week 5- molecular markers and allele dynamics

Question 1

what are molcular markers

Answer 1

They are specific sequences of DNA that can be used to identify individuals, populations or species

Question 2

what do molecular markers represent

Answer 2

variations in the genetic code that can be tracked and analysed

Question 3

what do molecular markers allow us to observe

Answer 3

alleles; information that is used to understand gentic diversity, inheritance patterns and evolutionary relationships

Question 4

what can seperate molecular markers provide

Answer 4

independent tests of hypotheses, thus using many together can provide more sensitivity

Question 5

what does direct DNA sequencing provide

Answer 5

direct observations of the DNA sequence and thus the alleles

Question 6

what technologies provide indirect observations of alleles

Answer 6

allozymes, RFLP and microsatellites

Question 7

molecular markers in 1960s

Answer 7

genomes variations assayed via proteins
1. protein immunology
2. Protein electrophoresis

Question 8

molecular markers 1970s-1990s

Answer 8

enter DNA
1. DNA-DNA hybridisation
2. restriction analyses including RFLP
3. minisatellites (DNA fingerprinting)

Question 9

molecular markers 1985 onwards

Answer 9

the polymerase chain reaction (PCR)
can now amplify, in vitro, assayable quantities of almost any desired piece of DNA from almost any biological source

Question 10

molecular approaches that depend on PCR

Answer 10

1. random amplified polymporphic DNAs (RAPDs)
2. amplified fragment length polymorphism (ALFPs)
3. micro satellites (aka STRs, SSRs, SSLPs)
4. direct DNA sequencing
5. single nucleotide polymorphisms (SNPs)

Question 11

what did PCR enable

Answer 11

• analysis of ancient and other forensic quality samples
• non-invasive sampling

Question 12

random amplified polymorphic DNA (RAPD)

Answer 12

• a short PCR primer (8-10mer) of arbitary sequence is used to randomly amplify anonymous regions of the genome

Question 13

amplified fragment length polymorphism (AFLP)

Answer 13

-it combines RFLP and PCR to produce more replicable fingerprints that RAPD

Question 14

pros and cons or RAPD and AFLP

Answer 14

pros: quick, inexpensive, represent the entire genome
cons: dominant, lack of reproductivity

Question 15

what are microsatellites

Answer 15

• a very important marker class developed in 1990s
  -Assays 1- 6 nt tandem repeats distributed throughout the genome:
  -often non-coding regions eg. telomeres, centromeres, promoters
  -co-dominant mendelian markers, mutilocus genotypes
  -variability in microsatellite repeat number arises by slipped-strand mispairing during replication

Question 16

pros and cons or microsatellites

Answer 16

pros:
-profiles obtainable from trace amounts of degraded DNA
-genome-wide coverage, high variability
-can score many loci on many samples pretty quickly
-neutral
cons:
- isolating loci laborious and expensive, loci often species specific
-evolve too quickly to be useful above the population level
-mostly inappropriate for intraspecific phylogeny

Question 17

applications of micro satellites

Answer 17

-individual and population level analysis:
-population structure and demography
-mating
-parentage and relatedness
-forensics
-mapping
used to decide on breeding pairs in the captive breeding program of cuban amazon parrot

Question 18

what is direct DNA sequencing

Answer 18

-1990s DNA sequencing emerged as a powerful and versatile source of genetic variation
- widely used in evolutionary genetics only following the advent of PCR
-sanger enzymatic sequencing developed in 1977, data collection now automated

Question 19

three steps of sanger sequencing

Answer 19

1. PCR with flourescent chain-terminating ddNTPs
2. size separtion by capillary gel electrophoresis
3. Laser excitation and detection by the sequencing machine

Question 20

direct DNA sequencing pros and cons

Answer 20

pros:
-can address questions at any taxonomic level by choosing the right gene or gene region: protein-coding, intron, mtDNA, RNA
-can choose to analyse all variable sites, or a subset such as synonymous sites, or even predicted amino acid sequence
cons:
-only looking at one locus, can be costly and/or time consuming

Question 21

Molecular genetic approach to monitoring whaling

Answer 21

-tested potetnial of molecular genetic methods for identifying specis and probable geographic source of whale products
-used 16 samples purchased in retail markets in japan all labeled as whale
-used a portable laboratory in hotel room to avoid issues with exporting
-PCR amplified, purified and later sequenced 155 to 378 base pairs of the mitochondrial DNA (mtDNA) control region
-early example of molecular identification of species from unknown tissues

Question 22

molecular markers, modern era

Answer 22

single nucleotide polmorphisms (SNPs)
- SNPs distributed across the genome represent the most widespread and potentially valuable source of genetic variation, but finding and screening have, until recently, been prohibitively costly and time-consuming

Question 23

exaplain sanger sequencing maxam and gilbert sanger chain-termination

Answer 23

-infer nucleotide using dNTPs then visualise with electrophoresis
-500-1000 bp fragments
-short read sequencing (hard to assemble)

Question 24

explain 454, solexa, ion torrent illumina

Answer 24

-high throughput from the parallellisation of sequencing reactions
- -50-500 bp fragments
-short-read sequencing (hard to assemble)

Question 25

exaplin pacbio, Oxford nanopore

Answer 25

-sequence native DNA in real time with single-molecule resolution -tens of kb fragments, on average -single-molecule sequencing

Question 26

what is single-molecule sequencing characterised by

Answer 26

-the lack of DNA or RNA amplification in template library preparation -require less input genomic DNA -avoid polymerase chain reaction-introduced error and amplification bias -real time measurements -longer reads problem: high error reads

Question 27

important considerations in choosing genetic markers- sensitivty

Answer 27

a marker must have the correct sensitivity for the question at hand - it is possible to have too much information (analogy: trying to navigate from newcastle to rome using 1:25000 scale topographic maps) -or too little information (trying to navigate to a restaurant in Hexam using a map of the UK)

Question 28

important considerations in choosing genetic markers- coding versus non-coding

Answer 28

knowledge about DNA regions used as genetic markers can help predict their likely sensitivity -a gene coding for a structural function will usually be more conserved by evolution than a DNA region that is non-coding -within protein-coding genes, there is a strong pattern that nucleotides within codons are constrained third

Question 29

important considerations in choosing genetic markers- organelle (mitochondrial, chloroplast) as well as nuclear DNA

Answer 29

cells from most eukaryotes contain biparentally-inherited nuclear DNA, as well as DNA in organelles that is usually inherited uniparentally -mtDNA and nuclear DNA gene genealogies reflect different aspects of population biology and history -mtDNA has a lower effective population size (around 1/4 that of nDNA) than do nuclear markers, so variants become diagnostic of taxa more rapidly -comparison of nuclear and mitochondrial genotypes can help recognise hybrid individuals, asymmetrical mating preferences, etc.

Question 30

important considerations in choosing genetic markers- rapid development

Answer 30

some genetic marker systems are directly applicable or easily convertible for use on new species. other are far less transferable

Question 31

important considerations in choosing genetic markers- rapid screening

Answer 31

recent advances in technology have made screening of large population sample very rapid

Question 32

important considerations in choosing genetic markers- DNA or protein

Answer 32

protein electrophoresis examines genetically variable proteins, and yielded the first data about gentic variation in natural populations. they are generally cheap and convenient

Question 33

explain DNA advantages

Answer 33

-DNA is generally more variable than proteins and is thus more highly resolving -DNA markers make available all the information carried in DNA substitutions that are not detectable by protein electrophoresis -provides a range of sensitivities, allowing for examination of questions at the level of the individual, population, species and higher order

Question 34

explain non-invasive sampling

Answer 34

material may be obtained without harming individuals, and even without capturing them (e.g. hairs, scat/feces, saliva, blood, tissue samples)

Question 35

why is DNA good for samples

Answer 35

it is robust and PCR-assayable so that small and degraded samples can be used

Question 36

what do nucleur and plasmid genomes reflect

Answer 36

different aspects of population history e.g. mito-nuclear discordance

Question 37

what is the frequency of an allele equal to:

Answer 37

p= the number of copies of the allele in the population/ total number of copies of all alleles in the population

Question 38

in a population what do frequency of alleles sum to

Answer 38

must sum to 1 p+q=1

Question 39

what is the hardy-weinberg principle

Answer 39

p2 + 2pq + q2 = 1

Question 40

what can genetic variation provide

Answer 40

can provide insight into the "health" of populations - low genetic diversity can result lower fitness, increased susceptibility to disease and reduced capacity to adapt to change can provide information on the ecology of the population -high genetic diversity can be a sign of a large population or lots of movement among population can provide information on the history of the population -low diveristy populations might be recently founded

Question 41

how is genetic variation measured and issues with it- the proportion of polymorphic loci

Answer 41

- the proportion of polymorphic loci (P) P= the number of loci that are polymorphic/total number of loci studied issues: -not very sensitive -no distinction between loci with e.g., 2 alleles or 20 alleles

Question 42

how is genetic variation measured and issues with it- the average heterozygosity (H) look at lecture slides

Answer 42

--proportion of heterozygotes, averaged over all loci -if their are n loci Hobs=1/n (sum of n, i=1) Hi where Hi is the observed frequency of heterozygotes at the ith locus, and the Hobs us the average of Hi over all loci studied n is the number of loci

Question 43

why does average heterozygosity mean different things (look at lecture slides)

Answer 43

when it is calculated from observed or expected values average Hobs= mean propotion of individuals that would be heterozygous at a locus average Hexp= the probability that two randomly chosen copies of a gene would be different alleles Hexp is more commonly used to describe the level of genetic variation in a population, often called 'gene diversity'

Question 44

what does mutation in a germ cell give rise to

Answer 44

a new allele the allele may be passed to offspring

Question 45

what two forces are at play in determining the fate of the new mutation

Answer 45

selection random genetic drift

Question 46

what is selection

Answer 46

fitness of a genotype determines whether alleles are passed on to next generation, i.e. whether there is selection against or for that genotype

Question 47

types of mutation and impact

Answer 47

-deleterious (lower fitness of genotype) -neutral (no effect on fitnes individual) -advantageous (increase fitness genotype, better adaptation) The relative impact on fitness will dictate the change in expected allele frequency from one generation to the next

Question 48

simple model of selection

Answer 48

look at ppt

Question 49

example of mode of selection

Answer 49

a heterozygote is twice as fit as either type of homozygote. One generation of selection can dramatically alter genotype frequencies

Question 50

what is genetic drift

Answer 50

changes in allele frquencies due to random sampling of gametes across generations chance events: random mortality has larger impacts in small populations

Question 51

what is the concept of effective population size

Answer 51

the degree to which a population experiences genetic drift can be described using the concept

Question 52

what is census population size

Answer 52

a count of the number of individuals

Question 53

what happens in a theoretical ideal population

Answer 53

(no migration, no selection, equal fitness, etc) census population size (N) will equal effective population size (Ne)

Question 54

what happens in real populations and why

Answer 54

effective population size is much smaller than census population size due to: -unequal contribution to next generation, e.g. dominant males, litter size differences -unequal sex ratios -bottleneck- changes in population size over time

Question 55

what effective population size is safe for a population

Answer 55

50/500 rule: -to avoid inbreeding depression (i.e., loss of 'fitness' due to genetic problems), Ne of at least 50 individuals in a population is required -to avoid eroding evolutionary potential (the ability of a population to evolve to cope with environmental changes), effective population size of at least 500 is required -typical ratio suggests 10:1 is very common

Question 56

in practive why is effective population size hard to calculate for a population

Answer 56

-dont know the numbers of breeders or offspring -inbreeding is hard to observe -can use gentic markers to estimate but is very complicated

Question 57

when does gene substitution occur

Answer 57

when the mutant completely replaces the 'old' or 'wild-type' allele -fixation probability: how likely -fixation time: how long does it take -rate of gene substitution: number of fixations of new alleles per unit time

Question 58

explain fixation probability

Answer 58

depends on: -present frequency -selective disadvantage -Ne if neutral: P = its frequency new allele: initial frequency of 1/ (2N): P = 1/2N - if selection is positive and the population size is large: P=2s - where s is the selective advantage if the selective advantage is weak then P=2%

Question 59

explain fixation time

Answer 59

depends on: -present frequency -selective (dis) advantage -Ne codominant with strong selection neutral or weak selection look at ppt

Question 60

explain gene substitution rate

Answer 60

K mutations reaching fixation per unit time neutral: -rate of substitutions= mutation propability in population with size N x probability of fixation i.e. mutation rate advantageous: -rate of substitution = mutation probability in population with size N x porbabilty of fixation i.e. population size, selective advantage and mutation rate