Transcript Profiling in Maize Inflorescences using...
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Transcript of Transcript Profiling in Maize Inflorescences using...
Andrea L. Eveland
Transcript Profiling in Maize Inflorescences using
High-throughput RNA-Sequencing
Cold Spring Harbor Laboratory
D. Jackson lab, Plant Genetics
D. Ware lab, Bioinformatics & Genomics
Corn Breeders’ School
March 7-8, 2011
Champaign, IL
Maize inflorescence development:
meristem determinacy and branch patterning
* * * *
*
Tassel Ear
Inflorescence meristem (IM)
2 Spikelet meristems (SM)
4 Floral meristems (FM)
Spikelet pair meristem (SPM)
Branch meristem (BM)
(tassel only) *
*
ramosa (ra) mutants of maize:
• display highly branched phenotypes
• indeterminacy in SPM
• influenced by genetic factors
• RA1 implicated in evolution of inflorescence
architecture in grasses Axillary Meristem
Determinacy
RA1
trehalose-P
phosphatase LOB TF
RA2 RA3
Zn finger
TF
ramosa1 ramosa2 ramosa3
• RA1 and RA3 co-localize to arc of cells just below SPM
• Suggested to act non-cell autonomously,
possibly by a mobile signal
• RA1 is epistatic to RA2 and RA3
RA1 RA3
RA3 = black
KN1 = red
Satoh-Nagasawa et al.
Axillary Meristem
Determinacy
RA1
trehalose-P
phosphatase LOB TF
RA2 RA3
Zn finger
TF
Overview
• Genome-wide transcript profiling using RNA-seq
• Systems level analyses
• Comparative genomics
• Moving forward: Integrating heterogeneous data types
• Co-expression
• Developmental series
• Genetic perturbation
• Prioritizing candidate gene selection: branch architecture
• Integration of RNA-seq data sets and genetic information
Overview
• Genome-wide transcript profiling using RNA-seq
• Systems level analyses
• Comparative genomics
• Moving forward: Integrating heterogeneous data types
• Co-expression
• Developmental series
• Genetic perturbation
• Prioritizing candidate gene selection: branch architecture
• Integration of RNA-seq data sets and genetic information
Developmental progression along maize inflorescence: capture
expression profiles in time and space
Experimental System: Sampling for RNA-seq
1mm
2mm
3mm
discard
discard
discard
IM/SPM (Tip)
SM (Mid)
FM (Base)
*R. Schmidt, S. Stanfield
10mm ear:
dissected specific meristem types Developing tassel libraries:
≤2mm, 3-4mm, 5-7mm
• whole tassel samples
*E. Vollbrecht, E. Unger-Wallace
• pools of ~10 tassels
wild-type ra1 ra2 ra3
1 m
m
2 m
m
Mutant ears collected at 2 developmental stages
*with Sasha Goldshmidt
Staging before and after visible phenotype
• Genotyped in field
• 5-10 ears per sample
• 3 biological replicates
• Validation by Q-PCR
RNA-seq library construction
AAAA
200nt
50nt
50nt
DS cDNA fragments
Random priming
PE sequencing
Shear RNAs
Isolate poly-A RNA
RNA-seq PE library construction:
Tophat
Cufflinks
Cuffcompare
Cuffdiff
Bowtie
Known maize gene models (build 5a.59)
Reference genome: AGP_v2
*Map paired reads to genome
*Assemble consensus of coverage
*Generate splice site index based on gene models
*Map to splice junctions
*Stitch consensus regions into transcripts
*Build structural equivalence classes of transcripts
*Determine differential expression
RNA-seq pipeline using the open-source “Tuxedo suite”:
**Trapnell et al., 2010
*Fragments Per Kilobase exon per Million reads
Category Sample reps
Ear (10mm)
developmental
series
Ear tip IM/SM 2
Ear mid SPM 2
Ear base FM 2
Tassel
developmental
series
Tassel < 2mm 1
Tassel 3-4mm 1
Tassel 5-7mm 1
Mutant
(1mm ear) series
wt 1mm ear 2
ra1 1mm ear 2
ra2 1mm ear 2
ra3 1mm ear 3
Mutant
(2mm ear) series
wt 2mm ear 3
ra1 2mm ear 3
ra2 2mm ear 3
ra3 2mm ear 3
Inflorescence 2mm 1
Vegetative SAM 1
Current RNA-seq
datasets:
Maize
Sorghum
Mapping Summary Statistics
Sample reps mapped
reads
#
genes
# transcripts > 5 FPKM > 2
FPKM
Ear tip (IM/SPM) 2 18.8M 20,040 37,679 14,988 22,396
Ear mid (SM) 2 21.9M 22,260 42,051 16,994 24,927
Ear base (FM) 2 21.3M 22,318 42,020 17,136 25,046
Tassel ≥ 2mm 1 14.5M 22,725 42,216 15,153 22,423
Tassel 3-4mm 1 14.7M 23,605 43,846 15,971 23,520
Tassel 5-7mm 1 14.5M 24,631 45,662 18,170 26,181
r = 0.89 r = 0.75
ear tip_1 (FPKM)
ea
r tip
_2
(F
PK
M)
ea
r b
ase
_1
(F
PK
M)
ear tip_1 (FPKM)
Working Gene Set: 10,411 additional
genes expressed:
Maize Filtered Gene Set (FGS; gene build 5a):
Transcripts per gene: 1.60
Total high-confidence genes: 39,656
Exons per transcript: 5.21
Genes identified as expressed in ear and tassel developmental series:
0
3000
6000
9000
12000
15000
>1000 100-1000 30-100 2to30 <2
FGS: 26,319 expressed
(99% protein coding):
0
2000
4000
6000
>1000 100-1000 30-100 2to30 <2
protein coding (66%)
pseudogene (15%)
transposable element (19%)
To
tal #
ge
ne
s e
xp
resse
d
FPKM FPKM
Overview
• Genome-wide transcript profiling using RNA-seq
• Systems level analyses
• Comparative genomics
• Moving forward: Integrating heterogeneous data types
• Co-expression
• Developmental series
• Genetic perturbation
• Prioritizing candidate gene selection: branch architecture
• Integration of RNA-seq data sets and genetic information
Organ-specificity
27,468 405 669
ear tassel
94 16,137 28,401
inflorescence vegetative
(shoot apical meristem+leaf)
• tassel-specific genes may help identify
key regulators in branching
• inflorescence libraries compared with
publically available RNA-seq data sets
Maize-specific (3%)
*EnsemblCompara gene trees: gramene.org; maizesequence.org
Using comparative genomics to classify expressed genes
and prioritize candidates:
Embryophyta (47%)
Poaceae (6.5%)
Eukaryota (32%)
Magnoliophyta (11%)
Andropogoneae (0.5%)
Genes expressed in inflorescences:
17,513 genes expressed in sorghum libraries > 0.5 FPKM:
Comparative profiling in Sorghum
339 735 16,439
shoot apical meristem inflorescence
• Highly branched inflorescence
• Model for drought tolerance
• Comparative genomics
tissue PE reads % mapped >2FPKM >5FPKM
inflorescence 32M 86% 80% 62%
SAM 22.7M 89% 78% 60%
*22 sorghum orthologs of tassel-
specific genes are expressed
exclusively in the inflorescence
compared to SAM
Transcript Isoform specificity
ear tassel
*~450 loci with tassel-specific transcripts > 10 FPKM
45,671 6,445 1,405
ARF8
*Differentially expressed during development
TF expression signatures in ear meristem types:
IM/SPM SM FM # TFs
13
0
1005
53
2
15
2
*
IM/SPM SM FM # TFs
21
134
11
Differentially expressed TFs:
**Enrichment: 0.5 FPKM > < 0.05 FPKM
TF Gene id Functional description IM/SPM SM FM
AUX-IAA GRMZM2G159285 IAA13
AUX-IAA GRMZM2G163848 IAA5
ARF GRMZM2G078274 uncharacterized
C2H2 GRMZM2G134759 uncharacterized
C2H2 GRMZM2G100146 uncharacterized
C2C2-GATA GRMZM2G397616 TSH1-duplicate
C3H GRMZM2G025014 Splicing factor U2af 38kDa
C3H GRMZM2G086614 RNA binding protein
HB GRMZM2G001289 uncharacterized
HB GRMZM2G154641 uncharacterized
HB GRMZM2G056600 uncharacterized
LFY GRMZM2G180190 Floricaula/leafy-like 2
SBP GRMZM2G113779 uncharacterized
SBP GRMZM2G101511 Teosinte glume architecture1
Differentially expressed TFs during ear development
high low expression:
**validation based on marker gene expression
Overview
• Genome-wide transcript profiling using RNA-seq
• Systems level analyses
• Comparative genomics
• Moving forward: Integrating heterogeneous data types
• Co-expression
• Developmental series
• Genetic perturbation
• Prioritizing candidate gene selection: branch architecture
• Integration of RNA-seq data sets and genetic information
Axillary Meristem
Determinacy
RA1
RA3 RA2
trehalose-P
phosphatase LOB TF
Zn finger
TF
Cluster genes by expression profiles in the different genotypes
wt ra1 ra2 ra3
*identify putative genes related to a RA1-RA3-dependent pathway
773 ra3
1,642
ra1
1,641
ra2
1,619
ra3
1,033 663 170
ra1
3,301
ra2
2,155
Differentially expressed genes in ramosa mutants
377
209
158
333
260 502
1,156
64
1,312
272 136
1mm: 2mm:
*322 genes differentially expressed in all mutants at 1mm + 2mm
1mm up (%) down (%)
ra1 53 47
ra2 56 44
ra3 65 35
2mm up (%) down (%)
ra1 59 41
ra2 51 49
ra3 46 54
% up- and down-regulated in ramosa mutants:
K-means co-expression clusters with significant enrichment
for genes differentially in ra1 and ra3 mutants
2
1
0
-1
-2
Re
lative
exp
ressio
n
38%
tip mid base stg1 stg2 stg3
ear development tassel development
2
1
0
-1
-2
Rela
tive e
xpre
ssio
n
tip mid base stg1 stg2 stg3
ear development tassel development
2
1
0
-1
-2
Re
lative
exp
ressio
n
tip mid base stg1 stg2 stg3
55%
47%
Gene Ontology Class P-value
Protein complex assembly 0.0001
Response to freezing 0.0017
Multicellular homeostasis
Signaling pathway 0.002
G-protein receptor signaling 0.008
RA1
RA3
TCP
C3H
ZAP1
HB-trihelix 0
100
200
300
wt ra1 ra3
0
100
200
300
wt ra1 ra3
bzip
TFs that are co-expressed with RA1 and RA3 and DE in mutants
* (up-regulated); * (down-regulated) = p < 0.001
0
1.5
3
4.5
wt ra1 ra3ra1 ra3 wt
* * *
*
0
20
40
60
80
wt ra1 ra3ra1 ra3 wt
* * * *
ra1 ra3 wt
* * *
ra1 ra3 wt
* * *
0
40
80
120
160
200
wt ra1 ra3ra1 ra3 wt
* * *
FP
KM
F
PK
M
1mm 2mm
FP
KM
F
PK
M
Differentially expressed GRAS family TFs
GRMZM2G079470
GRMZM2G024973:
Dwarf9
GRMZM2G144744:
Dwarf8
0
40
80
120
wt ra1 ra2 ra3ra3 wt ra2 ra1
* *
*
*
0
50
100
150
200
wt ra1 ra2 ra3ra3 wt ra2 ra1
* * *
0
100
200
300
400
500
wt ra1 ra2 ra3ra3 wt ra2 ra1
* * *
* *
* (up-regulated); * (down-regulated)
= p < 0.001
1mm 2mm
FP
KM
FP
KM
• Genes involved in GA signaling and
at intersect of multiple hormone pathways
TF-family up down alt
HB 5 1 2
AP2-EREBP 4 2
C2H2 1 2 1
C3H 1 2 1
TCP 3
MYB 1 2
HMG 1 2
AUX-IAA 1 1
MADS 2 0
NAC 1 1
ABI3-VP1 1 1
bZIP 1 1
bHLH 2
WRKY 1 1
SBP 1
BES 1
LFY 1
GRAS 1
TFs differentially expressed in both ra1 and ra3 mutants
TF-family up down alt
bZIP 2 3 1
HB 4 1
C3H 2 2
LUG 2 1
AUX-IAA 2
MADS 2
BES1 1 1
C2H2-Dof 1 1
AP2-EREBP 1 1
MYB 2
LFY 2
HMG 1 1
SBP 1
GRAS 1
WRKY 1
LOB 1
1mm: 64 TFs 2mm: 49 TFs
GRMZM2G069082
Clustering of differentially expressed AP2-EREBP genes
*capture quantitative differences in TF gene family expression
1mm 2mm 1mm 2mm 1mm 2mm 1mm 2mm wild-type ramosa1 ramosa2 ramosa3
wt ra1 ra3 TFs
6
1
918
3
0
5
10
GRMZM2G124011 DDF;CBF; AP2-EREBP CBF6
GRMZM2G002894 EMB1967; FHA uncharacterized
GRMZM2G466044 AP2-EREBP uncharacterized
GRMZM2G404375 Trihelix uncharacterized
GRMZM2G059102 MADS MADS Box TF 47
ra3 specific TFs:
TF expression in ra mutants (1mm):
GRMZM2G124011 DDF;CBF;ERF/AP2 CBF6
*Dehydration responsive element
*Expression of these TFs enriched in tassel
GRMZM2G466044
GRMZM2G466044 AP2-EREBP uncharacterized
GRMZM2G069082
GRMZM2G124011: CBF6
GRMZM2G069082 GRMZM2G069146
GRMZM2G124037: CBF3
Chr7:
Chr2:
Expression of tandem AP-EREBP genes enriched in tassel
GRMZM2G069082
*
*
GRMZM2G124011: CBF6
GRMZM2G069082 GRMZM2G069146
GRMZM2G124037: CBF3
Chr7:
Chr2:
0
20
40
60
80
tip mid base stg1 stg2 stg3
GRMZM2G069146
GRMZM2G124011
GRMZM2G069082
GRMZM2G124037 *
* F
PK
M
0
20
40
60
80
wt ra1 ra2 ra3
1mm
2mm
FP
KM
0
5
10
15
20
wt ra1 ra2 ra3
1mm
2mm
FP
KM
ear tassel
GRMZM2G069082
Maize chr 2
Maize chr 7
Rice chr 9
Sorghum chr 2
*Ortholog of differentially expressed CBF genes in sorghum is inflorescence-specific
* EnsemblCompara gene trees **Josh Stein
Summary
• AP2-EREPB TFs: possible intersects with development and stress
• Integrate large data sets to extract biological information
• Genome-wide expression profiles: build framework for exploratory
research and continuous refinement
• Further refine the system through integration of heterogeneous data
• Prioritize candidates for downstream analyses
• Model system: developing maize inflorescence primordia
- RA1 transgene (both GFP and HA tagged) introduced into ra1
- Combinatorial binding of TFs
- Gene expression influenced by both cis- and trans-acting factors
- Determine targets for key transcriptional regulators
Moving Forward: Integrating heterogeneous data types
• Genome-wide TF occupancy information: ChIP-seq
• Enrichment of cis-regulatory elements
• Small RNA profiles
• Variation data: associate genotype-to-phenotype
**example KN1 interacts with RA1 in Y2H
- Overlay RNA-seq data on variation maps
Acknowledgements
David Jackson Doreen Ware
• Sasha Goldshmidt • Jer-ming Chia
• Andrew Olson
• Sunita Kumari
• Molly Hammell
• Elena Ghiban
• Richard McCombie
• Tom Brutnell, BTI
• Lin Wang
• Robert Schmidt, UCSD
• Jerry Lu
• Shiran Pasternak
• Michael Regulski
• Michael Pautler
• Josh Stein
• Erik Vollbrecht, Iowa State
CSHL
Maize inflorescence project
NSF Postdoctoral Fellowship
in Biological Informatics
• Aaron Chuah
• Laura Gelley NSF PGRP