENCED BY DNA METHYLATION haven't actually fully reversed the process Assisted reproductive technologies- large offspring from inappropriate methylation/expression - same as human Beckwith-Wieserman syndrome - LOS IS ASSOCIATED WITH INAPPROPRIATE METHYLATION AND EXPRESSION OF IMPRINTED GENES
What are the 5 characteristics that should be considered when planning what a breeding program?
Specific - improve repro efficiency Meaningful - how effect herd - economics, health, welfare Actionable - net merit, fertility index, individual traits, genetic conditions Realistic - what endpoint is given with the rescources? Trackable - pregnancy rate, calf survival,
What is the difference between stabilizing selection, directional selection, and diversifying selection?
Stabilizing selection - middle traits and breed out extremes Directional selection - reducing one extreme Diversifying selection - breeding extremes to create less heterozygous
What role do the major stakeholders play or fail to play in establishing and maintaing genetic health in purebreed animals?
Stakeholders: pet owners, animal breeders, registry bodies and vets Geneticists and epidemiologist have historically played only small roles but their expertise needed in future
What is the terminology used with stem cell types? What is a transmit-amplifying cell? Progenitor cell? Oligopotent?
Stem cell - a cell that can continuously produce unaltered progeny cells (self-renewal) and also has the ability to produce progeny cells that have different, more restricted properties Transmit-amplifying cell: proliferative stem progeny fated for differentiation (temporary phase of proliferation) Progenitor cell - generic term for any dividing cell with the capacity to differentiate. Includes putative stem cells in which self-renewal hasn't yet been demonstrated Totipotent: sufficient to form entire organism (zygote - quintuplets) Pluripotent: all body's cell lineages including germ cells and some/all extraembryonic cell types (placenta) Multipotent: can form multiple lineages that constitue an entire tissue or tissues Oligopotent: able to form 2+ lineages within a tissue Unipotent - forms a single lineage
What are the Insertion-deletion polymorphism (Indels) and their differences with respect to abundance, utility for mapping and functional changes that may impact gene expression and/or phenotype? Disease?
Typically is not known which allele state is ancestral and which is derived Less frequent then SNPs Ancestral and derived alleles can be identified by comparison with an out group 2 mutations in whippets get muscle cramps from truncation - single mutation allows to run faster than wild type - allele frequency and Indel mutation
What are the X-linked dominance and what they look like in pedigrees?
X chromosome is much larger Disease not obscured in pedigrees - typically every affected animal has an affected parent Male to male transmission NOT possible All female offspring of an affected male will be affected Female equivalent to autosomal dominant inheritance X-linked dominant traits are more frequently observed in females, although heterozygous females are usually more mildly affected than hemizygous males
What is not included in merit indexes?
genetic conditions are NOT included in the merit indexes -> doesnt change reproduction factors or ranking
What is a metric trait? binary trait? factor trait? threshold? major/minor allele? meristic character? phenocopy? populaion structure?
metric trait - character can take on an infinite number of different values, at least within some range binary trait - can take on one of only two different values short/tall factor trait - a set of discrete categories we assign out of convergence or based on the model model of underlying biology Threshold - too much do to random chance for a p< value is an alpha value Major vs minor allele - if have high minor allele means both about the same frequency Meristic character - a count of something - ex. Your eyebrow hairs Phenocopy - when the enviroment causes a mimic of a phenotype, and therefore hard to identify correctly Population structure - a trait that is genetically complex in a large diverse population may behave simpler in an isolated population
What do large genomes (like those of mammals) have?
millions of sequence variations, most of which are presumed to be non-functional for any phenotype.
What are the implication that population genetic theory has for recessive disease alleles within a population:
when rare they are likely to be found in heterozygotes and selection against affected animals will be inefficient at removing the allele. Rare alleles almost never homozygous - .01% frequency - Can't select against phenotype so that you remove the heterozygous slowly must select against population
What are the 3 key components of genetic evaluations?
- conformation/type traits - production traits - health traits
What are the four components of genetic merit?
1. Genotype value - value of an individual's genes on their OWN performance 2. Breeding value- value of an individual's genes on their PROGENY's performance (EBV), direct phenotypic measures of performance or progeny testing Genomic prediction is based on gEBV 3. Progeny differenece = transmitting ability: expectation of what the progeny inherits from the parent (PTA) - .5 breeding value, commonly based on phenotypic measures (PTA), genomic prediction (gPTA) 4. Producing ability (PA) - performance potential of an individual for a repeated trait (consistentcy of a particular individual to repeatedly produce at a certain level)
What are the 3 factors to consider in selection of animals?
1. determine inividuals used for mating 2. determine number of offsping individual has 3.determine how long the individuals remain in the breeding population
What is the different types of measurement data collected a phenotypes and recognize when certain collection strategies may be feasible for some phenotypes but not others?
Phenotypes allow to evaluate quantititative data Collect tissues Look at some trait May be hard to ascertain from casual mutants (appear to have similar phenotypes from enviromental factors
What is a haplotype? and how do they change as recombination occurs and variants arise or are lost in populations? How often do recombinations occur? How does it affect LD?
A particular sequence of alleles along a chromosome Get one from dam and other from sire "half the genotype" - pg 4 A set of alleles together define a haplotype Haplotypes vary in genetic frequency do to genetic drift and are created by crossovers between pairs of markers Average only 1 crossover event per year between megabase per generation Haplotype diversity higher in large and old populations May lead to populations being less fit in future generations - takes long time to rebuild Recombination generates new combinations of alleles (new haplotypes) by crossing over Erodes LD More likely to occur for markers far apart LD between young or recently created alleles in high
Be comfortable calculating allele and genotype frequencies, especially under the assumption of Hardy-Weinberg equilibrium.
A single locus has 2 alleles and based on Mendel's rules one of 3 genotypes that exist at certain frequencies in the population Hardy-Weinberg equilibirum states The allele frequencies p and q will not change over time Expected genotype frequencies of a1, a2, a3 will be p2, 2pq, and q2
What is candidate gene screen and What are the requirements and benefits associated with each?
A technique widely used in the past due to expense and difficulty of whole genome mapping. Requires extensive prior knowledge about candidate gene biology - won't learn much new. Sequencing more efficient Negative - may have other genes associated with the trait Collect DNA and phenotypes from members of the population that aren't close relative, design PCR primers spanning exons of candidate, sequence each in case and control smaple to look for sequence variations, tatistically test for association
What is the phenomenon of imprinting in relation to the conflict hypothesis?
ALLELIC EXPRESSION DEPENDS ON THE PARENT OF ORIGIN MATERNALLY IMPRINTED - SUPPRESS FETAL GROWTH, BEST ISE MOTHER'S FOR ALL YOUNG PATERNALLY IMPRINTED ENCOURAGE FETAL GROWTH AT THE EXPENSE OF THE MOTHER'S RESOURCES Imprinted genes are protected from demethylation during embryogenesis and undergo their own epigenetic erasure and reprogramming in the germline Imprinting implies monoallelic expression of a gene and expressed allele dependent on parent inherited form "paternally imprinted" - expressed from the paternal allele - enhance fetal growth "maternally imprinted" - expressed from the paternal allele - repress fetal growth Methylation patterns retained and treated preferentially and erased and reestablished at the level of the gamete While hybrids like mules, liger, and tigon, carry same genetic info they are physically and behaviorally different - and these phenotypic variations associated with gene ecpression
What is selective sweep? What is a haplotype? What is hitchhiking?
All variants in the genome are physically linked to other variants which are on the same chromosome - variants can be reshuffled but generally only 1-2 per generation Haplotype - half an individual organisms genome from one parent It is when a positive selective variant reaches high frequency and drags along linked variants (genetic hitchhiking aka genetic draft) which reduces diversity and heterozygosity SIZE of the footprint of the selective sweep along the chromosome depends on the SPEED with which the sweep occurred Partial sweep occurs where sweep is still in progress or where it has stalled (not universally beneficial) Extreme for Fst outliers are a signal of recent adaption Deleterious mutations otherwise lost can rise to high frequency by hitchhiking if linked to positive trait Pleiotropy - variants can have multiple phenotypic effects, or even different effects in different environments Thrifty genes are favored by natural selection for survival during famines (genes x environment interactions)
What is an allele? Using genetic raw data to calculate allele and genotype frequencies.
Alleles - sequence variation at a site in the genome Evaluate by fractions to determine major and minor alleles - review hereditary lecture for example Evaluate gels on page 7 for dominant, recessive, heterozygous
What is the difference between natural selection, sexual selection, and artificial selection?
Selection acts on specific loci Natural selection: improve fitness by acting on survivorship, fecundity (fertility), mating success (sexual selection) Artificial selection - human-directed breeding for certain traits some of which would be disfavored otherwise
Which horse breeds are most susceptible to PSSM Type 1 and which breeds are known to have an additional mutation that results in more severe disease phenotype? What type of trait is this?
Autosomal dominant Affect 20 breeds esp. Draft, Belgian + Percheron (~25-35%), QH + paint (~6-7%) for GYS1 mutation, RYR1 relating to malignant hyperthermia (QH, paint, appaloosa - increases severity) Tyiing up - reduced ability to move after exercise Grade 1 - increased glycogen storage Grade 2 - PAS-positive glycogen inclusions, typically amylase-resistant Evaluate by GWAS -> GYS1 (Glycogen synthase) candidate gene -> sequencing -> identification of missense mutation Treat with pain management if needed + low non-structural carbohydrate (from thrifty GYS1)
How would you advise a client on how reduce risk of diabetes mellitus in his or her next cat?
Burmese cats and DSH high risk of diabetes mellitus from MC4R allele found in people as well Environmental control - wt. Management, reduce carbs, meal feeding, increase activity by 10 min/day Medical recommendations - avoid corticosteroids, progestins, prevent dental disease
How can stem cells and reprogramming-approaches be used for therapeutic approaches? How do you characterize adult stem cells? What are the components needed to induce pluripotency?
Can generate new replacement tissues from pluripotent cells - epidermis + cornea Can transfer through germ line if inserted in blastocyst stage of embryo if true pluripotent cell Characterize adult stem cells by serially diluting and observe for self-renewal + reconstitution of complete mammalian tissue following transplantation into a host Cell lineages can be traced by pluripotent stem cells expressing key factors over time iPs - induced pluripotent stem cells - adult fibroblast combined with Oct4, Sox2, hKif4, c-Myc 1. Repair dz mutations 2. Transplant genetically matched healthy cells 3. Design dz specific drugs and screen for therapeautic compounds 4. Transdifferentiation
What is whole genome sequencing and What are the requirements and benefits associated with each?
Can look at entire genome Avoid ascertainment bias in the marker set and can make better comparisons
What are the four methods of genetic mapping?
Candidate gene screen linkage analysis genome wide association whole genome sequencing
What are examples of functional changes and clinical impact caused by the various types of sequence variation.
Canine epilepsy - microsatellite - which inserts in coding region of gene
What are the basic test methods and indications for both test categories?
Categories of Genetic tests Identity testing - parentage, individual "barcoding", forensics, ancestry, species Marker and mutation test for specific loci 0 vet med diagnostics, dz diagnosis + carrier detection, genotyping, selection tools Combined genotypic for hereditary traits and identity - "DNA fingerprinting often incorporated as reduction to human error Can use gene testing to diagnose homozygous males/females prevent breeding: Avoid production of affected animals Reduce freq. Of deleterious mutations in breeding stock Maintain genetic diversity DNA and Dz registries
What are the Structural variations and their differences with respect to abundance, utility for mapping and functional changes that may impact gene expression and/or phenotype? DIsease?
Copy number variation (CNV) large-scale differences in genome content < kb Pervasive in genomes - humans have 11,700 CNVs of 440 bp or larger - affects .4% of human genome Bias for CNvs to affect non-genic sequences but many genes are affected Genetic studies on phenotype Chinese Shar-pei - GWAS study - increased fevers in relevance to wrinkly skin - which associate with a COPY NUMBER VARIATION associated with fever frequency - multi-factorial traits - 1 dominant - ameliorated by others (enhancers/inhibitors) - SAW Shar-pei's with high number CNV as animal with illness - indicated either epigenetic or phenotype error depending on age - multifactorial Horses and melanoma - horses go grey from CNV with 4.6-kb duplication in intron 6 of STX17 - leads to increase in expression of 2 genes STX17 and NR4A3 - highly coordinated with phenotypic trait Chondrodysplasia dogs - expressed Fgf4 retrogene - CNV - extra gene which inactivates Fgf4 gene SINEs (depend on LINEs for copying inserted) or LINEs can be inserted - "jumping genes" - canine has about 87,000 SINEs Sleep disorder narcolepsy - mutation in the hypocretin - acquisition of SINE element and leads to absence of receptor - disrupting slpicing of RNA polymerase and abnormal production
How is genetic sample collection related to mapping? How does a family-based vs. a population-based collection differ?
DNA is needed to enable genotyping and pedigrees are needed to check for "blood" relationships Compare genotype and phenotype but doesn't necessarily mean a direct relation Compare to a reference genome and collect tissues to compare to functional studies Family - looks at a small select set of related genomes Population - looks collects sample from the genomes as a whole through 3rd, 8th, 12th cousins
What are the concerns regarding anaphalaxis in dog breeds?
Dachshunds high risk Legality: pre-tx, plan to stay-in or near clinic, detailed record of where vx given, rabies vx exempt in some states, antibody titers, if not vaccinating reduce risk of exposure
What is epigenetics and what are the three type of epigenetic modifications?
Definition: Heritable changes in phenotype without changes to genotype which can arise over one generation and can be erased in the next DNA methylation- occurs at cytosine residues in cytosine guanine dinucleotides - promoter methylation = silencing and methylation of enhancers = gene activation Histone modifications - Histone tails, mainly histones 3 + 4 and can be covalently modified via acetylation or methylation at various lysine residues (which are highly replicable - associated w/ active and inactive elements DNA elements - histone code Chromatin domains - non-contiguous stretches of chromatin that are transcriptionally "synced" - domains assoc. With nucelar envelope, namely lamin proteins (Lamin Associated Domains) and Topologically Associated Domains (long-range chromatin interactions)
How is genetics a powerful tool for understanding aspects of domestication, such as phylogeny of breed relationships and the mapping of certain kind of traits? In particular, genetic analysis has revealed the genetic basis of many traits that have become highly diverged under artificial selection (color coat, fur length...) of dogs?
Descended from Eurasion grey wolves First human domesticate likely 15,000-20,000 yrs ago Possible early "aborted domestication" events Pre-dates plant domestication by more than 2000 years Likely asian origin Initially likely self-domestication as wolves would scavenge Eventually accepted into communities with specific roles - livestock, sporting, service, and protect (selective breeding) Most dogs still live as semi-feral comensals (village dogs) Earlier age of reproduction probably evolved with tameness as wolf-dogs because obligate scavengers Barking probably arose early but absent from dingoes, basenjis, and new guinea singing dogs Tremendous phenotypic diversity, > 400 breeds worldwide - intensified by positive selection
What is linkage analysis and What are the requirements and benefits associated with each? How many markers are needed? What does it detect and what does it make it difficult to detect?
Detects linkage between marker alleles and trait states. Difficult to collect need sample set for linkage (need to collect 3+ generations. Allows to detect several recombination events that occur within a small number (~2-3 generations) - only require 300-500 markers to cover genome. Job of fine mapping becomes argous Detects linkage between traits - traits near each other remain together Dificult to id casual mutants
What are some of the ways domesticated species differ genetically from wild species and the components of "domestication syndrome?"
Domestication syndrome Animals Reduction in neural crest cell migration may underlie most of the conditions in domesticated animals (white fur) Small body size Increased docility Coat color changes Reduced tooth size Craniofacial changes Ear/tail modifications Nonseasonal estrous Hormonal changes Prologed juvenile behavior Reduced brain size
What are the breeds and medical situations for which you would be most concerned about a dog's MDR1 status?
Dominant inheritance Leads to hindrance of outflow of potential drugs which can lead to seizures and potential death IVERMECTIN Collie, long-haired whippets, Australian shepherd, silken windhound "WHITE FEET, DON'T TREAT) Heartworm safe for dogs even with 2 copies of mutant
What are the genetics associated with transgene expression, including lineage- specific promoter activity, exon splicing, and effects on individual alleles?
Lineage-specific promoter activity - promoter is transferred from one generation to the next Exon splicing - replace exon with transgenics
What are the differences between embryonic and adult stem cells?
EMBRYONIC STEM CELLS pluripoent stem-cell lines derived from early embryos before formation of the tissue germ layers From inner cell mass of blastocyst + primordial germ cells of aborted fetus Pluripotent Difficult to collect large number of blastocysts to establish new embryonic stem cell lines, ethical concerns ADULT STEM CELLS: derived from, or resident in, a fetal or adult tissue, with potency limited to cells of that tissue. Sustain turnover and repair throughout life in some tissues Primordial germ cells (derived in pluripotency from blastocyst) can produce embryonic germ cells Sources: brain, heart, bone marrow, hematopoietic (long and short term stem cells), stromal Obtained from specific fetal and postnatal tissues - placenta and umbilical cord + placenta Multipotent Limited protential for differentiation - difficult for sufficient amount of cells form some organs
What elements are necessary in a transgene? What cells are used to make transgenic animals and why are these cells necessary?
Elements necessary in a transgene CDNA no genomic DNA Promoter considerations - enhances gene expression, determine timing, pattern, and extent, sequences far removed from gene may play a critical role Poly adenylation signal Embryonic stem cells or iPS are used bc they still have pluripotency in knockins
What are the 3 classes of drugs that alter epigenetic state?
Epigenetic chemotherapy - HDAC INHIBITORS - bromodomain-containing inhibitors - histone methyltransferase - DNA methylation modulators - HDAC inhibitors methyltransferase INHIBITORS, RNA INTERFERENCE
What does epigenomics evaluate? How can epigenetics lead to modification of gene expression? What is the differnece between imprinted and non-imprinted genes?
Epigenomics look at how differences in epigenetic regulation can affect the expression of various genes Example of epigenetic regulation is the phenomena of imprinted genes Methylation, miRNA, siRNA, paternal gene expressed, mother methylated Imprinted genes have more copies of a transcript from one parent allele vs another, non-imprinted fenes have equal number of transcripts from either parent allele
What is epistasis? Predictable?
Epistasis - selected variants often interact with each other Predictable - mutations in the same genes and gene pathways are found acoss species when selection is acting on similar traits (animals model human dz)
How is genetics a powerful tool for understanding aspects of domestication, such as phylogeny of breed relationships and the mapping of certain kind of traits? In particular, genetic analysis has revealed the genetic basis of many traits that have become highly diverged under artificial selection (color coat, fur length...) of camels?
Evolved in N. America during Eocene (~45 million years ago) Old world camels diverged form New World camels ~11 million years ago Dromedaries (one hump) Asia, Bactiran (two hump) central asia Uses: draft animal, saddle animal, milk, dung, hair, blood North American camels arrived in South America ~3 million years ago Llama were domesticated from guanaco and alpaca and used for meat and pack animals
What is genome wide association and What are the requirements and benefits associated with each?
FAVORED method - allows to assay thousands of SNPs at a time makes feasible, population-based sample easier to collect. Let recombination proceed for many more generations so that the chromosomes marker alleles shuffled - need more markers - easier to fine map detected regions Allows to evaluate a smaller region og association than does linkage but need many more markers Extract DNA, amplify, array, insert in bead and hybridize, add single base extension with labeled nucleotides and evaluate by scanner
How is genetics a powerful tool for understanding aspects of domestication, such as phylogeny of breed relationships and the mapping of certain kind of traits? In particular, genetic analysis has revealed the genetic basis of many traits that have become highly diverged under artificial selection (color coat, fur length...) of cats?
Fertile crescent around 10,000 years ago Selection mainly for coat variations Mainly for rodent reduction Genome sequenced in 2007, 60K SNP array now avaliable and have some hybrids
How does the strength of selection and drift depend on effective population size?
Fluctuating population size occurs from founder effects or bottlenecks and sex ratio distortion and non-randoming mating reduce Ne Drift leads to different allele frequencies between populations -may be fixed for 2 alleles Wahlund effect loss of heterozygotes where Fst (measure of genetic differentiation) AA = p2 + Fstpq AB = 2pq(1-Fst) BB = q2+ Fstpq Selection stronger in populations with larger effective sizes and strongest when the variant is at intermediate frequency - dominant mutations rise most rapidly but additive mutation reach fixation the fastest Selection is inefficient when dominant mutations are at high frequency or recessive mutations are at low frequency Drift - small Ne leads to strong drift - all but the most strongly deleterious mutations may be chance rise to high frequency in small population Rare variants may be moderately deleterious to fitness since they tend to be young (recent mutations) and selection hasn't had time to weed out
What is the utility of epigenomic characterization in the context of disease?
GENE PROMOTER METHYLATION IS TYPICALLY ASSOCIATED WITH REDUCED EXPRESSION OF A GENE - methylation is really stable SHORT ncRNAS TYPICALLY DOWNREGULATE GENE EXPRESSION - make into therapies Nutritional epigenetics - the Dutch hunger winter - exposed to famine - increased risk to metabolic conditions Agouti locus - genistein - hypermethylation Endocrine disrupters
Why might "hybrid vigor" might not apply as a new designer mixed breed becomes popular?
Heighten certain traits Genetic testing even more important - rapidly expand the breed, trend toward crossing labradoodles, tend to breed to young to know future status Popular designer dogs + no recognized breed standards
What are the autosomal dominance and what they look like in pedigrees?
Heterozygous also show - 1 copy causes disease Obscured in pedigrees with every affected animal having an affected parent Male to male transmission possible Homozygotes may be rare as disease may be more severe than heterozygotes
What are the main properties of stem cells?
Long-term self-renewal Increased proliferative potential Capacity to multilineage differentiation Multi-drug resistance Telomerase expression
How is knockin different from a pronuclear injection? How is gene targeting achieved? What do you need to create pure lines?
Knockin and knockout process Make construct - replace DNA in genome at precise loci - allows specific mutations/deletions in alleles where replacement involves delivering into the cell and selecting for VERY low probability homologous recombination events Neomycin - positive selection (checked by southern blotting) Have-TK cassette - negative selection - eliminates random recombinations Transfer into ES cells (propagated inn culture without losing their ability to differentiate) for homologous recombination- expand and select Inject into blastocyst Transfer to pseudopregnant female Breed chimeras -> F1 heterozygotes -> homozygous with gene Features: selectively eliminate gene, targeted placement of subtle mutations, easier injections but more laborious overall, replace a gene with marker to identify timing and location of expression in-vivo, adaption during development can complicate interpretation
How do you make a transgenic animals?
Making a pronuclear injection Mate at night - Harvest embryos next morning Culture briefly and inject transgenic DNA into pronucleus of the embryos Culture overnight and transfer blastocyst stage eggs into oviduct of pseudopregnant mouse who has been mated with vasectomized male Genotype offspring (somatic cells) from tail clips Features: multiple copies of head/tail arrays; random integration which may cause rearrangements and deletions to occur with random insertions may disrupt an endogenous Commonly use to examine over expression phenotype or lineage tracing Excellent for determining promoter/structure activity Can be used for loss-of-function phenotype by dominant negative expression, siRNA expression, or insertional mutation Cell/tissue - specific promoters confer selective gene expression
What is the nature and depth of info given to clients when they order and submit a Wisdom Panel and/or an Embark test?
Many variants and chromosomal segments are shared between breeds May give insight into issues (medical), training needs, size, and dietary considerations Embark evaluates 200,000+ genes, estimates age, evaluates 160+ dz at no additional charge, reports to vet, updates results, allows for contribution to science Wisdom Panel - evaluates up to 2,000 genetic markers, charges $50 per dz, NO reports to vet, NO updates results, DOESN'T allow for contribution to science
What are the two types of genetic tests: identity testing and genotyping for hereditary traits? Types of markers?
Marker test - test for sequence variations that are normal variations themselves and are located near a mutation locus Microsatellites markers - can see different size and based on size of actual alleles (polymorphism at one locus) SNP markers - normal one base pair mutations that are inherited and can be assessed with microarrays Copper Toxicosis in Bedlington Terrier - not accurate bc didn't know mutation Mutation test- test for actual mutation that causes a specific disorder More reliable than marker test for identifying carriers and affecteds
What is the process of cloning?
Nuclear transfer involves the transfer of a diploid nucleus from a cell to an enucleated unfertilized egg Uses chemicals or shock Germline access to virtually all species Major problems with epigenetics Process is inheritantly inefficient until er understand more about nuclear reprogramming
What is the key player in the process of X-chromosome inactivation?
ONE HETERPCHROMATIC (INACTIVE) - X-CHROMOSOME XCl requires transcription of the long non-coding RNA Xist In the inner cell mass (ICM) of early embryo, XCl occurs in random pattern Each cell has an equal chance (RANDOM) of inactivating either X-chromosomes Xist COATS AND SILENCES ONE ENTIRE chrX BY INITIATING A CASCADE OF CHROMATIN MODIFYING MECHANISMS
What is the genetic basis of variations in pancreatic amylase activity in dogs?
More pancreatic amylase in domesticated dogs allows them to digest grain diets as compared to wolves
What does the future maintenance of genetic health in purebred populations of animals require?
More thinking at the level of populations rather than simply individuals Many experts and motivated parties could participate in theory - each may have reason not to
What does mutation rate mean? Selective coefficient? What is the relative contribution of beneficial, deleterious, and neutral mutations contributing to new mutations, genetic variation within a species, and genetic divergence within a species?
Mutation rate: μ, quite small (μ ~ 2x10-8 per bp per generation) compared to large genome ~ (3x109 per bp) - about 60 new mutations per person with all positions likely being mutated Mutations and migrations can be only way to regain lost genetic variation Selection coefficient (s) - the effect on fitness (number of offspring an individual has surviving at maturity) of a variant: Fitness normalized to background population of 1 Homozygous mutant - 1+2s Heterozygous mutant - depends on dominance - 1 + 2h x s - Recessive - h=0, Dominant - h=1, Additive variants - h=0.5 Neutral - mutations unselected and often appear as -1/Ne < s < 1/Ne Deleterious mutations - harmful to organize Beneficial mutations - good in organism
What are several current options that owners or breeders of purebred dogs have for genetic diversity/inbreeding testing and multi-dz risk info?
Mydogdna - allows to determine diversity of animal and whether it is carrying certain traits/dz UC Davis - diversity test Mars/Wisdom Panel - breed specific, no simple table of common diseases + individual/mating pair risk Show chromosomes with both common haplotypes for breed and individual dog's haplotype Key on website for which risk alleles for certain dz in certain breeds Tells breed homozygosity: If more homozygous less likely to breed
What would an internet search for rescources provide in regards to commerical, academic, or government websites as rescources for information on veterinary genetic testing?
NCBI (diagnose disorders), PubMed (learn about dz), Academic institutions (genetic testing) AKC DNA certification - evaluates 17 microsatellite markers across genome for paternity conformation Microsatellite paternity in cattle - determines which sire parent of offspring No regulations requiring a lab to prove that a genetic alteration causes a dz before offering a veterinary genetic test Governmental certification of labs not equivalent to human genetic labs AVMA certification process for genetic counselors NOT AVALIABLE
What are the autosomal recessive and what they look like in pedigrees?
Only seem in autosomal recessive Affected animals may not have an affected parent 2 copies of the mutation needed - ONLY HOMOZYGOTES Inherit one mutation from each parent and parents may inherit the allele from the same source due to inbreeding
What is the role that population events such a bottlenecks play in changing the haplotype diversity and linkage disequilibrium within domesticate populations? What role do these forces play in mapping experiments in domestic populations?
Partly driven by population events such as bottlenecks Purebred population undergoes through a bottlenecks - Look at slides Breed dogs have high levels of LD Mapping - long distances - allow to find many LD Short distances - fine-mapping resolution and may need to sequence ALL the animals - readily identify causal mutation Sometimes start in an inbred population that then relate out - so can more quickly isolate Bernese Mounatain dog, akita (us isolated population), pekingese (bottlenecks) LD both limits and empowers association mapping - better to used combiend techniques by mapping multiple populations
How do you evaluate a population with long distances or short distances between LD?
Populations with LD long distances Reuires few markers to adequately cover the entire genome for genome-wide association Populations with LD short distances Fine-mapping resolution will be higher (but depends on selective sweeps) May more readily identify the casual mutation
How do we use genetic tools to asses gene expression and differences? What is the concept of pharmacogenomics/personalized medicine?
Previously by microarrays but now sequencing Microarrays have been used to look at differences in response of various tumors to treatment - analysis revealed that it was possible to categorize different-subgroupings of the tumors based on differential gene expression signature Possibility that microarrays can become used as a clinical diagnostic tool allowing us to determine what therapeutics approach will have the best results aka pharmocogenetics Sequencing allows to literally count how many transcripts in a given sample - how many times did you obtain sequence info for a particular gene, count up the number, and compare that against another sample - digital gene expression (RNA-seq approach)
How can pedigrees, Punnett squares and laws of probability allow you to infer modes of inheritance, genotypes and recurrence risks? Be able to evaluate data. What is haploinsufficiency? Dominant negative effect? Gain of function?
Probability laws Mutually exclusion - P(A or B) = P(A) + P(B) Independent - P(A and B) = P(A) x P(B) Dominance can arise from: Haploinsufficiency = single normal allele isn't sufficient to produce the normal phenotype Dominant negative effect = the altered gene product antagonizes the normal product Gain of function = changing the gene product's specificity or expression pattern rather than reducing or eliminating it Pedigrees - charts (practice inheritance lecture) Male - square Dead - line through Not dz - no fill Add dash when unsure like "R-"
What are the current limitations of stem cell technology?
Problems with human embryonic stem cells Difficult to collect a large number of blastocysts and to establish new embryonic stem cell lines Limited poor quality of available cell lines Possibility of immune rejection Ethical concerns iPS: Human iPs and ES not necessarily identical Malignant transformation possible Ethical concerns
What are the types of transgenesis and the species for which these techniques are applicable?
Pronuclear injection of DNA ` Mice, rabbits, pigs, sheep, cows, goats, rats, fish, frogs, flies - NOT DOGS Gene targeting by homologous recombination in ES (and iPS) cells - Mouse Nuclear Transfer aka cloning All species but not perfectly identical and may have instabilities in DNA - dz DNA editing - CRISPR/Cas9 Not used as of yet but potential to be used in all species Cas9 - finds and cleaves CRISPR sequences
What forces have shaped the populations, genomes and traits of our domesticated species from past to the present? What is the differentiation between village dog populations and breed dogs?
Relatively large amount of differentiation between dog and wolves (domestication; mainly drift except at certain loci) Little differentiation between village dog populations (Fst < or equal to 0.1) High differentiation between breeds (Fst > or equal to 0.2), with slightly higher differentiation between breeds from different groups Small effective populaion in breed dogs (vs village dogs) drastically accelerated drift in these lineages Breed structure contributes to mapping studies Skeletal and coat variation in the dog (other domesticates) is much older than breed barriers - breeds that share trait also share variants of trait Skeletal and coat traits are under intense selection - alleles rise/become fixed Mapping skeletal and coat traits is feasible in dogs bc there's tremendous "phenotypic signal", huge number of breeds selected on traits, individual breeds are subject to selection and drift and are likely to be fixed for just one or a few loci contributing to the trait - reduces diversity Borzoi - giant size chinese crested - small size Basset hound - chondrodysplasia and ear carriage Bulldog - thick bone and brachycephaly Small breeds share same sweep haplotype IGF1
How do we use genomic tools in SNP genome wide association studies? What is the difference between a nucleotide and a SNP How common are rare variants and what is their effect?
SNP create great genome variation in individual genomes - up to 20 million SNP differences Change in a nucleotide > 1% is a SNP, <1% is a mutation Genome-wide association studies - Allows to offer genotyping technologies Rare variants <0.5% of population, often less (found in 1 individual per 14,002, in one study) and are frequently in the coding region, while SNPs are typically in intergenic regions Each person has 25-30 coding region rare variants and Many coding region rare variants are missense mutations, and often nonsense mutations Rare variants therefore have large effects while SNPs have small effects and These variants are likely to have a major effect on disease susceptibility - recent variations have not had time to be "weeded out" But to assess their impact will require sequencing 1000's of people to get a large enough sample size Ex. Angus beef
What are the major types of genetic variation in mammalian genomes
SNPs - single nucleotide polymorphism Insertion-deletion polymorphism (Indels) Microsatellites Structural variations
How is genetics a powerful tool for understanding aspects of domestication, such as phylogeny of breed relationships and the mapping of certain kind of traits? In particular, genetic analysis has revealed the genetic basis of many traits that have become highly diverged under artificial selection (color coat, fur length...) of sheep and cattle?
Sheep/goats Domesticated about 9000 years ago Cattle Taurine ~10,000 years ago Bos indicus - domesticated separately in indus valley Selective sweeps for size, color, hornlessness and not shared between breeds - marker-assisted selection crucial for cattle improvement
What are 3 differences between short and long ncRNAs?
Short non-coding RNAs <200 nt long, several classes with well-known structures, processing, and functions!!!!! PiRNAs (transposon repression and DNA methylation) and miRNAs (targeting of mRNAs and many other organs) Long non-coding RNAs - longer (>200nt), variably spliced/polyadenylated and highly heterogenous in structure and function!!!!
What does it mean that traits are genetically simple or complex?
Simple inheritance Phenotypic outcome is the result of a single gene - "Mendelian" Many genetic diseases in companion Complex inheritance Multiple genes AND enviromental factors all contribute to make the trait value - "polygenic" Majority of all traits Often quantitative traits that are measured as a continuum - dysplasia, cancer risk... etc
What are the Microsatellites and their differences with respect to abundance, utility for mapping and functional changes that may impact gene expression and/or phenotype? Disease?
Simple tandem repeats (STRs) - thought "slippage" in DNA Mutate at a higher frequency (~102 to 10-6) than SNPs (~2 x 10-8) Many alleles at each marker so lots of info per marker Many genome scans performed with these markers High mutation rate such that it obscures relationship among allele population (many alleles are identical by state but not descent) PCR - DNA agarose gels Canine epilepsy - microsatellite - which inserts in coding region of gene
What are the reasons why transgenic animals are produced and what are the capabilities provided by this technology?
Transgenic - one whose genome has been experimentally altered by the stable introduction of exogenous DNA, or modification of endogenous DNA, in cells that form the animal's germline WHy? Determine function of individual genes Produce models of naturally occurring disease Produce proteins of medical or commercial value Improve health and productivity in domestic animals Examine cellular processes by introduction of signaling molecules Species preservation
How does the discovery of genetic variants influence complex diseases?
The discovery indicated that complex diseases is much more difficult and may be intractable in some cases, although this in an area of very active and ongoing research Risk alleles may be fixed in disease particularly prevalent - unrelated beeds not share same alleles Cross-breed mapping designs needed!
What are the assumptions of the Hardy-Weinberg model and how they are they violated or fulfilled by domesticated populations?
The is not under selection (all genotypes are equally fit) No new mutations occur at the locus (no new alleles) No migrants enter the populations (no new alleles) The population size is infinite (no genetic drift) Individuals mate randomly (no inbreeding or assortative mating) Domestic animal populations violate all assumptions : Inbreeding of domestic animals - increases homozygosity in most populations which results in identity by descent regions in their genome Inbreeding coefficient - (how related were the parents related) measures loss of heterozygosity in population at a locus - f= (H0-H)/H0 50% chance in inbreeding for identical twins - inbreeding coefficient half that coefficient of relatedness Increases the number of homozygous genotypes - esp. When allele frequency is low
What is the fundamental relationship between linkage mapping and genome wide association mapping?
They both rely on the same basic machinery of chromosome recombination
What is the underlying rationale for any genetic mapping project?
To understand biology related to some specific trait of interest or importance To provide genetic marker tests in a population so breeding decisions can be made before a phenotype is evident or heterozygous carriers of a recessive trait can be detected
What are the X-linked recessive and what they look like in pedigrees?
Trait is seen more frequently in males than females Male to male transmission NOT possible Female offspring of affected males are obligate carriers Affected females are homozygous - mating of affected sire and carrier dam
What is the concept of linkage disequilibrium and be familiar with how it is calculated?
When alleles A and B tend to co-occur and can occur in non-random mating and can occur if 2 loci occur very closely on same chromosome (physical linkage) Generally only see 2 main phenotypes as linked Linkage equilibrium when results equivalent to Hardy-Weinberg expectations Correlation between alleles and involved in mapping
What are the main reasons genomics works dairy cattle?
extensive historical background, well-developed genetic evaluation program, widespread use of AI in sires, progeny-test programs, high-value animals worth the cost of genotyping, long generation interval reduced by genotyping
What is an example of the limitations and cautions of selective breeding?
always improving with new research but has varying degrees of reliability such as bovine weaver syndrome in brown swiss
What are the four basic patterns of inheritance for simple traits?
autosomal dominant autosomal recessive X-linked dominant X-linked recessive
What are the types of animals that should be considered for use in genetic evaluations?
bull selection, repro strategies, culling decisions, marketing of animals
