Lab 11 :Test of Neutrality and Evidence for Selection

Goals: Calculate the expected number of different alleles for a given locus in a population. Detect departure from neutrality using: Ewens- Watterson test. Tajima’s D test. HKA test and Synonymous and Nonsynonymous nucleotide substitution test

Infinite Alleles Model (IAM) Each mutation produces a new allele At equilibrium: number of alleles – constant shape of allele frequency distribution – constant frequency of any given allele – not constant lost alleles replaced by new mutations

Ewens -Watterson test Expected homozygosity under mutation-drift equilibrium and assuming IAM: Expected homozygosity under HWE: P-value < 0.025: Too even -> Balancing selection or recent bottleneck P-value > 0.975: Too uneven -> Directional selection or population growth

Problem 1. Estimates of the long-term effective population size of human populations vary widely, ranging from as low as ~3,000 to as high as ~100,000. To estimate allele frequencies for a forensic identification study, you are genotyping individuals selected at random from a population with an estimated Ne = 7,500. You are using one allozyme and one microsatellite marker, with estimated mutation rates  = 0.810-6 and  = 9.210-2, respectively. How many different alleles do you expect to find for each marker in a sample of: 7 people? 12 people? List assumptions that need to be made for these calculations to be valid. Discuss which of these assumptions (if any) seem reasonable.

Pairwise DNA sequence diversity

Tajima’s D Under neutrality, we expect the following: Two different ways of estimating the same parameter Deviation of these two indicates deviation from neutral expectations Assumes neutral alleles and constant population size (Based on nucleotide diversity) (Based on number of polymorphic sites)

Tajima’s D test . Under neutrality: d =  − S = 0 D =

plantsciences.ucdavis.edu

Problem 2. File aspen_phy Problem 2. File aspen_phy.arp (which is already in Arlequin format) contains sequence data from exon 1 of the phytochrome B2 (phyB2) gene of 24 aspen (Populus tremula) trees sampled along a wide latitudinal gradient in Europe. Use Arlequin to: Determine the number of polymorphic sites (S) and calculate the nucleotide diversity () based on these sequences. Perform the tests of neutrality developed by Ewens-Watterson and Tajima and interpret the results. Provide a statistical and a biological interpretation of the results from the two neutrality tests. Is selection the only possible reason for significant discrepancies between  and S? Describe at least one other scenario in which a force other than selection would make D significantly different from 0. Include only a summary of the Arlequin output in your report (i.e., values of S and , as well as test statistics and p-values for the two neutrality tests). Do not include the entire output.

Hudson-Kreitman-Aguade (HKA) test p437 Hedrick 2005   Adh Control locus Polymorphism within species a 0.101 0.022 Divergence between species b 0.056 0.052 Ratio (within/between) 1.80 0.42 χ2 6.09 p-value 0.016

For each of these regions of tb1 and for each subspecies: Problem 3. Files utr_mays.arp, utr_par.arp, exon_mays.arp, and exon_par.arp contain sequence data from the 5’ untranslated region and from an exon of the teosinte branched1(tb1) gene of maize (Zea mays ssp. mays) and its most likely wild progenitor Zea mays ssp. parviglumis. For each of these regions of tb1 and for each subspecies: a) Use Arlequin to determine the number of segregating sites (S) and calculate the nucleotide diversity (). What can you infer by comparing nucleotide diversity between the two species for each region? b) Use Arlequin to perform the tests of neutrality developed by Ewens-Watterson and Tajima. Interpret and discuss the results both statistically and biologically. File Region of tb1 Subspecies utr_mays.arp 5’ untranslated region mays utr_par.arp parviglumis exon_mays.arp exon exon_par.arp

Problem 3 continue. C) Interpret and discuss the results from the following 2 HKA tests: Test A tb1 5’ untranslated region Average of control loci Polymorphism within subspecies 0.00093 0.01996 Divergence between subspecies 0.05255 0.02242 χ2 13.58   p-value 0.001 Test B tb1 translated region Average of control loci Polymorphism within subspecies 0.00243 0.01996 Divergence between subspecies 0.01273 0.02242 χ2 2.70   p-value 0.26

Synonymous and Nonsynonymous Nucleotide Substitutions

Actual synonymous substitutions: 1 seq1 Sj Nj seq2 AGT 0.333 2.667 AGC TTT TTA 0.667 2.333 CCA 1 2 GGG GCG CTA 1.333 1.667 Total 3.999 11.001 4.333 10.667 S F P G L 5’-AGT TTT CCA GGG CTA-3’ 5’-AGC TTA CCA GCG CTA-3’ S L P A L Actual synonymous substitutions: 1 Actual nonsynonymous substitutions: 2 𝑑𝑆= 1 (3.999+4.333)/2 =0.24 𝑑𝑁= 2 (11.001+10.667)/2 =0.1846 ω= 𝑑𝑁 𝑑𝑆 =0.1846/0.24= 0.77

Problem 4. Calculate the ω = dN/dS ratio based on the following 2 DNA sequences:   5’-ATG GTT CAT TTT ACC GGA CGA AGT CGA TTA-3’ 5’-ATG GAT CAC TTG ACC GCA CGA AGT AGA TTA-3’ What does the value of ω indicate?

Problem 5. GRADUATE STUDENTS ONLY: Search the literature for an example of an application of one of the tests for departures from neutrality. Describe the question that the test is addressing, the results, and the authors’ interpretation of the results. Receive two points of extra credit if you can find a case in which the test is inappropriately applied and/or interpreted. Please send the paper to Ran when you submit your report.