Molecular markers

Question 1

What are the three hierarchical levels in molecular markers

Answer 1

Molecular phenotype - Allozymes and isozymes

Transcription of DNA into RNA - RNA, micro-RNAs

DNA, the genetic code
- Nuclear DNA and DNA found in organelles (mtDNA, chloroplast DNA)

Question 2

What are the 4 main types of marker

Answer 2

DNA sequence variation
Microsatellites (or SSR, Simple sequence repeats)
SNP (or Single nucleotide polymorphism)
RAD markers (or Restriction site associated DNA markers)

Question 3

What needs to be considered when choosing a marker

Answer 3

Cost of development and screening
Have primers / protocols been developed?
Density of marker loci
Level of polymorphism
Mutation rate
Dominant or co-dominant
Accuracy and bias

Question 4

What is a linkage map

Answer 4

A map of the genes on a chromosome based on linkage analysis. It does not show the physical distances between genes but rather their relative positions, as determined by how often two gene loci are inherited together. The closer two genes are (the more tightly they are linked), the more often they will be inherited together

Question 5

What is genetic distance and how is it measured

Answer 5

= recombination frequencies (measured in centiMorgan, cM)

Question 6

What is a linkage group

Answer 6

linkage group is a group of markers that are all significantly linked (recombination ratio: r<0.5)

Question 7

What does PCR stand for

Answer 7

polymerase chain reaction

Question 8

What does PCR allow you to do

Answer 8

allows you to sequence a single part of the genome

Question 9

What are the advantages to PCR

Answer 9

Specificity - Primers (short DNA fragments) containing sequences complementary to the target region
Amplification allows minute quantity of DNA to be examined
Allows us to study population genetics from a mndellianvprspective

Question 10

Why are micro-satellites useful in conservation

Answer 10

You can look at the recent population history (100-1000 gens) because the mutation rate is so high

Question 11

Pros and cons of microsatellites high polymerism

Answer 11

It provides high resolution for near past, but low resolution for distant past. (homoplasy)

Question 12

Which methods avoids microsatellites resolution of the distant past

Answer 12

SNPs have a lower mutation rate so you can look deeper into coalescence and further back in the past

Question 13

Which marker is becoming more popular and why

Answer 13

RAD markers as it still gives lots of variation without needing to make a proper genome assembly

Question 14

What is a general problem with markers

Answer 14

trying to detemine if it is a heterozygous or homozygous individual (solution - use a codominant marker to see both forms)

Question 15

What does co dominant mean

Answer 15

It means neither allele can mask the expression of the other allele. e.g homo is with red of white but the hetero is pink

Question 16

Example of population genetics before molecular markers

Answer 16

genetics relied on studying phenotypic changes in drosophilia, looking at eye shape/colour to understand how alleles segregated and genotypes were selected

Question 17

Which species was used to create a complete linkage map

Answer 17

guppies

Question 18

`what is a centrimorgan

Answer 18

a unit of genetic length

Question 19

What is used more commonly than centimorgans

Answer 19

now we can measure physical distance - Kb

Question 20

When does linkage disequilibrium occur

Answer 20

when some genes work very well together, linkage blocks of low recombination can form, even is those genes are far from each other on the chromosome

Question 21

Where are linkage blocks common

Answer 21

in heterodimers such as the MHC

Question 22

What can be cause linkage disequilibrium

Answer 22

selection and bottlenecks

Question 23

how can linkage disequilibrium be caused by a bottleneck

Answer 23

after a bottleneck , a few genotypes survive with a particular combination of alleles.
recombination takes time to decouple those alleles
so straight after the bottleneck you will keep seeing the same combination of alleles (haplotype)
eventually those genes will dis-aggregate
so it is linkage disequilibrium by chance

Question 24

How can population sub-structuring lead to misinterpreting linkage disequilibrium

Answer 24

if you don’t have gene flow between populations you might get combination of alleles recurring by chance
the pops have differentiated, not undergoing linkage disequilibrium

Question 25

What is a limitation of using microsatellities,

Answer 25

F statistics are not very powerful, esp. Fst since you get so much polymorphism, you have high heterozygosity which means the Fst will will be incredibly low as the homozygotes will be low. The value isn't meaningful (F = fixation)

Question 26

When should microsatilites be used

Answer 26

paternity analysis in zoos to create pedigrees good for population geentic studis into microevolution (migration, inbreeding, pop structure), but not when there is lots of diversity, the Fst doesn't work well

Question 27

What if the mutation rate of snps

Answer 27

Base substitution mutation rate equals ~10−8 per base pair per generation mutation rate is low

Question 28

do snps undergo selection?

Answer 28

Many SNPs are silent and are under no (or negligible) selection

Question 29

disadvantages of microsatellites

Answer 29

Relatively few loci (generally N = 6 to 30) Very high heterozygosity for Fst based stats High mutation rate causes size homoplasy Expensive and labour intensive screening Out-of-fashion and likely to be replaced by next generation type of markers, e.g. SNPs

Question 30

How common are snps

Answer 30

very

Question 31

3 ways snps associated with complex diseases are identified

Answer 31

Candidate Gene Association Studies(CGAS) Genome-Wide Association Studies (GWAS) Pedigree studies

Question 32

advantages to snps

Answer 32

many useable loci to provide complete genome coverage Bi-allelic marker ideal for Fst based analyses Rapid screening using Next Generation Sequencing technologies e.g. Illumina Can be used on both coding and non-coding dna so it can look at functional variants and neutral variation

Question 33

Why does snps work well with Fst

Answer 33

unlike other methods, small sample size does not lead to overestimation of genetic differentiation You can have small sample sizes with snps as so many loci can be used

Question 34

How do snps form and how does this show relatedness

Answer 34

snps are a copying error that can be passed on. | the more closely related, the more snps you'll share

Question 35

what does RAD stand for

Answer 35

Restriction site Associated DNA

Question 36

what is the process of rad sequencing

Answer 36

Cut a genome with one or more restriction enzymes Ligate the first adapter to the overhangs Randomly shear the DNA into small fragments Ligate with the second adapter PCR amplify fragments that contain both adapters Sequence the resulting fragments using an Illumina Sequencing System This will give you data on SNPs and restriction sites

Question 37

what is an advantage of RAD

Answer 37

you can start looking at the whole genome and see how particular areas of the genome are behaving (e.g. diff parts will respond to inbreeding differently)

Question 38

What is RAD

Answer 38

randomly shearing DNA, amplifying and sequencing small parts of it and comparing SNPs

Question 39

When should you use RAD

Answer 39

Excellent for population genetics, paternity, disease association etc cus it covers the entire genome, and produces many markers andhigh density linkage maps

Question 40

disadvantage to RAD

Answer 40

still expensive with relatively few papers published

Question 41

what is targeted sequencing (Mybaits)

Answer 41

rather than looking at a single organism, eg plants, you can create baits which pull out DNA exactly matching the RNA sequence so you can capture the pathogens also inside the plant

Question 42

how do you calculate Fis

Answer 42

1-Ho/Hs

Question 43

What does Fis measure

Answer 43

measures the ratio of the observed and expected heterozygosity, and is used to measure inbreeding.

Question 44

how to calculate Fst

Answer 44

1 – Hs/Ht

Question 45

What does Fst measure

Answer 45

measures genetic drift and subpopulation divergence

Question 46

what is the symbol for nucleotide diversity

Answer 46

pi or π

Question 47

What does pi show

Answer 47

expresses the proportion of polymorphic sites within a genome.

Question 48

what does dN/dS measure

Answer 48

measures natural selection

Question 49

If the ratio dN/dS is low, what does that mean

Answer 49

The gene is undergoing purifying selection; only synonymous mutations are tolereated

Question 50

If the ratio dN/dS is high, what does that mean

Answer 50

the gene is undergoing positive selection; selection favours changes in amino acids

Question 51

what unit is used to measure recombination

Answer 51

Rho or ρ/μ

Question 52

What does a high Rho mean

Answer 52

A high value means a high level of recombination compared to the mutation rate.

Question 53

What is Theta

Answer 53

It is the the drift-mutation parameter (4Ne𝛍).

Question 54

What does Tajima' D capture

Answer 54

it capture demographic effects and measures selection and population size expansion/contraction

Question 55

Why must you not look at Tajima's D alone

Answer 55

you can see alleles in intermediate frequencies which suggest balancing selection, but actually there was a bottleneck and the population is expanding and there is no selection

Question 56

What does a high Tajima's D mean

Answer 56

A high value means a lack of rare alleles, e.g. due to positive selection or a recent population size contraction.