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Analytical Genomics of Dormancy in Seeds. Jimmy Hoang Professor Erica Bakker Department of Horticulture Howard Hughes Medical Institute University Honors College. GA3 β ox. Germination Promoting Factors. GA. ?. Germination. Light. Cold. GA.
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Analytical Genomics of Dormancy in Seeds Jimmy Hoang Professor Erica Bakker Department of Horticulture Howard Hughes Medical Institute University Honors College
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
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
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
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
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 CCT CCG Proline Proline Nonsynonymous Mutation CCT CCT CAT Proline Histidine
RGL2 Synonymous Nonsynonymous
DNAsp Analyze Nucleotide Diversity, Ka/Ks values
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
Association Mapping Determine if there is any statistical significance between: • Single nucleotide polymorphism data (SNP) • Seed germination phenotype data • 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 SNP at Site 496 CGG CAG Electrically Charged Polar
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
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