1. Analytical Genomics of Dormancy in Seeds Jimmy Hoang Professor Erica Bakker Department of Horticulture Howard Hughes Medical Institute University Honors College

2. Light Cold Germination 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

3. RGL2GASeed 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: 1120-1130 Courtesy of Hiro Nonogaki GA Signal Transduction

4. GA inducible gene Motif ? RGL2 “De-Repression” SCF complex C S F-box (SLY1) 26S Proteasome Ub Courtesy of Hiro Nonogaki De-Repression at the Protein Level

5. 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

6. 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

7. The Accessions

8. Analyzing the Genetic Data through Bioinformatics

9. Contig Express Edit the raw gene sequence

10. ClustalX Align multiple DNA fragments

11. Filter and remove any “incorrect” sequences BioEdit

12. MEGA Create phylogeny trees to show relationships between accessions (Molecular Evolutionary Genetics Analysis)

13. Synonymous Mutation CCT Nonsynonymous Mutation CATCATCCT CCGCCT CCTCCT Proline Histidine

14. RGL2 SynonymousNonsynonymous

15. Analyze Nucleotide Diversity, K a /K s values DNAsp

16. Ka/KsPi ATG1D1A<10.00037 PIL5<10.0207 SLEEPY1<10.00011 RGL2>10.00257

17. 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

18. Seed Germination Study

19. Determine if there is any statistical significance between: Single nucleotide polymorphism data (SNP) Seed germination phenotype data Population structure data Association Mapping

20. H 0 : Null Hypothesis = No association H 1 : Alternative Hypothesis = Association P-value : Determines if H 0 is true or false If p < 0.05, then H 0 is false and there is significant statistical association

21. TASSEL Trait Analysis by aSSociation Evolution and Linkage

22. RGL2 P-values Trial 1 TraitSiteP-value 74960.0061 Trial 2 TraitSiteP-value 54960.016 74960.035 Trial 3 TraitSiteP-value 67230.007 65590.013

23. RGL2 SNP at Site 496 CGGCGGCAGCAG Electrically ChargedPolar

24. IIIIIIIVV RGL2, GRAS superfamily) VIVII DELLA SH2-like VHYNP GA signaling 5471447081111166172218234286290385389406 443 Hussain et al., 2005; Plant J 44: 88-99 Peng et al., 1999; Nature 400: 256-261 ** * * * * I II III IV V VIVII LXXLL DomainsVHIIDNLS Characteristic to GRAS Q Courtesy of Hiro Nonogaki

25. 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

26. Sources & Acknowledgements Contig Express http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Vector-Design-Software/ 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/ http://bips.u-strasbg.fr/fr/Documentation/ClustalX/ BioEdit http://www.mbio.ncsu.edu/BioEdit/BioEdit.html http://www.mbio.ncsu.edu/BioEdit/BioEdit.html MEGA http://www.megasoftware.net/ http://www.megasoftware.net/ DNAsp http://www.ub.es/dnasp/ http://www.ub.es/dnasp/ TASSEL http://www.maizegenetics.net/index.php?option=com_content&task=view&id=89&Itemid= 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