Analytical Genomics of Dormancy in Seeds

1
Analytical Genomics of Dormancy in Seeds

• Jimmy Hoang
• Professor Erica Bakker
• Department of Horticulture
• Howard Hughes Medical Institute
• University Honors College

2
Germination Promoting Factors
GA
?
Light
Germination
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
GA Signal Transduction
Repression
De-Repression
RGL2
GA
Seed Germination
Activator
(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
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
CCG
CCT
Proline
Proline
Nonsynonymous Mutation
CAT
CCT
CCT
Proline
Histidine
14
RGL2
Synonymous
Nonsynonymous
15
DNAsp
Analyze Nucleotide Diversity, Ka/Ks values
16
DNAsp
  Ka/Ks Pi
ATG1D1A lt1 0.00037
PIL5 lt1 0.0207
SLEEPY1 lt1 0.00011
RGL2 gt1 0.00257
17
Seed Germination Phenotype Data

• Three Petri dishes each with 100 seeds for every
  accession
• Initial 24 hour cold treatment at 4C
• Incubated at 21-22C 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 lt 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
Trial 1 Trial 1 Trial 1
Trait Site P-value
7 496 0.0061
     
Trial 2 Trial 2 Trial 2
Trait Site P-value
5 496 0.016
7 496 0.035
     
Trial 3 Trial 3 Trial 3
Trait Site P-value
6 723 0.007
6 559 0.013
23
RGL2 SNP at Site 496
CGG
CAG
Electrically Charged
Polar
24
RGL2, GRAS superfamily)
443
547
1
44
70
81
111
166
172
218
234
286
290
385
389
406
I
II
III
IV
V
VI
VII
DELLA
SH2-like
VHYNP
LXXLL
Domains
VHIID
NLS
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/e
  n/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.h
  tml
• MEGA http//www.megasoftware.net/
• DNAsp http//www.ub.es/dnasp/
• TASSEL http//www.maizegenetics.net/index.php?opti
  oncom_contenttaskviewid89Itemid
• The Pattern of Polymorphism in Arabidopsis
  thaliana
• http//biology.plosjournals.org/perlserv/?request
  get-documentdoi10.1371/journal.pbio.0030196ct1
  
• 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