Lecture 21: Association Studies and Signatures of Selection. November 6, 2006

Transcription

1 Lecture 21: Association Studies and Signatures of Selection November 6, 2006

2 Announcements Outline due today (10 points) Only one reading for Wednesday: Nielsen, Molecular Signatures of Natural Selection Term paper draft due November 17 (10 points)

3 Last Time QTL examples and limitations Linkage Disequilibrium and Association studies

4 Today Signatures of selection Reverse genetics

5 Modes of selection on single genes s AA < s Aa < s aa or s aa < s Aa < s AA Directional One extreme genotype has the highest fitness (purifying selection) w AA Aa aa Overdominance An intermediate genotype has the highest fitness (balancing selection) Underdominance The two extreme genotypes have the highest fitness (diversifying selection) w w w = fitness, s i =selection against genotype i s Aa < s aa & s AA AA Aa aa s Aa > s aa & s AA AA Aa aa

6 What are the Signatures of Selection? Compare patterns of phenotypic variation to patterns of neutral genetic variation among populations Neutral genetic variation: F ST and analogs σ = a 2 where σ a = variance in allele frequency among populations F ST = 2 2 σ Analog of F ST for adaptive traits: Q ST Howe and Aitken

7 Comparing Q ST to F ST Howe et al. Q ST generally > F ST (allozymes) for adaptive traits Relative importance of adaptive traits can be inferred from difference

8 Inferring Selection from Patterns of Diversity Purifying or directional selection should reduce diversity due to 'Selective Sweep' Reduces number of alleles present in population compared to expectations Generate expectations based on comparisons to closely related species or neutral theory Directional selection should also increase differentiation of populations: unusual pairwise F ST values

9 Detecting Selection in European beech Common beech (Fagus sylvatica) occurs throughout most of western Europe Sampled 210 trees along elevational gradient at 3 study sites Scanned genome with 254 AFLP loci

10 Selection in Fagus One AFLP locus had unusually high F st Frequency of allele decreased with temperature at time of establishment of individual trees Jump et al MOLECULAR ECOLOGY 15 (11):

11 Linking sequence to phenotypes QTL I Candidate Region Candidate Gene Identification Candidate Gene Assessment COARSE ROOT ABOVE:BELOW P_204_C S8_32 P_2385_C P_2385_A T4_10 S15_8S5_37 T4_7S6_12 S8_29 P_2786_A S12_18 T1_13 T7_4 T3_13 T3_36 S17_21 S15_16T12_15 T2_30 S13_20 S1_20 T9_1 S1_19 S3_13 S1_24 S2_7 P_575_A T12_22 S2_32 T7_9 S2_6 S13_16 T5_25 T5_12 T10_4 T1_26 T7_13 P_93_A S4_20 S7_13 S7_12 T12_4 S4_24T3_10 S6_4 P_2852_A S3_1 S6_20 S13_31 T7_15 T2_31 S8_4 S8_28 O_30_A T5_4 T3_17 T12_12 S5_29 P_2789_A P_634_A S17_43 S17_33 S17_12 S4_19 S17_26 Homology- Based Selection SNP Association Studies Metabolic QTL Expression QTL Mutants: Knockouts Overexpression Complementation

12 Case Study: Disease Resistance Melampsora spp. Is a leaf rust of great commercial importance in poplar culture MXC3 confers major gene resistance to Populus trichocarpa Positional cloning and chromosome walking failed: suppressed recombination Attempt to identify candidate genes by examining genomic scaffolds in this region Stirling et al TAG 103:1121

13 Candidate Gene Identification Examined 6.65 Mb of sequence linked to marker: 1530 genes No NBS-LRR genes, typical of gene-for-gene interactions. (NBS-LRR closely linked to another rust locus in poplar (Lescot et al. 2004)) Closest putative resistance genes were thaumatin-like pathogenesisrelated proteins: A new mechanism for plant disease resistance? I STK II C-C NBS LRR III IV TIR NBS LRR LRR TM Thau TM STK V LRR TM STK Yin et al., 2004 Sauter et al. Proteins 48: 146 (2002)

14 No Genome Sequence? Use Expressed Sequence Tags from enriched libraries and/or differential display to identify genes up-regulated in contrasting individuals Creation of large insert DNA libraries and screening for chromosomal regions containing markers Bacterial Artificial Chromosome contains kb fragments Redundant coverage of genome required (50,000 BACs required for Populus genome) Complex pooling and PCRscreening strategies can be efficient

15 Proving genes cause phenotypes Forward Genetics: Start with the phenotype and try to find the gene QTL mapping, association studies, random mutagenesis Usually correlative and therefore lacking experimental proof Reverse Genetics: Start with the gene and try to find the phenotype Association studies with candidate genes in populations with low LD Signatures of selection within candidate genes: SNPs

16 SNPs A Single Nucleotide Polymorphism (SNP) is a single base mutation in DNA. The most common source of genetic polymorphism (e.g., 90% of all human DNA polymorphisms). Two types of nucleotide base substitutions resulting in SNPs: transitions and transversions

17 First and second position SNP often changes amino acid Third position SNP often synonymous.

18 SNP Characteristics Two Major Classes in Coding Regions: Synonymous and Nonsynonymous Nonsynonymous substitution: Thr Tyr Leu Leu ACC TAT T T TTG CTG Synonymous substitution: Thr Tyr Leu Leu ACC TAT TTG CTG ACC TCT T T TTG CTG Thr Ser Leu Leu ACC TCC TTG CTG Thr Tyr Leu Leu The rates of nucleotide substitutions in the third position are much higher than in the first and second positions, due to redundancy in the third position:

19 Synonymous & Nonsynonymous Substitutions K S is the relative rate of synonymous mutations per synonymous site K A is the relative rate of nonsynonymous mutations per non-synonymous site ω = K A /K S If ω = 1, neutral selection If ω < 1, purifying selection If ω > 1, positive Darwinian selection For human genes, ω 0.1 Neutral selection: no selection for or against amino acid changes; generally, K A = K S = µ (genome-wide mutation rate, heavily influenced by non-coding regions) Purifying selection: amino acid changes are selected against; only synonymous substitutions persist Positive (Darwinian) selection: amino acid changes are advantageous; this is relatively rare Often, different sections of a gene will have different K A /K S McDonald-Kreitman test can be used to determine significance of K A /K S ratios: compares rates within and between species

20 Allele Frequency Spectra and Signatures of Selection Statistical tests for departure from neutrality: Tajima's D is essentially an estimate of whether there is an excess of low frequency alleles Nielsen 2005

21 Reverse Genetics and Standards of Proof for Gene Function The best way to prove a gene is associated with a phenotype is to mutate the gene and show that the phenotype is eliminated Simplest for Mendelian traits, but also applies to quantitative traits 'Gold Standard' is complementation of mutant with wild-type gene Mutagenesis and genetic engineering are the primary approaches

22 What is Genetic Engineering? Gene isolation, configuration, and asexual transfer Genes isolated from any organism, introduced into cell in tissue culture Whole plants or animals regenerated from single cell Transgenic organisms contain DNA introduced by genetic engineering Allows isolation of effect of single gene by turning up or turning down expression

23 Agrobacterium is a natural plant genetic engineer Courtesy of Steve Strauss, Oregon State University

24 The Ti-plasmid is required for crown gall disease T-DNA = Transferred DNA T-DNA Ti plasmid Ti = Tumor inducing Courtesy of Terri Lomax, Oregon State University

25 Disarming the T-DNA Border Hormones Food Border Auxin Synthesis Cytokinin Synthesis Opine Synthesis Cut and replace Antibiotic Resistance Gene of Interest Reporter Gene Courtesy of Steve Strauss, Oregon State University

26 Insertion of DNA into cells via biolistics ( gene gun ) Courtesy of Terri Lomax, Oregon State University

27 Plate of regenerating Golden Promise Process of Genetic Engineering of Plants Create rdna with gene from same or different organism Transfer DNA to plant cell; allow plant cells to divide under selection Cue cells to reform plant every cell will have new DNA Confirm introduced DNA and expression of foreign protein in plants Selection rdna Introduce DNA Cells dividing Source of gene Hormones Regenerating barley plants in Magenta box Check for introduced trait Put in soil Remove hormones Courtesy of Peggy Lemaux, University of California, Berkeley

28 Next Time Landscape genetics