Reseach Training Presentation

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Reseach Training Presentation

• By Yanhong Zhao
• Department of Evolutionary Functional Genomics,
  Uppsala University, Sweden
• Supervisor Prof. Ulf Lagercrantz

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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

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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

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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

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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

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Methods

• PCR of STY1, STY2, SHI, SRS4 genes 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

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Method 1ExoSAP
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Method 2MegaBACE 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 95C 2min for the unfoldment of
  templates

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Result 1 Sliding window plots of Ka/ Ks for
pairwise comparisons between STY1 and SHI in
A.thaliana (DnaSP program)
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Sliding window plots of Ka/ Ks for pairwise
comparisons between STY2 and SRS4 in A.thaliana
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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.

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Result 2polymorphism table for STY2 (9 sequences)
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Summary of Nucleotide Diversity
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• S Number of polymorphic (segregating) sites
• ps estimated pairwise silent-site diversity
• psyn estimated pairwise synonymous-site
  diversity
• pnonsyn estimated pairwise nonsynonymous-site
  diversity
• ?w estimated nucleotide diversity based on
  number of segregating sites
• NS not significant

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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 (TajimasD,
  Fay and Wus H and MacDonald Kreitman) were
  significant

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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 (TajimasD, Fay and
  Wus H and MacDonald Kreitman) were significant

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Thanks!
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McDonald and Kreitman test
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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.

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• 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 (TajimasD, Fay and
  Wus H and MacDonald Kreitman) were significant

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• This is a polymorphism table for STY2. The
  nucleotide position and region of each
  polymorphism are indicated (Ppromoter, EXexon,
  and INintron). A dot represent an equivalent
  base relative to the reference sequence. A minus
  means a gap.