Study guide #2 Genetics and Conservation
73) What is the range of possible values for Fst
0-1
30) In a population, if four different alleles are present in a population (e.g. 233, 236, 239, and 241), how many individual diploid genotypes are possible (list them)
10 genotypes 233 -233 233-236 233 -239 233-241 236 -236 236-239 236- 241 239-239 239-241 241-241
21) If given a double stranded DNA sequence, be able to specify a primer on each end that will amplify the middle. Be able to specify 5' and 3' ends on primers and template molecules
3' end is always on the inside. GC content must be similar. Base pair number limitation must be at least a specific number of base pairs so that it does stick to the end of the target region for specificity and it doesn't stick to itself.
7) What is a staggered cut. How does this produce 'sticky ends'
A staggered cut is when a restriction enzyme cuts a piece of DNA between two differing base pairs on the two strands of DNA. Since the restrictions sites are usually palindromic it cuts between the same base pairs on both strands but that cut is on different lengths of the DNA strand. When it cuts there are base pairs that don't have complimentary base because of the cut this creates a sticky end.
19) What are some of the advantages and disadvantages of RFLP analysis
Advantages: Simple to do in the lab Inexpensive Good for species ID Good for gender ID Disadvantages Not good for detection of genomic differences between individuals of the same species Can miss sequence variation if only looking at the fragment lengths
94) Which will be more impacted by a population bottleneck, observed heterozygosity or allelic richness., Explain
Allelic richness. Because rare alleles will be gone first and they don't contribute much to heterozygosity.
60) Why might genetic drift interfere with a population's ability to evolve in response to selection
Because genetic drift can be stronger in small populations the population will not evolve in a beneficial way.
58) Explain why the strength of genetic drift depends on both the population size and allele frequencies
Because genetic drift is a random process the alleles that are passed down are passed on by random chance. If you have a larger pool of alleles the next generation will look more like the parental allele frequency because the alleles conform to a binomial distribution. IF the population is smaller the probability of drift is larger because the smaller pool of alleles can change dramatically between parental and offspring frequencies. For example the more times that you flip a coin the closer the proportion of heads and tails will be to 50:50. If you flip it only 5 times you could end up with a proportion of 70:30 or 20:80. The coin flips are like the random individuals that mate and the alleles that are passed on are the heads or tails.
74) Why is an Fst value for a set of multiple populations useful
Because it can tell you if there is geneflow between all the populations for regional comparison. It's a crude number of geneflow.
55) Why is next generation sequencing so much more powerful than microsatellites in distinguishing individuals
Because it gives you actual sequencing data instead of fragment lengths. It allows you to do an analysis of many SNPs.
17) Why are RFLPs usually performed on PCR products and not genomic DNA
Because there would be too many fragments to infer on any change and compare among individuals or populations with genomic data. PCR uses a specific target regions and with the smaller DNA fragments you can compare and identify changes.
48) Be able to describe some specific applications of Sanger sequencing in wildlife
Can be sued for taxonomic identification and for finding SNPs among individuals.Better for identifying species because you are looking at one place on the genome.
41) How is capillary electrophoresis different from the usual 'slab' electrophoresis
Capillary electrophoresis can simultaneously run many samples in the same lanes as well as the ladder. It also uses a camera to detect the base pairs. The DNA fragments don't need to accumulate in one place on the gel.
56) Be able to define genetic drift
Changes in population allele frequencies from generation to generation due to random sampling of parental allels for next generation. It is due to random chance there is no direction to genetic drift.
43) What do you predict would happen in a Sanger sequencing reaction if the ratio of ddNTP:dNTP was 1:10 instead of 1:100. Explain what the results might look like
DNA polymerase will have a higher chance of adding a ddNTP instead of regular dNTP so the only thing you woud be able to sequence is the beginning portion of the sequence. The chain would stop early a lot more. The results would be a lot of short peaks on left of the chromatogram and nothing on the right side. And you might miss sequence base pairs. The sequences would be short because too many chains would have been terminated.
44) What do you predict would happen in a Sanger sequencing reaction if the ratio of ddNTP:dNTP was 1:10,000 instead of 1:100. Explain what the results might look like
DNA polymerase would have a very low chance of adding a ddNTP so you will miss a lot of sequence base pairs. The results would create a very short sequence. here would be gaps in lengths and poor signal; not all possible lengths would be represented. Not enough termination.
72) What does Fst measure
Differences in allele frequencies between populations.
88) What is overdominance, and heterosis
Dominance is a feature of a specific locus where heterozygotes have higher fitness than either homozygotes. heterosis is when the heterozygote advantage is seen across the genome at multiple loci. If you're always selecting for heterozygotes you will always have a variable population. If you have an individual that has a high level of heterozygosity it will be more fit. Natural selection will maintain genetic diversity.
87) Why is inbreeding depression generally due to recessive deleterious alleles instead of dominant recessive alleles
Dominant alleles would be selected against more strongly. Deleterious alleles are usually due to a loss of function so recessive to a functional allele.
18) If given a set of fragment sizes in a mixture, be able to draw a diagram of the resulting electrophoresis gel image
Done
24) Be able to draw a thermal profile over time for 3 full cycles of a PCR reaction. Label your axes
Done
36) If you were presented with a simple dataset of microsatellite alleles at three loci across a few individuals, could you draw out what you expect to see on an agarose gel or in a capillary gel system. Think about what the data would look like in a table
Done
93) Describe three metrics of genetic diversity that can be used in populations
Expected heterozygosity - can be rather insensitive with highly polymorphic markers Allelic richness - A or number of alleles at a locus in a population - Commonly used with msat data because there are only 4 possibilities per locus. - Need to adjust for sample size (rarefaction) when comparing populations - Good measure of evolutionary capacity Mitochondrial haplotype richness - Haplotypes undergo more drift so less diverse and less informative than nuclear DNA
89) What is the "genetic load" in a population. As a manager, how might you be able to minimize the genetic load accumulated by a population
Genetic load is the amount of recessive deleterious mutation carried in a population. Increase gene flow by building corridors and habitat improvement. Mitigate reasons for bottlenecks by habitat improvement or diversification and disease prevention. You can remove dominant breeders if it equalizes breeding opportunities. Selectively breed of genetically distinct individuals' supplementation to the extent possible from other populations.
70) Be able to fill in this graph, with three differently colored lines for each genotype frequency
Genotype graph.
95) What is outbreeding depression
Hybrid progeny have lower performance or fitness than either parent. Intrinsic = due to genetic incompatibilities. Extrinsic = due to reduced adaptation to environmental conditions also may not manifest until F2.
13) If you extracted DNA from your own cells and incubated it with a restriction enzyme, what would you expect to see on an electrophoresis gel
I would not made bands because there are a lot more lengths. It would look like a continuous smear down the gel.
3) If you are designing a set of genetic markers to tell you about genetic diversity in two different populations, do you want your markers to be in specific genes or not. Explain
If your question is about genetic diversity you want to know about the whole genome so if you sequence just one place you don't want them to be under selection because the differences are going to reflect patterns of selection. You want a neutral place on the genome because this can give you information about genetic drift. The finer scale question you want hypervariable alleles. Individual =microsatellites Species = you want a mitochondrial gene because across species that will be different but within species it will be the same.
4) Be able to name at least three different ways that tissue samples can be preserved for later genetic analysis
In liquid nitrogen, dry silica gel, or 95% ethanol.
62) In what kinds of populations would you expect Ne and Nc to be especially different, and why might that be a problem
In polygynous populations. This could be a problem because Ne is what determines the strength of drift so these populations have a very small Ne making drift stronger and the possibility to lose an allele greater.
81) What is the difference between inbreeding and inbreeding depression
Inbreeding depression has negative fitness consequences. Inbreeding is just the level of inbreeding.
32) Why are dye-labelled primers useful in microsatellite analysis
It allows you to combine different samples in the same lane because a different dye can be added to specific samples. Then you can compare multiple samples on the same chromatogram. And you can combine different loci in the same lane.
20) Why is PCR such valuable tool in molecular genetics (think about sampling the genome)
It allows you to sample just one target region and give you a lot of replication of the sample.
6) What is a hair snare
It can be used to get hair samples from animals by baiting them and getting them to walk by a wire that can catch hair.
54) How is a closely related reference genome useful in aligning fragments resulting from shotgun sequencing
It gives you a reference of what the genome would most likely look like and also provides a layer of depth.
14) What overall charge does DNA have, and what pole does it migrate to in electrophoresis
It has a negative charge and migrates to the positive side.
11) What is a molecular ladder, and how is it useful in electrophoresis
It is a control to compare your electrophoresis bands to. The bands made by the molecular ladder are made from predetermined number DNA base pairs. Comparing your results to the band can give you an idea of the number of base pairs in your fragments.
35) What are some of the downsides to choosing microsatellites as a marker system and some of the benefits?
It is expensive It doesn't use sequence data just lengths so it can miss sequence variation that does not change the fragment. Cannot infer on divergence so different lengths do not say anything about the relatedness of species. Benefits Nuclear markers hypervariable good for identifying individuals good for population-level gene flow and diversity
90) What is the 'extinction vortex'
It is positive feedback that happens when populations get small, fragmented and isolated. This leads to inbreeding and loss of genetic diversity, which leads to reduced adaptability and survival and reproduction, which leads to an even further population decline.
61) What is the "effective population size" and how does it differ from "census size"
It is the individuals that effectively reproduce in the population. It is much smaller than the census size because not all the adults reproduce and contribute to the next generation.
40) Explain why high-resolution electrophoresis is important in Sanger sequencing
It needs to be able to detect changes in a single base pair which is really small.
91) Without genetic information, what clues might you have about there being an inbreeding depression problem in your population
Lower litter size, clutch size, low birth weight, decreased fertility, immunological deficiencies, physical deformities, behavioral anomalies.
83) What does a positive FIS mean
Means there is a heterozygote deficit relative to the HWE expectations due to inbreeding.
82) What does FIS measure
Measures inbreeding within a local population based on deviations from expected heterozygosity of HWE
5) Do you expect a longer hair to give you more DNA than a shorter hair. Explain
No because the genetic material is in the follicle the hair is just proteins.
69) Be able to describe at least FIVE assumptions underlying HWE
Random mating No selection No mutations No emigration immigration Infinite population Equal sex ratios
86) What is a deleterious recessive allele, and why is it a problem
Recessive alleles that can hide from selection until the homozygous genotype is expressed and reduces the fitness of an individual generally the reduced fitness comes from multiple loci. This is a problem because natural selection does not act on them in heterozygotes so they can remain in populations.
1) What is a "genetic marker"
Samples of the genome at specific loci to describe sequence variation at the individual level.
57) What causes genetic drift, and why is it considered to be a stochastic process
Sampling error and the fact that not all reproducing individuals contribute to the next generation. Its stochastic because the changes are random and not predictable. Genetic drift is not directional.
9) Explain why restriction fragments are polymorphic among individuals or species
Since the sequence of DNA is not the same for individuals or populations the specific places in which the restriction enzyme will cut will be different creating different lengths.
29) What is it about microsatellite loci that make them more hypervariable than the rest of the genome
Slippage in replication leads to new msat alleles. Most are non-coding and there are no fitness repercussions.
22) Why should the melting point of both primers (based on GC content) be similar for a particular PCR reaction
So that the temperature in which they are denatured is the same and the PCR thermocycling is in sync.
37) what are the ingredients of PCR? How are the ingredients in a Sanger sequencing reaction different from a normal PCR reaction. Specifically, what is your 'template' and what is your 'nucleotide' in Sanger sequencing
Template (extracted genomic DNA sample) primers dNTPs taq polymerase MgCl Ultra pure water The template for sanger sequencing and PCR is the same (genomic DNA). Sanger sequencing uses only one not dye-labeled primer instead of two primers one of which can be dye labeled in PCR. The nucleotide is both dNTPs and ddNTPs and they are dye-labeled for sanger sequencing.
23) Which do you expect to be more conserved across populations or species when designing a PCR reaction - the target region or the primer binding sites Explain
The binding sites. If you want to compare the DNA then there must be some variation in the target region that you are looking at. The binding sites should be the same so that you only have to use one primer and it will bind to the same spot in the DNA of different populations or individuals.
92) Why is inbreeding depression often more pronounced in natural environments than in captive conditions
The captive individuals don't have a lot of environmental factors to deal with. So the reduced fitness is not as visible. They can look like their fine in captive conditions. THE CAPTIVE CONDITIONS can compensate for the inbreeding depression.
53) Explain how a series of images of the same flow cell can provide information about the sequence of the clusters
The color of the dot in the picture represents the base pair that comes next in the sequence. Each picture captures the color or base pair of each cluster. So after every cycle of adding a ddNTP and then removing it and adding a dNTP you will get a sequence from the pictures.
42) In Sanger sequencing, you need a single primer, while in normal PCR you need two primers. Why is this
The ddNTP added to the mixture will automatically stop the sequence so you don't need a second primer to stop it like in PCR.
12) How does gel electrophoresis separate molecules into "bands" of similar charge and size
The gel has a negative and positive pole. DNA is slightly negative so it will be attracted to the positive pole. The smaller fragments will move through the gel faster because they have less molecular weight. So smaller fragments will make bands closer to the positive pole and larger ones will stay closer to the negative side. The ones that are similar in length will reach the same spot on the gel and accumulate there.
78) In a Structure analysis, what is K
The most likely groupings of these individuals into a number of populations by minimizing departures from HWE.
15) What specific molecule gives DNA its charge
The phosphates in the backbone.
34) How does the ladder work with fragment analysis in capillary electrophoresis compared to gel electrophoresis
The predetermined lengths or number of basepair sample is also added but with a different dye. the camera is also picking up signal from these lengths but a different dye is added to them to be able to see both on a chromatogram. The peaks or the base pair number at which the fragment was detected acts like the bands in electrophoresis to tell you the fragment length.
33) Why do microsatellite chromatograms often show a 'stutter' pattern, with multiple peaks differing by the size of the motif
The slippage that happens when the msat is replicated in the organisms also happens in the PCR reaction. So the little peaks are the detection of fragments where this has happened. But there's not not a lot of them in which this happens. So they are small. Most of them are representative of the actual number of basepairs and the signal is strong.
16) In an electrophoresis gel looking at DNA fragments, which bands move fastest and slowest
The smaller fragments that have less base pairs move the fastest. The larger ones that have more base pairs move slower.
85) What does an FIS of zero mean
The subpopulation is in HWE.
84) What does a negative FIS mean
There are more heterozygotes than expected based on HWE random mating (excessive outbreeding)
45) What do you predict would happen in a Sanger sequencing reaction if the sequencing template (i.e. PCR product) contained two different sequences
There would be overlapping peaks.
28) Why are microsatellite markers especially useful for distinguishing closely related individuals or populations
They are hyper variable which means they have a higher rate of mutation and they have many different lengths at each locus this is helpful for distinguishing individuals at a fine scale. They are also found in large numbers in eukaryotic genomes.
39) How are dNTPs different from ddNTPs structurally, and why is that important
They are missing an oxygen on the 3'end which makes it so that DNA polymerase does cannot add any more nucleotides.
26) In terms of nucleotide sequence, what makes microsatellites different from other sequences in the genome
They are simple sequence repeats SSR. So they are non-coding and they are short nuclear sequences that have repeating motifs.
2) What are some applications for using genetic markers
They can tell you the demographic history (genetic drift), inbreeding, geneflow, evolutionary history, parentage, kinship, gender, mapping of loci that contribute to a trait. They can be sued as an indicator of genome-level processes.
8) Where do restriction enzymes come from
They come from bacteria.
27) How is a microsatellite allele different from a SNP allele
They have the same repeating sequence but different lengths SNP differ in sequence. With an SNP you have four different ways it could be different in microsatellites the differing base pairs could be 2 or three based on the motif.
1) Be able to describe some specific applications of microsatellites in wildlife management. Using Google Scholar to find a few papers might be insightful!
They were used to show population structure in fish for conservation purposes.
50) What purposes do the adapters serve. There are two
To stick to the flow cell and they also have the primer
79) Given a Structure diagram, as in the lecture notes, be able to explain what the narrow vertical lines represent, what the blocks of these lines represent, and what the colors represent and how they are related to a K value.
Vertical line are individuals and the colors are the number of genetic groups that the whole population is divided into which is K. K can be manipulated. The blocks are sampled populations.
63) What is a selective sweep
When an advantageous allele increases in frequency because of selection.
64) In an aligment of sequences from multiple individuals in a population, what is the 'signature' of a recent selective sweep. Be able to explain this
When individuals have this advantageous allele they reproduce and pass it on. But since there is recombination and this is a course process not just that allele gets passed on but chunks of the chromosome. Natural selection is acting on that allele but also the chunks that come with it during recombination. So this results in a part of the genome that is highly homogenized at that locus. You can see this through next gen sequencing as time passes this signature starts to fade because of mutations and more recombination.
31) In automated capillary electrophoresis, how are bands detected if they end up migrating out of the capillary and into the buffer reservoir during analysis. Explain in terms of the primers and the process
When the fragment passes through the capillary filled with gel the camera detects this fragment and its length. The first detected will be the shortest fragments. The forward primers have a florescent dye that the camera can detect. This data is converted into a chromatogram and the peaks are the detection of the fragment in relation to when the camera detected it. This is how you can tell the fragment lengths by when the fragment passed through the lane.
68) Be able to determine whether a population is in HWE, and if not, whether the deviations from HWE are statistically significant. For this you will be given a genotype dataset on a population sample with just one locus and two alleles, and you will be given a Chi-square formula, information on how to calculate the degrees of freedom, and a Chi-square table. There are several worked examples in your notes and on the video sets, so practice this. You should be able to do this using a simple calculator (available at the testing center)
When the predicted genotype frequency is not the same as the observed genotype frequency then the population is in disequilibrium.
77) What is the Wahlund Effect
When you sample two populations simultaneously that differ in allele frequencies and as a result you see an underrepresentation of heterozygotes.
52) Do the clusters represent different sequences, or are they all identical to each other
Within the cluster it is the same sequence but across clusters the sequence is different.
71) If you have two populations which are each in HWE, is it possible to get an Fst pooling these populations that is >0 Explain?
Yes. Because they can still differ in allele frequencies even though each one is in HWE the pooled expected value of heterozygotes can still be more than the observed just based on whether there is gene flow between the populations. You can have two population that are in HWE but they have different allele frequencies so they will have a deficit of heterozygotes.
75) Why is a set of pairwise Fst values (i.e. among all possible pairs of populations) more useful than the situation in the previous question
You can get indicator geneflow between each of the populations and not geneflow between the whole pooled population and gives you more information it also gives you an indicator for genetic distance and tells you where the divergence is among populations and which ones are more closely related.
49) Explain the sequence (ha-ha) of events in shotgun sequencing, starting with the DNA extraction
You extract the DNA then chop up the genome up into little pieces with sonification. Then you add adapters to the ends of the chopped up DNA these adapters stick to fragments of DNA on the flow cell. Then you run a PCR reaction on the flow cell to amplify the pieces of DNA that are stuck on the flow cell. This creates clusters made up of the same sequence of DNA. Then you denature the DNA so it is single stranded. You start to build the new strand of DNA by adding a florescent ddNTP taking a picture and then removing it. You add regular dNTPs to build the strand only by one base pair at a time. Then once the regular dNTP is added you add another round of ddNTPs and take a picture. The clusters will be the color of the base pair in the picture. Then you input that data into a computer for it to give you a sequence based on overlapping nucleotides in the sequence.
51) How do you go from one molecule on a flow cell to a cluster of identical molecules
You run a PCR reaction so that they are multiplied.
10) When you conduct a restriction analysis, you are adding enzymes (clear liquid) to a DNA extraction (clear liquid). How do you visualize the resulting restriction fragments in the tube of clear liquid that is your reaction
You use electrophoresis to separate the fragments by length and then this creates bands based on number of base pairs?
59) Why is genetic drift a problem in natural populations.(multiple reasons)
You will always lose genetic variation over time if no variation comes from mutation or migration. If you lose variation you can fix deleterious recessive alleles leading to inbreeding depression and reduced fitness The effect of drift on small populations is stronger than natural selection There is loss of adaptive potential.
47) Sanger sequencing is generally good for sequence lengths up to 600-800 bp. If you wanted to sequence a region of the genome that was 5000 bp long, how might you accomplish that with Sanger sequencing
You would have to have to get many shorter sequences using different primers and sequence them then put them together. Or you could start at different places with the primers.
80) If you have two populations (or species) which are not able to interbreed, but you have migration among populations, what do you expect your Structure diagram to look like
You would see all of the vertical lines be one color but they will be randomly interspersed.
66) HWE is an equilibrium between ______ ____ and _______
allele frequencies genotype frequencies
38) Be able to explain in detail why it is important to use ddNTPs and label them (fluorescently) differently depending on whether they are ddATP, ddGTP, ddTTP, and ddCTP
ddNTPs. Stop the sequence when added because they are missing an on the 3' end. The sequence is stopped at every base pair at the end of the reaction. To distinguish what the base pair is you need the florescent dye. Each type of base pair has a specific dye. The shortest or beginning base will pass through first and the camera detects the color of the dye that it has which starts the chromatogram as the next longest fragments go through the peaks will be color coded and gives you the sequence.