CH927 Quantitative Genomics What is the genetic basis of ...By the end of this lecture you should be...
Transcript of CH927 Quantitative Genomics What is the genetic basis of ...By the end of this lecture you should be...
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What is the genetic basis of complex traits?
One of the most enduring problemsin evolution and molecular biology
CH927 Quantitative Genomics
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What is the genetic basis of complex traits?
• Lecture 1 (Mon 9:30-10:30): markers, maps
• Lecture 2 (Mon 11:00-12:00): QTL methods
• Wet-bench practical (Mon 13:15-16:15): data for QTL mapping ** bus leaves to go to Warwick HRI at 12pm **
• Lecture 3 (Tues 9:30-10:30): Alternative methods: association mapping
• Lecture 4 (Tues 10:45-11:45): eQTL mapping
• Workshop (Tues 14:00-17:00): eQTL analysis using R-QTL
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By the end of this lecture you should be able to explain:
• Quantitative genetics: homozygotes, heterozygotes and inheritance
• The basis and features of quantitative vs. qualitative traits
• Why genetic markers are needed for QTL mapping
• How genetic maps are created
Lecture objectives
And know what you’ll be doing in this afternoon’s practical at Warwick HRI
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• Many sequenced genomes
• Huge cost!
• But still not easy to identify the right genes
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Genetics: the study of inheritance and its variations
Gene: the segment of DNA involved in producing a protein
Locus: a region of the genome, commonly a gene
Some definitions in molecular genetics
DNA promoter exon intron exon intron exon DNA
Chromosome: A linear end-to-end arrangement of genes and other DNA, sometimes with associated protein and RNA
Genome: the entire complement of genetic material in an organism
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Homozygosis vs. Heterozygosis
Self pollination Cross pollination
Plant A Plant B
♂ ♀ ♀ ♂e.g. one pair
of chromosomes
re-association (F1)
pair is split Meiosis
Different chromosomesDifferent genesheterozygous
Identical chromosomesIdentical geneshomozygous
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Diploid: pair of chromosomes from cross-
pollination
Duplication of the chromosomes
We can use this property to localise the parts of
chromosomes involved in a trait
Also during meiosis: crossing over occurs
Crossing-over
Separation of chromosomes
at end of meiosis
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Quantitative vs. Qualitative traits
• Qualitative traits follow ‘Mendelian’ inheritance
• Can predict the phenotype from the alleles carried
• Recessive allele: phenotypic effect is expressed in homozyous state but masked in heterozygous (Blue eyes in bb only)
• Dominant allele: same phenotypic character when heterozygous or homozygous (Brown eyes: Bb bB BB)
e.g. A locus for eye colour with 2 alleles, B and b
- four possible combinations: BB Bb bB bb
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Qualitative trait characteristics
• For qualitative traits you can predict the phenotype from the alleles being carried
• These traits are often encoded by single genes e.g. albinism
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Quantitative trait characteristics
• ‘Infinitesimal model’: genetic variation in a trait due to a large number of loci, each of small effect
• Many genotypes can produce the same phenotype
• Quantitative traits often vary along a continuous gradient
e.g. height, skin colour diseases such as cancer disorders such as epilepsy
non-Mendelian inheritance
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What is the genetic basis of complex traits?
• Complexity of these traits, esp. those involved in adaptation probably arises from segregation of alleles at many interacting loci
= Quantitative Trait Loci (QTL)
• Combination of molecular genetics and statistical techniques are needed to identify where these QTLs are located
• QTL effects are sensitive to the environment
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• No typical patterns of dominance and recessiveness• Locus contributions thought to be additive (assumed) = polygenic, or quantitative inheritance
Quantitative trait characteristics
threshold for disease to occur
increasingdisease
• The coefficients of the binomial expansion of (a + b)2n will give the frequency of distribution of all n allele combinations
• For a sufficiently high n, this binomial distribution will begin to be normal
• This can be explained as Mendelian inheritance at many loci (n)
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By the end of this lecture you should be able to explain:
• Quantitative genetics: homozygotes, heterozygotes and inheritance
• The basis and features of quantitative vs. qualitative traits
• Why genetic markers are needed for QTL mapping
• How genetic maps are created
Lecture objectives
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Objectives of QTL analysis
• The statistical study of the alleles that occur in a locus and the phenotypes (traits) that they produce
• Methods developed in the 1980s, perform on inbred strains of any species
1. Score a population for (i) a trait, and (ii) distribution of genome markers
2. Associate occurence of a marker with the phenotype
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• (iii) Markers over the genome to pinpoint QTL location - features to distinguish sequence from different origins
What do you need for QTL analysis?
• (i) A large population of individuals that you can score for phenotypes and genotypes: Recombinant Inbred Lines (RILs)
• (iv) A way to compare identify which markers from each parent have been inherited by the progeny
• (ii) A map of the genome to find out where you are (find out which chromosome the QTL is on)
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F1 =Heterozygous
at all loci
(i) A large population of mapping Recombinant Inbred Lines
A Bx
F2 =Heterozygousat some loci
Parents =Homozygous
crossing-over(recombination)
x
F7 RILs =Homozygous
at all loci& heterogeneous
x5Many different individuals are obtained & separately
selfed to develop RILs
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• Visible phenotypes or molecular markers (DNA sequence differences)
(ii) Markers to enable identification of which parental genome each part of the chromosomes of the progeny have come from
parent A
parent Bparent A
parent B
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Parent AChr 1
(iii) A map of the genome: anchor the markers
Different chromosomes Molecular markers = features of the DNA sequence
Parent AChr 2
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Markers differ between parents (natural variants)
Parent AChr 1
Parent BChr 1
Different species variants single nucleotide polymorphisms
GAATTC GATTTC
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(iv) You can distinguish these sequence differences using molecular techniques = molecular markers
• Restriction enzymes e.g. EcoRI cut DNA only at a specific recognition sequence
• Compare restriction patterns:
Parent A Parent B........GAATTC.......GAATTC.......GAATTC....... ........GAATTC.......GATTTC.......GAATTC.......
........GAATTC.......GAATTC.......GAATTC....... ........GAATTC.......GATTTC.......GAATTC.......
Second generation (F2)from selfing F1:
First generation (F1)
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There are many types of molecular markers
• Restriction Fragment Length Polymorphisms (RFLPs)
• Simple Sequence Length Polymorphisms (SSLPs)
• Cleaved Amplified Polymorphic Sequences (CAPS)
• Microsatellites (repeated sequences of 1-6 bases)
• Essentially, all of these are methods with which to detect sequence differences that have occured between two variants of a species
• They mostly differentiate single nucleotide polymorphisms (snps)
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By the end of this lecture you should be able to explain:
• Quantitative genetics: homozygotes, heterozygotes and inheritance
• The basis and features of quantitative vs. qualitative traits
• Why genetic markers are needed for QTL mapping
• How genetic maps are created
Lecture objectives
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Need to know the linkage order: making a genetic map
There are two types of maps:
• Physical map: lays out the sequence information and annotates it: promoters, genes etc.
• Linkage map: order of genetic markers and relative distances from each other - plus how much meiotic recombination (crossing over) there is between homologous chromosomes carrying alternative alleles (genetic markers)
a A
B b
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a A
B b
Rf = 0.5 (50%) = no linkage
• Are loci A and B linked (on same chromosome) or unlinked (different chromosomes)?
Genetic linkage is related to recombination frequency
Little recombinationso Rf = small= tight linkage
a AB b
aB, Ab, ab, and ABin equal proportions
Only aB and Ab
Some recombinationso Rf = medium
= quantifiable linkage
a AB b
More aB, Abthan ab, AB
More recombinationso Rf = high ( <0.5 )
= weak linkage
a A
B b
aB, Ab, ab, and ABin similar proportions
Rf = recombinationfrequency
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Map distances and genetic linkage
• A linkage map is made by characterising the recombination events that have taken place in a cross between two parental genotypes ** Every individual cross will have an individual linkage map **
• To make a map you need to score many markers in many individuals
• Recombination frequency of 0.01 (1%) = a genetic map unit of 1 cM
• Recombination events occur randomly, once or twice per chromosome
a AB b
• Assumes that linkage is the only cause of non-independence between markers and that segregation is Mendelian
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• Likelihood ODds ratio: likelihood of the observed linkage
• The higher the LOD score, the more closely linked the markers are
Determining map order
• Traditionally done by hand using e.g. the Chi-squared statistic to test for goodness of fit for the observed segregation ratios between markers
• Data on the presence/absence of 100s of markers in (F7) progeny population• Then you can use statistics to work out the marker order
• With even just 10 marker scores, this means looking at many combinations: 1 2 3 4 5 6... 1 3 2 4 5 6... 1 3 4 2 5 6... and so on... = (10 x 9 x 7 x 6 x 5 x 4 x 3 x 2 x 1)/2 = 1,814,400 possible orders!!
• That’s a lot of Chi-squared tests!• So we use mapping software e.g. Mapmaker, JoinMap
a AB b
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Determining map order
A a
B b
• Recombination fraction = n recombinant gametes total
• Haldane mapping function adjusts map distance to account for double crossovers that go undetected
• Kosambi mapping function also adjusts for crossover interference i.e. a crossover reduces the probability of a second crossover nearby
C c• Map distance ≈ (RAB + RAC - 2RABRBC) x 100 cM
• 2RABRAC is negligible for <10cM
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These should theoretically correspond to chromosomes, but if...
• Chromosomes very long
• Recombination frequency very high
• Mapping populations are not large enough
...one chromosome can statistically “break” into several linkage groups
• Also, centromeres and heterochromatin have supressed recombination
Linkage groups are the basis of genetic maps
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A genetic linkage map for broccoli1 2 3 4 5 6 7 8 9
map unitscM
• Recombination frequency of 0.01 (1%) = a genetic map unit of 1 cM