Lecture 12 Flashcards Preview

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Flashcards in Lecture 12 Deck (27):
1

Progeny with the parental type will have

- An intermediate trait score
- High variance

2

Progeny with the recombinant type will be

- Either heigh or low
- Little variance

3

LOD score (Z):

- Logarithm of Odds
- Probability of most likely recombination fraction/probability that they are unlinked
- If numerator - LODs are additive
- Used to answer whether the two genes of interested are linked

4

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

5

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)

6

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

7

LOD = 0

Ha = Ho (in other words beta base 1 = 0)

8

There are three parameters

- Beta base 1
- Beta base 0
- Variance

9

Beta base 1:

- The mean difference between the two populations

10

Beta base 0:

- The base level trait value
- The trait value that everyone has, eg) height is always 150cm (B0) + x (B1)

11

Trait value:

Y = B1X + B0 + e

12

Single marker analysis:

- T-tests marker by marker

13

Intverval mapping:

- Considers the likelihood of a QTL existing for every point in the linkage map
- Uses LOD analyses

14

Composite Interval mapping:

- Adjusts lokelihood based on the state of QTLs at other parts of the genome

15

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?

16

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

17

Pros of RAPDS:

- Not resource intensive
- Scattered throughout the genome
- Can use in uncharacterised organisms

18

Cons of RAPDS:

- Low reproducibility
- Bands of different intensity and sharpness
- Very sensitive to slight variations in reagent concentrations
- PCR artefacts
- PCR contamination

19

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.

20

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.

21

What have we discovered?

- Candidate genes can be identified
- Complementation test to see if natural alleles are complimented by artificial lab alleles
- Positional cloning

22

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

23

Chromosome walking:

1. isolate a series of overlapping clones
2. Determine the order of clones and select clone closest to target
3. Repeat to 'walk' along the chromosome bidirectionally

24

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

25

We can test candidate genes by..
(5)

- Homology with other systems
- Expressed in the correct tissue
- Obvious mutations
- Complementation
- Transgenic test

26

AFLPs (Amplified fragment length polymorphisms):

- Annonymous markers that are robust
- Can still score many polymorphisms
- Good for mapping uncharacterised genomes
- Easily visualised
- Reliale

27

Steps for generating AFLP gels

1. Take genomic DNA
2. Digest with 2 RE
3. Use two dslinkers complementary to the overhanging ends and hook onto the end
4. Perform PCR reaction
5. Determine which bands are polymorphisms - these are informative.