analytical genomics of dormancy in seeds jimmy hoang professor erica bakker department of...
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Analytical Genomics of Dormancy in Seeds
Jimmy HoangProfessor Erica Bakker
Department of HorticultureHoward Hughes Medical Institute
University Honors College
Light
Cold
Germination
GA
GA
GA3βox ?
Yamaguchi et al., 1998; Plant Cell 10: 2115-2126
Yamauchi et al., 2003; Plant Cell 16: 367-378 Courtesy of Hiro Nonogaki
Germination Promoting Factors
RGL2GA Seed GerminationActivatorSLY1
“Repression”“De-Repression”
Steber et al., 1998; Genetics 149: 509-521
(E3 ubiquitin ligase)
Lee et al., 2002; Genes & Development 16: 646-658
Silverstone et al., 2001; Plant Cell 13: 1555-1566
McGinnis et al., 2003; Plant Cell 15: 1120-1130Courtesy of Hiro Nonogaki
GA Signal Transduction
GA inducible geneMotif?
RGL2“De-Repression”
SCF complex
CS
F-box (SLY1)
26S Proteasome
Ub
UbUb
Ub
Courtesy of Hiro Nonogaki
De-Repression at the Protein Level
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
Filter and remove any “incorrect” sequences
BioEdit
MEGA
Create phylogeny trees to show relationships between accessions
(Molecular Evolutionary Genetics Analysis)
Synonymous Mutation
CCT
Nonsynonymous Mutation
CATCCT
CCGCCT
CCT
Proline
Proline
Proline
Histidine
RGL2 Br0 CIBC5 Ts5 Pna17 RRS10 Ga0 Ra0 Ms0 Lz0 Ren1 Col0 Got22 Se0
Sq1 Wei0 Sorbo Spr16 Kz1 NFA8
CS22491 Pu27
Fei0 Kz9 Bay0
Ull25 Uod1 Wa1 Yo0 Gu0 Lp22 Omo23 Zdr6 Uod7 Pu223 Spr12
C24 NFA10 HR5 Ler1 Sq8
Zdr1 Kin0 Wt5 Ei2 HR10 Mr0
Pro0 Tamm27 Cvi0 Fab2 Fab464
3489
95
62
63
65
36
22
0.002
Fab2 Fab4
Tamm27 Pro0 Cvi0
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
Mr0 Wei0 Br0 CS22491 Got22 Kin0 Lz0 Omo23 Ra0 Sorbo Ts5 Wa1 Wt5 Yo0 Zdr6
Kz1 Zdr1
0.002
Synonymous Nonsynonymous
Analyze Nucleotide Diversity, Ka/Ks values
DNAsp
DNAsp
Ka/Ks Pi
ATG1D1A <1 0.00037
PIL5 <1 0.0207
SLEEPY1 <1 0.00011
RGL2 >1 0.00257
Seed Germination Phenotype Data
• Three Petri dishes each with ~100 seeds for every accession
• Initial 24 hour cold treatment at 4°C• Incubated at 21-22°C for seven days• Record germinated seeds for each day
Seed Germination Study
Determine if there is any statistical significance between:
• Single nucleotide polymorphism data (SNP)
• Seed germination phenotype data• Population structure data
Association Mapping
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
TASSELTrait Analysis by aSSociation Evolution and Linkage
RGL2 P-valuesTrial 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 CAG
Electrically Charged Polar
I II III IV V
RGL2, GRAS superfamily)
VI VII
DELLA SH2-like VHYNP
GA signaling
5471 44 70 81 111 166 172 218 234 286 290 385 389 406 443
Hussain et al., 2005; Plant J 44: 88-99
Peng et al., 1999; Nature 400: 256-261
* *
MKRGYGETWDPPPKPLPASRSGEGPSMADKKKADDDNNNSNMDDELLAVLGYKVRSSEMAEVAQKLEQLE
MVLSNDDVGSTVLNDSVHYNPSDLSNWVESMLSELNNPASSDLDTTRSCVDRSEYDLRAIPGLSAFPKEE
EVFDEEASSKRIRLGSWCESSDESTRSVVLVDSQETGVRLVHALVACAEAIHQENLNLADALVKRVGTLA
GSQAGAMGKVATYFAQALARRIYRDYTAETDVCAAVNPSFEEVLEMHFYESCPYLKFAHFTANQAILEAV
TTARRVHVIDLGLNQGMQWPALMQALALRPGGPPSFRLTGIGPPQTENSDSLQQLGWKLAQFAQNMGVEF
EFKGLAAESLSDLEPEMFETRPESETLVVNSVFELHRLLARSGSIEKLLNTVKAIKPSIVTVVEQEANHN
GIVFLDRFNEALHYYSSLFDSLEDSYSLPSQDRVMSEVYLGRQILNVVAAEGSDRVERHETAAQWRIRMK
SAGFDPIHLGSSAFKQASMLLSLYATGDGYRVEENDGCLMIGWQTRPLITTSAWKLA
*
*
*
*
I
II
III
IV
V
VI VII
LXXLLDomains VHIIDNLS
Characteristic to GRAS
Q
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