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. Department of Evolutionary Functional Genomics. Patterns of genetic diversity, effects of the present and the past - PowerPoint PPT PresentationTRANSCRIPT
Reseach Training Presentation
By Yanhong Zhao
Department of Evolutionary Functional Genomics, Uppsala University, Sweden
Supervisor: Prof. Ulf Lagercrantz
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
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
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
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
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
Method 1:ExoSAP
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 appropri
ate loading solution for separation and detection Big difference from normal PCR: only on
e primer; without the step 95°C 2min for the unfoldment of templates
Result 1: Sliding window plots of Ka/ Ks for pairwise comparisons between STY1 and SHI in A.thaliana (DnaS
P program)
Sliding window plots of Ka/ Ks for pairwise comparisons between STY2 and SRS4 in A.thaliana
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.
Result 2:polymorphism table for STY2 (9 sequences)
E E I I I I I I E E
X X N N N N N N X X
P P P P P P P P P P P P P 1 1 1 1 1 1 1 1 2 2
1 1 1 1 1 1 1 1 2 2
2 2 2 3 4 5 6 6 7 8 8 4 5 5 5 5 5 7 8 0 2
4 5 3 4 5 7 5 4 3 3 3 1 1 8 1 3 3 3 3 5 5 6 8
4 9 4 4 4 9 0 5 0 8 1 4 6 4 5 0 1 3 7 7 6 4 4
Sty2_Bol1 A G C T G A A C A A A C C A C T T T T A G C G
Sty2_Bol21 . . . . . . . C . . A . . . C . . . . . . . -
Sty2_Tos99 . . . . T . . C C . G G G . C . A . . . . . .
STY2_TOS30 . . . . T . . G . . A . G . C A A A A . . . .
Sty2_Ale41 . . A . T . . C . C A . . T C . . . . . . . .
Sty2_Bel1 . . . . T . . C . . A . . . C . . . . . . . .
Sty2_Mel19 . . A . T . G G . . A . . T C . . . . . C . .
Sty2_Ale77 . . A . T . . G . . A . . T C . . . . . . . .
Sty_Bel15 G C . G T C . C . . A . . . G . . . . C C A T
Summary of Nucleotide Diversity
Gene N (sequences)
Length(bp)
S πs πsyn
πnonsyn
θw Tajima’s D Statistic
STY2 9 2360
23 0.00384
0.00109
0.00099
0.00365
-1.04178(NS)
S: Number of polymorphic (segregating) sites πs =estimated pairwise silent-site diversity πsyn= estimated pairwise synonymous-site di
versity πnonsyn =estimated pairwise nonsynonymou
s-site diversity θw =estimated nucleotide diversity based on
number of segregating sites NS = not significant
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
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
Thanks!
McDonald and Kreitman test
Type of change Fixed Polymorphic
Non-synonymous 18 3
Synonymous 10 1
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.
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
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.