Lecture 12 Flashcards
Progeny with the parental type will have
- An intermediate trait score
- High variance
Progeny with the recombinant type will be
- Either heigh or low
- Little variance
LOD score (Z):
- Logarithm of Odds
- Probability of most likely recombination fraction/probability that they are unlinked
- If numerator <denominator, the log will be negative
- LODs are additive
- Used to answer whether the two genes of interested are linked
Mapping markers by recombination:
- Let c be the actual recombination distance between markers
- We estimate c from the observed recombination fraction (r)
- Cest = no. of recombinants/total number
Maximum likelihood (ML) approach:
- Write likelihood L (probability of data) as a function of unknown c: L(c)
- CMLE (maximum likelihood estimate) of true c
- L(c) = (1 - c) to the power of 90 x c to the power of 10
(100 offspring, 90 parentals, 10 recombinants)
LOD = 3 means
- Linkage (Ha) is 1000 times more likely than free recombination (Ho) we are confident that we have linkage there.
- This is the standard figure threshold
LOD = 0
Ha = Ho (in other words beta base 1 = 0)
There are three parameters
- Beta base 1
- Beta base 0
- Variance
Beta base 1:
- The mean difference between the two populations
Beta base 0:
- The base level trait value
- The trait value that everyone has, eg) height is always 150cm (B0) + x (B1)
Trait value:
Y = B1X + B0 + e
Single marker analysis:
- T-tests marker by marker
Intverval mapping:
- Considers the likelihood of a QTL existing for every point in the linkage map
- Uses LOD analyses
Composite Interval mapping:
- Adjusts lokelihood based on the state of QTLs at other parts of the genome
Position and effect are somewhat confounded..
- The effect of the causal variant is likely to be underestimated (because recombination between the marker and the causal variant)
- In the causal variant distant with a big effect of closer with a smaller effect?
Randomly Amplified Polymorphic DNA (RAPDs)
- Synthesises 10mers, of any sequence, to use as PCR primers
- Low stringency CR with a single primer yields DNA fragments on a gel
- If every individual has it it is no informative
- If only some individuals have it polymorphisms can be identified which provide information about individuals
Pros of RAPDS:
- Not resource intensive
- Scattered throughout the genome
- Can use in uncharacterised organisms
Cons of RAPDS:
- Low reproducibility
- Bands of different intensity and sharpness
- Very sensitive to slight variations in reagent concentrations
- PCR artefacts
- PCR contamination
How do we calculate contribution to variance?
- Compare linkage groups
- Attribute the amount of phenotypic variance each group explains, by comparing LL and HH homozygotes.
Monkey flowers, and the YUP gene:
- Controls yellow carotenoid pigments
- Putting lewisii into a cardinalis background and vice versa showed that changing the colour of the flower changes the pollinators (birds or bees)
- Clear evidence of an adaptive trait being mapped and understood.
What have we discovered?
- Candidate genes can be identified
- Complementation test to see if natural alleles are complimented by artificial lab alleles
- Positional cloning
Positional cloning:
- The closest molecular markers to the trait in the linkage map
- This can be used to identify the region of the physical map
Chromosome walking:
- isolate a series of overlapping clones
- Determine the order of clones and select clone closest to target
- Repeat to ‘walk’ along the chromosome bidirectionally
QTL mapping to positionally clone, these steps can be replaced by sequencing the genome:
Steps 1 - 6 from last lecture
Step 7: Probe a genomic library with the molecular markers flanking the QTL
Step 8: Chromosomal walk to span the distance between markers
Step 9: Characterise the clones uncovered in the walk
Step 10: Identify and test candidate genes
We can test candidate genes by..
5
- Homology with other systems
- Expressed in the correct tissue
- Obvious mutations
- Complementation
- Transgenic test
AFLPs (Amplified fragment length polymorphisms):
- Annonymous markers that are robust
- Can still score many polymorphisms
- Good for mapping uncharacterised genomes
- Easily visualised
- Reliale
Steps for generating AFLP gels
- Take genomic DNA
- Digest with 2 RE
- Use two dslinkers complementary to the overhanging ends and hook onto the end
- Perform PCR reaction
- Determine which bands are polymorphisms - these are informative.
