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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
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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
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regulates gynoecium development, and shows a
remarkable functional conservation, although they
are highly divergent in sequences except in two
conserved region
Purpose
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
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
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PCR of STY1, STY2, SHI, SRS4 genes from 16
individuals
Clean the PCR production
MegaBASE sequencing to get the sequence
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Data Analysis using PHRED and
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PHRAP ,DnaSP program, Mega3.1 and so on
Method 1:ExoSAP
Method 2:MegaBACE
sequencing
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Principle: A sequencing reagent premix is
combined with template DNA and primer and thermally
cycled (Thermo Sequenase™ II DNA polymerase )
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Result: Samples are finally dissolved in an
appropriate loading solution for separation and
detection
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Big difference from normal PCR: only
one 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
(DnaSP program)
Sliding window plots of Ka/ Ks for pairwise
comparisons between STY2 and SRS4 in
A.thaliana
Ka/Ks
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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 STY1SHI 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)
P
P
P
P
P
P
P
P
P
P
P
P
P
E
E
I
I
I
I
I
I
E
E
X
X
N
N
N
N
N
N
X
X
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
(seq
uenc
es)
STY2 9
Len
gth(
bp)
S
236
0
23
πs
πsy
n
πno θw
nsyn
Tajima’
sD
Statisti
c
0.00 0.00 0.00 0.00 384 109 099 365 1.0417
8(NS)
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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
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 STY2SRS4, 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.