Analytical Genomics of
Dormancy in Seeds
Jimmy Hoang
Professor Erica Bakker
Department of Horticulture
Howard Hughes Medical Institute
University Honors College
Germination Promoting Factors
GA
GA3βox
Light
?
Germination
Cold
GA
Yamaguchi et al., 1998; Plant Cell 10: 2115-2126
Yamauchi et al., 2003; Plant Cell 16: 367-378
Courtesy of Hiro Nonogaki
GA Signal Transduction
“De-Repression”
GA
SLY1
Activator
RGL2
“Repression”
Seed Germination
(E3 ubiquitin ligase)
Lee et al., 2002; Genes & Development 16: 646-658
Silverstone et al., 2001; Plant Cell 13: 1555-1566
Steber et al., 1998; Genetics 149: 509-521
Courtesy of Hiro Nonogaki
McGinnis et al., 2003; Plant Cell 15: 1120-1130
De-Repression at the Protein Level
26S Proteasome
Ub
Ub
Ub
Ub
RGL2
Motif?
Courtesy of Hiro Nonogaki
F-box (SLY1)
S
C
SCF complex
“De-Repression”
GA inducible gene
The Germination Genes in
Arabidopsis thaliana
• RGL2 – Germination Repressor Protein
• PIL5 – Light Receptor; GA Producer
• ATG1D1A – GA Receptor, if GA present,
degradation begins
• SLEEPY1 – Adds a marker to RGL2
Steps
• Sequence the four genes through 96 accessions
– Accession =
• Same Species but from different parts of the world
• Similar but different genetic data
• Able to interbreed with each other
• Seed germination phenotype data
– Record rates of germination
• Find any significant association between the
two
The Accessions
Analyzing the Genetic Data
through Bioinformatics
Contig Express
Edit the raw gene sequence
ClustalX
Align multiple DNA fragments
BioEdit
Filter and remove any “incorrect” sequences
MEGA
(Molecular Evolutionary Genetics Analysis)
Create phylogeny trees to show relationships between
accessions
Synonymous Mutation
CCT
CCG
Proline
Proline
Nonsynonymous Mutation
CCT
CAT
Proline
Histidine
Sq1
Wei0
Sorbo
Spr16
Kz1
NFA8
Synonymous
Uod7
Sq1
Se0
Pu27
NFA8
Lp22
HR5
Ga0
Col0
Bay0
CIBC5
Fei0
HR10
Ler1
NFA10
Pu223
RRS10
Spr16
Sq8
Uod1
C24
Ei2
Gu0
Kz9
Ms0
Pna17
Ren1
Spr12
Ull25
Nonsynonymous
CS22491
62 Pu27
Fei0
Kz9
Bay0
22
C24
NFA10
HR5
Ler1
Sq8
36
RGL2
Br0
CIBC5
Ts5
Pna17
RRS10
Ga0
65 Ra0
Ms0
Lz0
Ren1
Col0
Got22
Se0
Fab4
Tamm27
Pro0
Cvi0
Ull25
Uod1
Wa1
Yo0
Gu0
Lp22
63 Omo23
Zdr6
Uod7
Pu223
Spr12
Zdr1
Kin0
Wt5
Ei2
HR10
Mr0
Pro0
95
Tamm27
Cvi0
89 Fab2
34
64 Fab4
0.002
Kz1
0.002
Wei0
Br0
CS22491
Got22
Kin0
Lz0
Omo23
Ra0
Sorbo
Ts5
Wa1
Wt5
Yo0
Zdr6
Fab2
Mr0
Zdr1
DNAsp
Analyze Nucleotide Diversity, Ka/Ks values
DNAsp
Ka/Ks
Pi
ATG1D1A
<1
0.00037
PIL5
<1
0.0207
SLEEPY1
<1
0.00011
RGL2
>1
0.00257
• Three Petri dishes each with ~100 seeds for
every accession
• Seed
Initial 24
hour cold treatment
at 4°C
Germination
Phenotype
• Incubated at 21-22°C for seven days
Data
• Record germinated seeds for each day
Seed Germination Study
100
90
Germination percentage
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Days since planting
5
6
7
8
Bil-5
Bor-1
Bur-0
CS22491
Ct-1
Cvi-0
Eden-2
Edi-0
Est-1
Fei-0
Gy-0
Kas-1
Kondara
Lov-1
Lp2-2
Lz-0
Ms-0
NFA-8
Nok-3
Oy-0
Pro-0
Pu2-7
RMX-A02
RRS-7
Se-0
Shahdara
Sorbo
Spr1-2
Tamm-2
Ts-1
Tsu-1
Var2-1
Wa-1
Ws-0
Ws-2
Wt-5
Zdr-1
Determine if there is any statistical significance
between:
• Single nucleotide polymorphism data (SNP)
• Seed germination
phenotype
data
Association
Mapping
• Population structure data
Association Mapping
• H0 : Null Hypothesis = No association
• H1 : Alternative Hypothesis = Association
• P-value : Determines if H0 is true or false
• If p < 0.05, then H0 is false and there is
significant statistical association
TASSEL
Trait Analysis by aSSociation Evolution and Linkage
RGL2 P-values
Trial 1
Trait
Site
P-value
7
496
0.0061
Trial 2
Trait
Site
P-value
5
496
0.016
7
496
0.035
Trial 3
Trait
Site
P-value
6
723
0.007
6
559
0.013
RGL2 SNP at Site 496
CGG
Electrically Charged
CAG
Polar
RGL2, GRAS superfamily)
1
44 70 81 111
I
Domains
II
166 172
218 234
286 290
III
IV
V
NLS
VHIID
DELLA VHYNP
GA signaling
385 389 406 443
VI
547
VII
LXXLL SH2-like
Characteristic to GRAS
MKRGYGETWDPPPKPLPASRSGEGPSMADKKKADDDNNNSNMDDELLAVLGYKVRSSEMAEVAQKLEQLE
I
MVLSNDDVGSTVLNDSVHYNPSDLSNWVESMLSELNNPASSDLDTTRSCVDRSEYDLRAIPGLSAFPKEE
II
EVFDEEASSKRIRLGSWCESSDESTRSVVLVDSQETGVRLVHALVACAEAIHQENLNLADALVKRVGTLA
Q
III
GSQAGAMGKVATYFAQALARRIYRDYTAETDVCAAVNPSFEEVLEMHFYESCPYLKFAHFTANQAILEAV
*
IV
TTARRVHVIDLGLNQGMQWPALMQALALRPGGPPSFRLTGIGPPQTENSDSLQQLGWKLAQFAQNMGVEF
*
V
EFKGLAAESLSDLEPEMFETRPESETLVVNSVFELHRLLARSGSIEKLLNTVKAIKPSIVTVVEQEANHN
*
VI
VII
GIVFLDRFNEALHYYSSLFDSLEDSYSLPSQDRVMSEVYLGRQILNVVAAEGSDRVERHETAAQWRIRMK
*
SAGFDPIHLGSSAFKQASMLLSLYATGDGYRVEENDGCLMIGWQTRPLITTSAWKLA
*
*
Peng et al., 1999; Nature 400: 256-261
Hussain et al., 2005; Plant J 44: 88-99
Courtesy of Hiro Nonogaki
Results
• RGL2 shows significant association between
the SNP and the seed germination phenotype
data
• Arginine
Glutamine
• SNP occurred in a functional domain
• PIL5, ATG1D1A, and SLEEPY1 does not show
any significant statistical association
Sources & Acknowledgements
•
•
•
•
•
•
Contig Express http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Vector-Design-Software/
ClustalX http://bips.u-strasbg.fr/fr/Documentation/ClustalX/
BioEdit http://www.mbio.ncsu.edu/BioEdit/BioEdit.html
MEGA http://www.megasoftware.net/
DNAsp http://www.ub.es/dnasp/
TASSEL http://www.maizegenetics.net/index.php?option=com_content&task=view&id=89&Itemid=
•
•
•
The Pattern of Polymorphism in Arabidopsis thaliana
http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0030196&ct=1
Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis
Eunkyoo Oh, Jonghyun Kim, Eunae Park, Jeong-Il Kim, Changwon Kang, and Giltsu Choi.
PIL5, a Phytochrome-Interacting Basic Helix-Loop-Helix Protein, Is a Key Negative Regulator of Seed Germination in Arabidopsis
thaliana. Eunkyoo Oh, Shinjiro Yamaguchi, Yuji Kamiya, Gabyong Bae, Won-Il Chung, and Giltsu Choi.
•
Mentor and Collaborator
• Erica Bakker
• Hiro Nonogaki
Howard Hughes Medical Institute / University Honors College