Molecular markers Flashcards
What are the three hierarchical levels in molecular markers
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)
What are the 4 main types of marker
DNA sequence variation
Microsatellites (or SSR, Simple sequence repeats)
SNP (or Single nucleotide polymorphism)
RAD markers (or Restriction site associated DNA markers)
What needs to be considered when choosing a marker
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
What is a linkage map
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
What is genetic distance and how is it measured
= recombination frequencies (measured in centiMorgan, cM)
What is a linkage group
linkage group is a group of markers that are all significantly linked (recombination ratio: r<0.5)
What does PCR stand for
polymerase chain reaction
What does PCR allow you to do
allows you to sequence a single part of the genome
What are the advantages to PCR
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
Why are micro-satellites useful in conservation
You can look at the recent population history (100-1000 gens) because the mutation rate is so high
Pros and cons of microsatellites high polymerism
It provides high resolution for near past, but low resolution for distant past. (homoplasy)
Which methods avoids microsatellites resolution of the distant past
SNPs have a lower mutation rate so you can look deeper into coalescence and further back in the past
Which marker is becoming more popular and why
RAD markers as it still gives lots of variation without needing to make a proper genome assembly
What is a general problem with markers
trying to detemine if it is a heterozygous or homozygous individual (solution - use a codominant marker to see both forms)
What does co dominant mean
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
Example of population genetics before molecular markers
genetics relied on studying phenotypic changes in drosophilia, looking at eye shape/colour to understand how alleles segregated and genotypes were selected
Which species was used to create a complete linkage map
guppies
`what is a centrimorgan
a unit of genetic length
What is used more commonly than centimorgans
now we can measure physical distance - Kb
When does linkage disequilibrium occur
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
Where are linkage blocks common
in heterodimers such as the MHC
What can be cause linkage disequilibrium
selection and bottlenecks
how can linkage disequilibrium be caused by a bottleneck
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
How can population sub-structuring lead to misinterpreting linkage disequilibrium
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
What is a limitation of using microsatellities,
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)
When should microsatilites be used
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
What if the mutation rate of snps
Base substitution mutation rate equals ~10−8 per base pair per generation
mutation rate is low
do snps undergo selection?
Many SNPs are silent and are under no (or negligible) selection
disadvantages of microsatellites
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
How common are snps
very
3 ways snps associated with complex diseases are identified
Candidate Gene Association Studies(CGAS)
Genome-Wide Association Studies (GWAS)
Pedigree studies
advantages to snps
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
Why does snps work well with Fst
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
How do snps form and how does this show relatedness
snps are a copying error that can be passed on.
the more closely related, the more snps you’ll share
what does RAD stand for
Restriction site Associated DNA
what is the process of rad sequencing
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
what is an advantage of RAD
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)
What is RAD
randomly shearing DNA, amplifying and sequencing small parts of it and comparing SNPs
When should you use RAD
Excellent for population genetics, paternity, disease association etc cus it covers the entire genome, and produces many markers andhigh density linkage maps
disadvantage to RAD
still expensive with relatively few papers published
what is targeted sequencing (Mybaits)
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
how do you calculate Fis
1-Ho/Hs
What does Fis measure
measures the ratio of the observed and expected heterozygosity, and is used to measure inbreeding.
how to calculate Fst
1 – Hs/Ht
What does Fst measure
measures genetic drift and subpopulation divergence
what is the symbol for nucleotide diversity
pi or π
What does pi show
expresses the proportion of polymorphic sites within a genome.
what does dN/dS measure
measures natural selection
If the ratio dN/dS is low, what does that mean
The gene is undergoing purifying selection; only synonymous mutations are tolereated
If the ratio dN/dS is high, what does that mean
the gene is undergoing positive selection; selection favours changes in amino acids
what unit is used to measure recombination
Rho or ρ/μ
What does a high Rho mean
A high value means a high level of recombination compared to the mutation rate.
What is Theta
It is the the drift-mutation parameter (4Ne𝛍).
What does Tajima’ D capture
it capture demographic effects and measures selection and population size expansion/contraction
Why must you not look at Tajima’s D alone
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
What does a high Tajima’s D mean
A high value means a lack of rare alleles, e.g. due to positive selection or a recent population size contraction.
