eXtreme Array Mapping and Haplotype analysis Using Arrays Justin Borevitz Salk Institute naturalvariation.org

1
eXtreme Array Mappingand Haplotype
analysisUsing ArraysJustin BorevitzSalk
Institutenaturalvariation.org
2
Talk Outline

• Bulk Segregant Mapping of
• Mendelian mutations
• eXtreme Array Mapping of QTL
• Kas x Col RILs and Simulations
• Haplotype analysis
• Patterns Global Variation Selection

3
Potential Deletions
4
False Discovery and Sensitivity
real data
Observed t statistics vs Null (permuted) t
statistics
5 FDR
Permuted data
5
Chip genotyping of a Recombinant Inbred Line
29kb interval
6
Potential Deletions
111 potential deletions 45 confirmed by Ler
sequence 23 (of 114) transposons Disease
Resistance (R) gene clusters Single R gene
deletions Genes involved in Secondary
metabolism Unknown genes
7
Potential Deletions Suggest Candidate Genes
Chr1 (bp)
8
Deletions between Accessions
9
Fast Neutron deletions
FKF1 80kb deletion CHR1
cry2 10kb deletion CHR1
10
100 bibb mutant plants
Map bibb
100 wt mutant plants
11
bibb mapping
Bulk segregant Mapping using Chip hybridization
bibb maps to Chromosome2 near ASYMETRIC LEAVES1
AS1
ChipMap
12
BIBB ASYMETRIC LEAVES1
AS1 (ASYMMETRIC LEAVES1) MYB closely related
to PHANTASTICA located at 64cM
as1
bibb
Sequenced AS1 coding region from bib-1 found g
-gt a change that would introduce a stop codon in
the MYB domain
bib-1 W49
as-101 Q107
bibb
as1-101
MYB
13
Other Mendelian mutations
aar21 arhythmic
ein6 ethylene insensitive (no een?)
Also aar90, aar60 and stamenstay
14
Short pool Tall pool Kas x Col RILs all
Features
RED2 QTL
15
eXtreme Array Mapping
Chromosome 2
LOD
16
12
RED2 QTL
LOD
8
4
0
0
20
40
60
80
100
cM
RED2 QTL
Composite Interval Mapping
15 tallest RILs pooled vs 15 shortest RILs pooled
QTL likelihood model using bulk segregant
analysis with SFP genotyping
Red light QTL RED2 from 100 Kas/ Col RILs
16
Simulation Genotypes
15 eXtreme RILs of 100 2 QTL chr2 37var chr5
13var
17
Simulation Genotypes
100 eXtreme RILs of 700 2 QTL
18
Simulation Genotypes
50 eXtreme F2s of 500 2 QTL
19
Simulation Chip Noise
50 eXtreme F2s of 500 2 QTL
20
Simulation Likelihood
50 eXtreme F2s of 500 2 QTL
21
Array Haplotyping

• Hybridize 48 arrays with 15 accessions
• 300ng DNAeasy MiniPrep leaf tissue
• Overnight Bioprime Klenow labeling 25C
• "col", "lz", "ler", "bay", "shah", "cvi",
• "kas", "c24", "est", "kendl", "mt", "nd",
  "sorbo", "van", "ws2"

22
Linkage Disequilibrium explained
1 SNP 2 haplotypes
Mutation 2 SNPs 3 haplotypes
2 SNPs 4 haplotypes
recombination
23
(No Transcript)
24
Pairwise Correlation between and within replicates
25
Feature Density chr1
26
Diversity measure
27
LIGHT1 tstat and raw data
28
LIGHT1 tstat and raw data
29
Array Haplotyping
Inbred lines Low effective recombination due to
partial selfing Extensive LD blocks
30
Quantitative Trait Loci
31
(No Transcript)
32
Feature level model
Gene Expression index that accounts for feature
effect and polymorphisms
FLC controls flowering time Difference detected
it 3 day old seedlings
33
PAG1 down regulated in Cvi
PLALE GREEN1 knock out has long hypocotyl in red
light
34
Review

• Single Feature Polymorphisms (SFPs) can be used
  to identify recombination breakpoints, potential
  deletions
• Bulk segregant mapping, and
• eXtreme Array Mapping of QTL
• Haplotyping Diversity scans

35
NaturalVariation.org
NaturalVariation.org
Salk Jon Werner Sam Hazen Sarah Liljegren Ramlah
Nehring Joanne Chory Joseph Ecker UC San
Diego Charles Berry Scripps Elizabeth Winzeler
Salk Jon Werner Sam Hazen Sarah Liljegren Ramlah
Nehring Joanne Chory Joseph Ecker UC San
Diego Charles Berry Scripps Elizabeth Winzeler
Syngenta Hur-Song Chang Tong Zhu