Reseach Training Presentation

1. Reseach Training Presentation • By Yanhong Zhao • Department of Evolutionary Functional Genomics, Uppsala University, Sweden • Supervisor: Prof. Ulf Lagercrantz

2. Department of Evolutionary Functional Genomics • Patterns of genetic diversity, effects of the present and the past • Genetics of climatic adaptation in trees • Genetics of flowering time variation • Evolution of plant genomes • Evolution of gene expression

3. Sequence analysis of genes in the highly redundant SHI family of Arabidopsis thaliana • Functional Redundancy • invoked when a gene is knocked out, which gives no discernable mutant phenotype, especially when a related duplicate gene exists • STY1, STY2, SHI, SRS4, in the SHI gene family in Arabidopsis thaliana • regulates gynoecium development, and shows a remarkable functional conservation, although they are highly divergent in sequences except in two conserved region

4. Purpose • To characterise the evolutionary forces that act on the highly redundant SHI genes • I will perform analysis of sequence divergence between SHI paralogs, and of patterns of within-species variation in A. thaliana

5. Materials • Materials: Leaf samples of Arabiopsis thaliana are collected from 16 individuals which originally belong to 8 populations from Scandinavia (Norway and Sweden) and Italy

6. Methods • PCR of STY1, STY2, SHI, SRS4genes from 16 individuals • Clean the PCR production • MegaBASE sequencing to get the sequence • Data Analysis using PHRED and PHRAP ,DnaSP program, Mega3.1 and so on

7. Method 1:ExoSAP

8. Method 2:MegaBACE sequencing • Principle: A sequencing reagent premix is combined with template DNA and primer and thermally cycled (Thermo Sequenase™ II DNA polymerase ) • Result: Samples are finally dissolved in an appropriate loading solution for separation and detection • Big difference from normal PCR: only one primer; without the step 95°C 2min for the unfoldment of templates

9. Result 1: Sliding window plots of Ka/ Ks for pairwise comparisons between STY1 and SHI in A.thaliana (DnaSPprogram)

10. Sliding window plots of Ka/ Ks for pairwise comparisons between STY2 and SRS4 in A.thaliana

11. Ka/Ks • Ks = average distance between genes at synonymous sites; Ka = average distance between genes at nonsynonymous sites • The average ratio was 0.134 and 0.198 for STY1-SHI and STY2-SRS4, respectively indicating mainly purifying selection (ratio less then one). • A Ka/Ks ratio above one is indicative of positive selection. These regions could potentially be involved in diverged function of the paralogous genes.

12. Result 2:polymorphism table for STY2 (9 sequences)

13. Summary of Nucleotide Diversity

14. S: Number of polymorphic (segregating) sites • πs =estimated pairwise silent-site diversity • πsyn= estimated pairwise synonymous-site diversity • πnonsyn =estimated pairwise nonsynonymous-site diversity • θw =estimated nucleotide diversity based on number of segregating sites • NS = not significant

15. Nucleotide diversity is therefore reduced • Estimate of silent-site (synonymous and non-coding) nucleotide diversity for STY2 is 0.00384. It is lower than the mean nucleotide diversity of 0.0074 reported for other A.thaliana genes (Yoshida et al. 2003). • Nucleotide diversity is therefore reduced for that gene. One explanation for this reduction is positive selection for an advantageous haplotype although none of the neutrality tests (Tajima’sD, Fay and Wu’s H and MacDonald Kreitman) were significant

16. Conclusion • mainly purifying selection for STY1-SHI and STY2-SRS4 • The average ratio was 0.134 and 0.198 for STY1-SHI and STY2-SRS4, respectively indicating mainly purifying selection although they have one or two peaks which is indicative of positive selection • Nucleotide diversity is therefore reduced for that gene • Estimate of silent-site (synonymous and non-coding) nucleotide diversity for STY2 is 0.00384. It is lower than the mean nucleotide diversity of 0.0074 reported for other A.thaliana genes (Yoshida et al. 2003). Nucleotide diversity is therefore reduced for that gene. One explanation for this reduction is positive selection for an advantageous haplotype although none of the neutrality tests (Tajima’sD, Fay and Wu’s H and MacDonald Kreitman) were significant

17. Thanks!

18. McDonald and Kreitman test

19. McDonald and Kreitman test • Principle: If polymorphism within species and divergence between species are both the result of neutral mutations, the ratio of synonymous to replacement (non-synonymous) within species should be the same as the ratio between species. • Result:no significant difference in the ratio of synonymous to replacement substitutions was found between fixed and polymorphic site, so we can not reject neutral evolution. However, the limited polymorphism and small sample of genes results in a low power of this test.

20. Estimate of silent-site (synonymous and non-coding) nucleotide diversity for STY2 is 0.00384. It is lower than the mean nucleotide diversity of 0.0074 reported for other A.thaliana genes (Yoshida et al. 2003). Nucleotide diversity is therefore reduced for that gene. One explanation for this reduction is positive selection for an advantageous haplotype although none of the neutrality tests (Tajima’sD, Fay and Wu’s H and MacDonald Kreitman) were significant

21. This is a polymorphism table for STY2. The nucleotide position and region of each polymorphism are indicated (P=promoter, EX=exon, and IN=intron). A dot represent an equivalent base relative to the reference sequence. A minus means a gap.