SAB presentation to Jim narrated

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SAB presentation to Jim narrated

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  • SAB presentation to Jim narrated

    1. 1. Ecological and evolutionary functional genomics: finding & studying genes that matter Christopher W. Wheat
    2. 2. Ecological and Evolutionary Functional Genomics <ul><li>Integrative Research </li></ul><ul><ul><li>Ecology (Hanski) + Molecular Biology (Frilander) + Evolution Genetics (Wheat) + Physiology (Marden) </li></ul></ul><ul><li>Need genomic tools </li></ul><ul><ul><li>Access to the coding genes </li></ul></ul><ul><ul><li>1000’s of SNPs &/or 100’s of microsatellites </li></ul></ul><ul><ul><li>Microarrays for gene expression </li></ul></ul><ul><ul><li>QTL, association mapping, and outlier analyses </li></ul></ul><ul><li>Need to sequence the genes </li></ul>
    3. 3. Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing C. Vera and C. Wheat, Fescemyer, Frilander, Crawford, Hanski, Marden 2008
    4. 4. 48k contigs
    5. 5. First de novo transcriptome assembly using 454 pyrosequencing SNP’s
    6. 6. Annotation of 45,000 contigs + singletons Predicted genes: D. melanogaster = 13,379 B. mori = 18,510 Estimated coverage : 70% D. mel estimate 50% B. mori estimate Lower estimate 50% genes
    7. 7. Upper estimate of 70% = 13,142 genes
    8. 8. Metabolic Map Comparison <ul><li>M. cinxia with 454 seq. </li></ul>Bombyx mori with WGS
    9. 9. Increasing 454 sequencing (Aland + China & France) <ul><li>Find & cover more genes </li></ul><ul><ul><li>Get full length contigs </li></ul></ul><ul><li>SNPs </li></ul><ul><ul><li>Reconfirm previous </li></ul></ul><ul><ul><li>Identify population specific SNPs </li></ul></ul><ul><ul><li>Map genes & compare with other species (synteny) </li></ul></ul><ul><ul><li>Finding genes of interest </li></ul></ul><ul><ul><ul><li>QTL and association mapping </li></ul></ul></ul><ul><ul><ul><li>Outlier analyses (Fst) </li></ul></ul></ul><ul><ul><li>Demographics </li></ul></ul><ul><ul><ul><li>Population structure effects on genetic diversity </li></ul></ul></ul><ul><ul><ul><li>Colonization history </li></ul></ul></ul>
    10. 10. <ul><li>Custom Designed Microarrays (Agilent) : </li></ul><ul><li>Using 13,780 assembled contigs </li></ul><ul><li>Used validated probes (selected best of 6 per contig) </li></ul><ul><li>60 bp probes randomly printed at least in triplicate </li></ul><ul><li>44,000 feature array, with 4 arrays per slide. </li></ul><ul><li>2 dyes hybridized per array </li></ul><ul><li>randomized across population type </li></ul><ul><li>amplified RNA indirectly labeled using Alexiflour dyes (555 & 647) coupled to aaUTP incorporated during synthesis. </li></ul><ul><li>Scanned using Genepix 4000B </li></ul>
    11. 11. <ul><li>2 day old females, 25 families from 25 different local populations (n = 65) </li></ul><ul><li>Common garden reared for 2 generations </li></ul>Wheat et al., in prep. Population age: P = 0.02
    12. 12. <ul><li>Assessing expression variation </li></ul><ul><li>across  12,000 genes in : </li></ul><ul><li>Abdomen (n = 20) </li></ul><ul><li>Thorax (n = 34) </li></ul><ul><li>Head (n = 18) analysis in progress </li></ul><ul><li>What are the expression differences: </li></ul><ul><li>between new and old populations? </li></ul><ul><li>across PMR performance variation? </li></ul>
    13. 13. Mixed Model Analysis : popage & peak metabolic rate Popage fixed effects: dye popage bodymass popage*bodymass PMR fixed effect: dye PMR Random effects: slide array(slide) spot spot*array individual (FDR threshold = 5%) Using JMP Genomics (SAS)
    14. 14. Mixed model analysis of Abdomen ANOVA normalized (Multiple test correction used FDR @ 5% = 3.16 -log10P) <ul><li>221 genes differentially expressed </li></ul><ul><li>(≈ 11 false positives) </li></ul>-log 10 P-value
    15. 15. Significantly different genes (FDR threshold = 5%) Peak Metabolic Rate Continuous Wheat et al., in prep. 623 33 188 656 Population Age 221 Categorical
    16. 16. <ul><li>Abdomen: </li></ul><ul><li>33 genes overlap population age and PMR </li></ul><ul><ul><li>Egg development </li></ul></ul><ul><ul><ul><li>Ecdysteroid 22-phosphate </li></ul></ul></ul><ul><ul><ul><li>3-dehydroecdysone 3alpha-reductase </li></ul></ul></ul><ul><ul><li>Metabolism </li></ul></ul><ul><ul><ul><li>Fructose 1,6-bisphosphate aldolase </li></ul></ul></ul><ul><ul><ul><li>Gallerin </li></ul></ul></ul><ul><ul><ul><li>Tyrosine aminotransferase </li></ul></ul></ul><ul><ul><li>Only 12 of 33 with homology inferred function </li></ul></ul><ul><li>Thorax: No sig. popage effects expression </li></ul>
    17. 17. Biological validation: vitellogenin <ul><li>Egg yolk precursor protein </li></ul><ul><li>Higher protein = more eggs </li></ul><ul><li>Protein levels </li></ul><ul><ul><li>new > old (P < 0.05) </li></ul></ul><ul><li>Microarray (mRNA levels) </li></ul><ul><ul><li>new > old (P = 0.02) </li></ul></ul>
    18. 18. Temperature treatments Last instar larvae Reared in lab Study gene expression Kvist et al., in prep. 20 o C 26 o C 35 o C Temperature maximum Cold Standard Hot Treatments Hot Standard Cold
    19. 19. Summary <ul><li>We’ve built, and are improving, our genomic tools : </li></ul><ul><ul><li>Sequenced transcriptome & aiming for full length, better coverage </li></ul></ul><ul><ul><li>1000’s of SNPs identified and being prep’d for high throughput analysis </li></ul></ul><ul><ul><li>Microarray validated and used to address ecological hypotheses </li></ul></ul><ul><li>Using tools to test hypotheses about expression differences </li></ul><ul><ul><li>between population types? </li></ul></ul><ul><ul><li>larval temperature experience? </li></ul></ul><ul><li>Preliminary findings: </li></ul><ul><ul><li>Metapopulation dynamics sort/maintain expression variation </li></ul></ul><ul><ul><li>Larval thermal experience matters, some shared responses to cold and heat </li></ul></ul><ul><li>Dispersal variation appears to arise from </li></ul><ul><ul><li>abdomen fuel supply variation rather than thorax structural variation </li></ul></ul>

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