Lab 11 :Test of Neutrality and Evidence

for Selection

Goals: 1.Calculate exp. # of different allele in a population for different marker.

2.Detect departure from neutrality using-

1. Ewens- Watterson test.2. Tajima’s D test.3. HKA test and4. Synonymous and Nonsynonymous

nucleotide substitution test

Infinite Alleles Model (IAM)

• Each mutation produces a new allele• At equilibrium, number of alleles and shape of

allele frequency distribution remain constant• Lost alleles replaced by new mutations

Ewens -Watterson test

12...

211)(

12

0

NikE

N

i

NeuWhere 4,

14

1

ee N

f

2iHW pf

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 = 0.810-6 and = 9.210-2, respectively. How many different alleles do you expect to find for each marker in a sample of:

•7 people?•12 people?•What assumptions were made for these calculations to be valid?

Tajima’s D

• Under neutrality, we expect the following:

• Test of the coalescent model– Assumes neutral alleles and constant population

size

1 1n

i

S

i

S m

Tajima’s D test

d = − S = 0Under neutrality

D = )(dSE

d

.

bottleneckrecent or selection Balancing0

growth populationor selection positivePurifying/0

D

D

(Hamilton 270)

plantsciences.ucdavis.edu

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:

a.Determine the number of polymorphic sites (S) and calculate the nucleotide diversity () based on these sequences.

b.Perform the tests of neutrality developed by Ewens-Watterson and Tajima and interpret the results.

c. Provide a statistical and a biological interpretation of the results from the two neutrality tests.

Hudson-Kreitman- Aguade(HKA) test

(Hamilton 266)

Hudson-Kreitman- Aguade(HKA) test

Adh Control locusPolymorphism within species (S/m)

0.101 0.022

Divergence between Species(D/m)

0.056 0.052

Ratio (within/between)

1.80 0.42

χ2 6.09p-value 0.016

File Region of tb1 Subspeciesutr_mays.arp 5’ untranslated region maysutr_par.arp 5’ untranslated region parviglumisexon_mays.arp exon maysexon_par.arp exon parviglumis

Test A tb1 5’ untranslated region Average of control lociPolymorphism within subspecies

0.00093 0.01996

Divergence between subspecies

0.05255 0.02242

χ2 13.58p-value 0.001

Test B tb1 translated region Average of control lociPolymorphism within subspecies

0.00243 0.01996

Divergence between subspecies

0.01273 0.02242

χ2 2.70p-value 0.26

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:•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? •Use Arlequin to perform the tests of neutrality developed by Ewens-Watterson and Tajima. Interpret and discuss the results.•Interpret and discuss the results from the following 2 HKA tests:•GRADUATE STUDENTS ONLY: Download and read the paper describing this study (Wang et al. 1999), which is uploaded on the lab page of the class website, and provide an extended biological interpretation of the results of a) – c).

Synonymous and Nonsynonymous Nucleotide Substitution test

S

N

d

d

dN = Observed # nonsynonymous substitutions/nonsynonymous sitedS= Observed # synonymous subsitutions/synonymous site

5’-ATT GTT CAT CGT ACC CAT CGA-3’5’-ATT GTT CAT CGC ACC CAA CGA-3’

Synonymous siteSynonymous mutation

Nonsynonymous siteNonsynonymous mutation

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 GTT CAC TTG ACC GCA CGA AGT AGA TTA-3’

Seq 1 Codon No. potential synonymous sites (sj)

No. potential nonsynonymous sites (nj)

Seq 2 Codon No. potential synonymous sites (sj)

No. potential nonsynonymous sites (nj)

ATG 0 3 ATG 0 3GTT GTT

Total