Studies of Genome Wide Molecular Variation in Arabidopsis thaliana using Arrays
Justin BorevitzSalk Institutenaturalvariation.org
Talk Outline
• Natural Variation in Light Response
• Transcriptional profiling– for QTL candidate genes
• Single Feature Polymorphisms (SFPs)– Potential deletions
• Bulk Segregant Mapping– Extreme Array Mapping
• Haplotype analysis
Natural Mutation in PHYA photoreceptorNatural Mutation in PHYA photoreceptor
Quantitative Trait Loci
differences may be due to expression or hybridization
Feature level model
FLC controls flowering time Difference detected it 3 day old seedlings
Gene Expression index that accounts for feature effect and polymorphisms
PAG1 down regulated in Cvi
PLALE GREEN1 knock out has long hypocotyl in red light
Potential Deletions
False Discovery and Sensitivity
Permuted data
real data
5% FDR
PM only SAM threshold
5% FDR
GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity
Polymorphic 340 117 223 34% Non-polymorphic 477 4 473
False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p- value = 1.845e- 40
Observed t statistics vsNull (permuted) t statistics
Chip genotyping of a Recombinant Inbred Line
29kb interval
Potential Deletions
111 potential deletions45 confirmed by Ler sequence
23 (of 114) transposons
Disease Resistance(R) gene clusters
Single R gene deletions
Genes involved in Secondary metabolism
Unknown genes
Potential Deletions Suggest Candidate Genes
deletion of MAF1
FLOWERING1 QTL
Chr1 (bp)
Flowering Time QTL caused by a natural deletion in MAF1
MAF1
Fast Neutron deletions
FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1
Map bibb100 bibb mutant plants100 wt mutant plants
bibb mapping
ChipMapAS1
Bulk segregantMapping usingChip hybridization
bibb maps toChromosome2 near ASYMETRIC LEAVES1
BIBB = ASYMETRIC LEAVES1
Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain
bibb as1-101
MYB
bib-1W49*
as-101Q107*
as1bibb
AS1 (ASYMMETRIC LEAVES1) =MYB closely related toPHANTASTICA located at 64cM
LOD
eXtreme Array Mapping
Red light QTL RED2 from 100 Kas/ Col RILs
QTL likelihood model using bulk segregant analysis with SFP genotyping
0
4
8
12
16
0 20 40 60 80 100cM
LO
D
Composite Interval MappingRED2 QTL
RED2 QTL
Chromosome 2Chromosome 2Chromosome 1
Chromosome 4
15 tallest RILs pooled vs15 shortest RILs pooled
Array Haplotyping
Inbred lines
Low effectiverecombinationdue to partialselfing
Extensive LDblocks
Col Ler Cvi Kas Bay Shah Lz Nd
Chr
omos
ome1
~50
0kb
Review
• Transcriptional profiling to identify candidate genes and downstream responses that consider polymorphisms
• Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for bulk segregant mapping, and haplotyping
NaturalVariation.org
SyngentaHur-Song ChangTong Zhu
SyngentaHur-Song ChangTong Zhu
SalkJon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker
UC DavisJulin Maloof
UC San DiegoCharles Berry
ScrippsElizabeth Winzeler
SalkJon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker
UC DavisJulin Maloof
UC San DiegoCharles Berry
ScrippsElizabeth Winzeler
University of Guelph, CanadaDave WolynUniversity of Guelph, CanadaDave Wolyn