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Analytical Genomics of Dormancy in Seeds

Analytical Genomics of Dormancy in Seeds

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Analytical Genomics of Dormancy in Seeds

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

  2. GA3βox Germination Promoting Factors GA ? Germination Light Cold GA Yamaguchi et al., 1998; Plant Cell 10: 2115-2126 Yamauchi et al., 2003; Plant Cell 16: 367-378 Courtesy of Hiro Nonogaki

  3. SLY1 Lee et al., 2002; Genes & Development 16: 646-658 Silverstone et al., 2001; Plant Cell 13: 1555-1566 GA Signal Transduction “De-Repression” “Repression” GA Activator RGL2 Seed Germination (E3 ubiquitin ligase) Steber et al., 1998; Genetics 149: 509-521 McGinnis et al., 2003; Plant Cell 15: 1120-1130 Courtesy of Hiro Nonogaki

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

  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. BioEdit Filter and remove any “incorrect” sequences

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

  13. Synonymous Mutation CCT CCT CCG Proline Proline Nonsynonymous Mutation CCT CCT CAT Proline Histidine

  14. RGL2 Synonymous Nonsynonymous

  15. DNAsp Analyze Nucleotide Diversity, Ka/Ks values

  16. DNAsp

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

  20. 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

  21. TASSEL Trait Analysis by aSSociation Evolution and Linkage

  22. RGL2 P-values

  23. RGL2 SNP at Site 496 CGG CAG Electrically Charged Polar

  24. I II III * IV * V * VI VII * RGL2, GRAS superfamily) 443 1 44 70 81 111 166 172 218 234 286 290 385 389 406 547 I II III IV V VI VII SH2-like DELLA VHYNP LXXLL Domains NLS VHIID GA signaling Characteristic to GRAS Q * * Peng et al., 1999; Nature 400: 256-261 Hussain et al., 2005; Plant J 44: 88-99 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/ • 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