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