Lecture 14: Linkage and Mapping II Flashcards
explain Tetrad analysis in Neurospora
- In most eukaryotes recombination analysis cannot be used to map centromeres.
Few markers that display heterozygosity near centromere.
How can we define the position of the centromere genetically?
2. Fungi and algae produce either unordered or linear (ordered) tetrads. Neurospora is haploid and produces linear tetrads.
explain The linear (ordered) meiosis of Neurospora = 4
- Haploid fungi unique in that for any given meiocyte the spores are products of meiosis and held together in membranous sac called ascus.
2 Four products of meiosis lie in straight row.
- Four products of a single meiosis undergo mitosis to produce octad.
- Position of spores within ascus reflects chromatid segregation during meiosis
First division (MI) segregation pattern (no crossing over) = 4
1 * A and a segregate into separate nuclei at the 1st meiotic division
2 * A/a meiocyte undergoes meiosis, then mitosis, resulting in equal numbers of A and a products.
3 * Two adjacent blocks of four spores:
AAAAaaaa
4 * Indicative of no crossing over between centromere and A at prophase I
The second division (MII) segregation pattern = 4
1 * A and a segregate into separate nuclei at the 2nd meiotic division
2 * A/a meiocyte undergoes meiosis then mitosis
3 * Alternate pairs of spores: AAaaAAaa
4 * Indicative of crossing over between A and centromere
Four second-division (MII) segregation patterns are equally possible - explain how? = 3
- Homologous chromosomes attach randomly to the spindle pole, so sister chromatids segregate at random to give four different patterns
- Crossovers only happen between non-sister chromatids on homologous chromosome arms (they cannot span a centromere)
- Frequency of second-division segregation patterns is proportional to distance between A and its centromere, which always segregates in a predictable pattern.
Centromere mapping: the steps = 3
- Identify the two classes of divisions
MI patterns, two types, alleles in two groups of 4:
AAAAaaaa or aaaaAAAA
MII patterns, four types, alleles in four groups of 2:
AAaaAAaa, aaAAaaAA, AAaaaaAA, aaAAAAaa - Sum up MII octads and divide by the total, to work out the frequency
of meioses that underwent recombination - Divide this frequency by 2 to get map units from locus to centromere. (map units are defined by the number of recombinant chromosomes from meiosis – 50% of chromosomes from these meioses will be recombinant)
Tetrad analysis is useful because: 3
- We can see the products of individual meioses and crossing over – visual confirmation (rather than inference) of equal segregation of chromatids
- We can map centromeres genetically, very hard in most eukaryotes (and therefore the order of loci relative to the telomeres and the centromere)
- Centromere mapping allows the integration of genetic, cytological and physical maps
What gene is responsible for a given disease (phenotype)? = 3
– Cannot perform controlled crosses
– Small numbers of progeny (sampling error
becomes high)
– Testcross equivalents (e.g. A/a x a/a) are very rare
- Remember the basis of mapping: looking for genetic linkage between traits = 2
– Nail-patella syndrome is autosomal dominant (Nn) – abnormal fingernails and kneecaps. N is rare allele
– N is linked to the ABO blood-type locus (I), which has multiple alleles: IA, IB, and i (null, group O). IA and IB are co-dominant
Identifying underlying gene for a disease =4
1 * Two loci are linked if they appear nearby on the same chromosome.
2 * The task of linkage analysis is to find molecular markers that are TIGHTLY linked to the hypothetical disease locus (loci)
3 * When markers segregate very tightly with the disease phenotype, the underlying gene is likely to be nearby
4 * Integration of Genetic & Physical Maps:
– DNA polymorphisms provide
markers for the genetic trait
– DNA markers on a chromosome can help to locate a disease or other gene – by linkage and physical location
Linkage mapping: Is a marker linked to the disease gene? = 3
1 * Collect families with affected individuals (more the merrier, but 1000+ families)
2 * Genome scan – test everyone for markers evenly spaced across the genome approx. every 10 cM, 400 markers (determine marker genotype)
– rough map to large region, can fine map later to improve resolution
– Calculate LOD score (“log of the odds”): a statistic that describes the strength of evidence for linkage
- “Given the family data, how likely is it that there is linkage between my gene of interest and a given marker?”
Determining LOD score = 6.
1 * The LOD score is an example of a maximum likelihood procedure (estimate the value of a parameter that cannot be directly observed, i.e. θ)
2 * The parameter value that gives the maximum likelihood is taken as the best estimate of the parameter.
3 * Calculate two different probabilities for obtaining the given set of recombinants observed in a family:
1. Assuming independent assortment (θ = 0.5)
2. Assuming a specific degree of linkage (θ = x)
4 * Calculate the ratio (odds) of these two probabilities
5 * Compute log10 of ratio – this is the LOD score
6 * Repeat calculation for range of different values of x, and compare: what value of θ gives highest LOD score?
Determining LOD score; equation
Test to estimate whether the likelihood that two loci are linked is greater than the likelihood that two loci are unlinked.
Z = log10 (LIKELIHOOD OF LINKAGE (θ < 0.5) [A]/ LIKELIHOOD LOCI ARE UNLINKED (θ = 0.5) [B])
EXAMPLE:
A and B are probabilities between 0 and 1
If A = B, then Z = 0.
If A > B, Z becomes positive (linkage more likely) If A < B, Z becomes negative (linkage less likely) Z = 1 means 10:1, 2 = 100:1, 3 = 1000:1 etc
Interpreting LOD scores
Z > +3 means
Conclusive evidence for linkage (higher is better)
+2 < Z < +3
Suggestive evidence for linkage, but relatively weak (inadequate)
Z < -2
Can confidently exclude linkage
-2 < Z < +2
Uninformative linkage analysis (not enough data)
Fill in the blanks
1. During meiosis, …. keeps genes together, but crossing over breaks genes apart.
- The proportion of recombinant gametes produced from a single crossover during meiosis is…
- The maximum recombination frequency between any two loci is … and the minimum frequency is…
- Recombination frequency is half the frequency of …
- Completely linked genes show a recombination frequency of…
- Two loci …. apart exhibit a recombination frequency of 25 %.
- During meiosis, ***linkage keeps genes together, but crossing over breaks genes apart.
- The proportion of recombinant gametes produced from a single crossover during meiosis is ***50 %.
- The maximum recombination frequency between any two loci is 50%, and the minimum frequency is 0%
- Recombination frequency is half the frequency of ***crossovers
- Completely linked genes show a recombination frequency of ***0%
- Two loci ***25cM apart exhibit a recombination frequency of 25 %.