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Natural Variation in Arabidopsis thalianaLight Response:Genomic Approaches
Justin BorevitzSalk Institutenaturalvariation.org
Talk Outline
• Survey of light response in
Arabidopsis thaliana accessions
• QTL mapping of light response in
Ler/Cvi Recombinant Inbred Lines
• Transcriptional profiling for QTL candidate genes
• Analysis of Single Feature Polymorphisms
(SFPs)
Epistasis scan
Chr1 Chr2 Chr3 Chr4 Chr5
Chr
1
C
hr2
Chr
3
Chr
4
Chr
5
BQTLhttp://hacuna.ucsd.edu/bqtl43,956 pair-wise tests 163 markers and 133 intervalsPermutation threshold p < 0.05 (5000 permutations)
replacementsynonymous Between Species =Within Species =
McDonald-Kreitmanp = 0.027
2362
858
= 0.23Ka
Ks
Review Natural Variation
• Light response variation is correlated with latitude
• Functional variation in Photoreceptors PHYA, PHYB, CRY2 (El-Din El-Assal et al 2001)
• New QTL identified in common and unique photoreceptor pathways
• QTL may be under selection?
False Discovery and Sensitivity
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 SAM threshold 18% FDR
GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity
Polymorphic 340 195 145 57% Non-polymorphic 477 28 449
False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, df = 1, p-value = 1.309e-59
90% 80% 70%
41% 53% 85%
90% 80% 70%
67% 85% 100%
Cereonmay be asequencingError
TIGRmatch isa match
bibb mutant phenotypes
Col bib-1 det2-1
23 days
bib-3
• three independent recessive alleles • medial sepals remain attached• “cabbage-like” rosette leaves • flowers open prematurely• fruit appear more slender
bib-3
Ler
bib-3
bibb mapping
ChipMapAS1
Bulk segregantMapping usingChip hybridization
bibb maps toChromosome2 near ASYMETRIC LEAVES1
BIBB=AS1
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 to PHANTASTICAlocated at 64cM
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
Gene expression revisited
FLC controls flowering time Difference detected at 3 day old seedling stage
Gene Expression index that accounts for feature effect and polymorphisms
~100 genes @ 20% FDR
Review
• Transcriptional profiling to identify candidate genes and downstream responses
• Single Feature Polymorphisms (SFPs) can be used for identifying recombination breakpoints, bulk segregant mapping, and haplotyping
• Consider polymorphisms for expression analysis
25 bp 25 bp
25 bp 25 bp16 bp
25 bp 25 bp8 bp
1st Set
2nd Set
3rd Set
Complete Genome Tiling Chip
Polymorphisms (re-sequencing)Global methylation (Methylome)Comparative Genomics (Brassica)
New Gene DiscoveryImprove AnnotationAlternative SplicingMicro RNAs
9 Whole-Genome Expression Chips2 Splicing Chips2 5’ Mapping Chips
Validate features
Extra Chips:
ChIP – Chip (DNA binding sites)
ChipViewer: Mapping of transcriptional units of ORFeome
From 2000v At1g09750 (MIPS) to the latest AGI At1g09750
2000 v Annotation (MIPS)
The latest AGI Annotation
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