Download Lotus japonicus

Legume genomics
Lotus japonicus as model
Wheat & barley, P&T
Lolium EST seguencing
Potato SAGE & P
Metabolomics sec metabolism
Arabidopsis functional genomics
Jens Stougaard Department of Molecular Biology
University of Aarhus Denmark
Lotus japonicus as model
Characteristics
Primary plant small
Bushy plant after secondary shoot formation, and branching
Perennial
Period from seed to flowering: 7 weeks
Generation time, seed to seed: 3-4 months
Small seeds: 1.2 g / 1000 seeds
Regrowth from stem base/tap root
Propagation
Large Flowers
Self-fertile
Approximately 20 seeds per pod
Approximately 6000 seeds per plant
No seed scattering
Hand Pollination possible
Genome
Diploid, 2n=12
Genome size 472 Mb
Tissue Culture
Regeneration from callus
Agrobacterium tumefaciens transformation
Regeneration of transgenic plants
Mendelian inheritance of transgene
Nodulation
Fast growing Rhizobium loti
Determinate nodules
Genetic background
two very different microsymbionts
Signal perception
Recognition of
Rhizobium and
mycorrhiza
Correct response
nodule development/
mycorrhization
Symbiont
or
pathogen?
Regulation of
developmental
changes
Reorganisation of metabolism:
symbiotic life.
Genetics and gene discovery
DISCIPLINES
APPROACHES &
TOOLS
Genomics
Reverse genetics & TILLING
Bioimaging
Transcriptomics
Physiology and biochemistry
Proteomics
Protein networks
Lotus japonicus
Metabolomics
Bioinformatics
Status of Lotus genome sequencing at Kazusa
Number of accumulated sequence files from random sequencing:
1,656,329 files Approximately 2.4-fold whole genome coverage
BAC by BAC sequencing:
Clones selected
Random library phase
Production phase
Phase 1
1802 (486 by walking)
35
5
1234
Phase 3 with
438
gene modeling & func. annotation
data released
(123 Mb)
(44.9 Mb)
4089 genes have been predicted
(Average gene density: 1 gene / 10.7 kb)
Features of the assigned protein-coding genes and
the functional classification
Amino acid biosynthesis
Biosynthesis of cofactors,
prosthetic groups, and carriers
Cell envelope
Cellular processes
No similarity
Similar to
genes of
known function
34%
31%
Central intermediary metabolism
Energy metabolism
Fatty acid, phospholipid and
sterol metabolism
Photosynthesis and respiration
Similar to
Hypothetical genes
35%
Purines, pyrimidines, nucleosides,
and nucleotides
Regulatory functions
DNA replication, recombination,
and repair
Transcription
BLAST cutoff of e-20
Translation
Transport and binding proteins
Other categories
Structural features of potential protein-coding genes
Features
Gene length (bp) including introns
L. japonicus
1,316 genes
A. thaliana
6,451 genes*
168-21,418 (2,581)
78-17,203 (1,918)
16-2,036 (433)
25-4,706 (427)
Genes with introns
960 (73%)
4,906 (76%)
Number of intron/gene
0-37 (3.4)
0-48 (4.0)
Exon length (bp)
3-5,604 (297)
2-5,966 (256)
Intron length (bp)
30-6,718 (377)
23-2,989 (157)
Product length (amino acids)
GC content of exons
45%
44%
GC content of Introns
33%
32%
10.7 kb
4.5 kb
Gene density (kb/gene)
*Structural features of the potential protein genes previously assigned
in Kazusa A. thaliana genome sequencing project are listed.
Lotus transcriptomics (cont.)
•
•
•
•
•
Lotus cDNA arrays
–
–
10K LjNEST macro-arrays
12K LjKDRI macro-arrays
High-throughput qRT-PCR
–
Approx. 500 PCR primer pairs for Lotus and M.
loti genes involved in metabolism and regulation
(TFs)
MAPMAN implementation for Lotus
–
Primary
Secondary
Intensity
R1 - r1
C2 - c1 10.18
R1 - r1
C2 - c2 71.02
R1 - r1
C2 - c3 64.46
R1 - r1
C2 - c4103.78
R1 - r2
C2 - c1198.39
R1 - r2
C2 - c2105.58
R1 - r2
C2 7.44
R1 - r2
C2 - c4 11.14
c3
R1 - r3
C2 - c1 17.26
R1 - r3
C2 - c2 9.98
R1 - r3
C2 - c3 50.78
R1 - r3
C2 - c4 10.14
R1 - r4
C2 - c1 14.26
R1 - r4
C2 - c2 72.93
R1 - r4
C2 - c3170.64
R1 - r4
C2 - c4 8.69
to project expression data onto metabolic
pathways etc.
Plan for Lotus Affy chip
Currently profiling various Lotus sym mutants and
symbioses formed with M. loti mutants
71 24
3 4 LB 6
65 17
D2 3 5
Global changes in gene
expression during nodulation
Root mean (n=5)
Transcript levels
Nodule mean (n=8)
870 genes induced >2-fold
in nodules (P<0.05)
Metabolism
Transport
Signalling
Transcription
Protein synthesis
Cell Division
Cell Biogenesis
Intracellular transport
Unknown Function
No homology
Colebatch et al. (2004) Plant J. 39, 487-512
Nodule up-regulated genes:
Carbon fixation
12
19
3.6
Lotus japonicus Metabolic Profiling
GC-MS profiles (A) and HCA
(B) of polar extracts from
different organs of Lotus
PCA of GC-MS profiles of
Lotus organs at 12 weeks
after germination
Distribution patterns of
representative metabolites
from HCA classes.
Desbrosses et al. (2005) Plant Physiol. 137, 1302-1318
Outline:
1. Early signalling events and the signal transduction pathway
5-deoxy-strigol
Nod factors (LCO)
Nod genes
Rhizobia
Mycorrhiza
Flavonoids
?
Working model for the early endosymbiotic signal perception in legumes
Rhizobium signal(s)
Nod-Factor
Mycorrhiza
signal(s)
SYMRK
Nuclear
NFR1
LjNup133
NFR5
LjCastor
LjPollux
LjSym24
Plastids
Membrane depolarisation
Early ion fluxes
Root tip swelling
Calcium spiking
LjCCaMK
LjCyclops
Root hair curling
Nin gene activation
Mycorrhiza
pathway
Position of 35 symbiotic loci on Lotus japonicus linkage map
Identified and published: 10. Cloned or actively worked on: >15
I
0
1
5
11
21
II
Nin
Nup133
Nfr1
Sym24
Alb1
Lot
Sym73
Sym35
Sym102
III
67
86
89
V
0
8
13
Sst
16
Sym8
15
25
Sym104
SymRK
Sym6
61
Nfr5
Ign
Sym10
Sym67
Sym7
Sym105
Sym15
Klavier
51
53
25
VI
Sen1
20
35
40
IV
43
44
50
30
Astray
37
Rhl
70
Pollux
Crinkle
Prh
Sym43
Fen1
Sym80
Castor
78
Har1
84
Srh
Sandal et al submitted
Sequenced clones located on the linkage map of L. japonicus (MG-20)
http://www.kazusa.or.jp/lotus
1
TM0088, ***
TM0145, *******
***************
*****
TM0507
TM0496, *
TM0039, *****
TM0125, ***
TM0036, *
TM0094, ***
TM0063
TM0982
TM0523
TM0050, *
TM0349
TM0133, **
TM0442, *
TM0154, *
TM0372, *
TM0487
TM0334
TM0670
TM0233
TM0195, **
TM0121, *
TM0178
TM0386
TM0101
TM0284
TM0393, *
TM0051
TM0199
TM0141, *
TM0187
TM0410
TM0675, *
TM0215
TM0688
TM0637
TM0220
TM0117, **
TM0017
TM0231
TM0240, ****
TM0147
TM0185, **
TM0800, *
TM0316
TM0001, *
TM0064
TM0356
TM0222
TM0236, *
TM0098, ****
TM0009, *
TM0276, **
TM0318, **
TM0332, *****
TM0033, **
TM0012, **
TM0010, ****
TM0029, *
TM0144, *****
TM0109, *
TM0600
TM0835, *
TM0105
2
TM0067
TM0237, ****
TM0838
TM0413
TM0134
TM0368
TM1171
TM0074, *
TM0383
TM0490, ***
TM0053
TM0254
TM0153, *
TM0610, *
TM0660
TM0065, ****
TM0263
TM0201, *
TM0400
TM0081
TM0225, *
TM0377
TM0338, *
TM0655
TM1263
TM0124
TM0120
TM0641
TM0608, *
TM0028, **
TM0008
TM0076, **
TM0541, ***
TM0020, **
TM0886
TM0695
TM0737
BM1206
TM0257
TM0374, *
TM0250, *
TM0060
TM0021, **
TM0018, *****
TM0588, *
TM0587, **
TM0504, ***
TM0058, ***
TM0011, *
TM0417
TM0002, ****
TM0031
TM0370
TM0621
TM0512, *
TM0191, **
TM0102, ***
3
TM0793, *
TM1208
TM0745,*
TM0106, *
TM0059, *
TM0340
TM0282, *
TM0436, *
TM0984
TM0080
TM0513
TM0574, **
TM0590,****
TM0450
TM0996
TM0279, **
TM0190
TM0111
TM0538
TM0035
TM0070, **
TM0196
TM0198, *
TM0116, *
TM0022, *
TM0159, *****
TM0005, *
TM0707
TM0047, *
TM0129, *
TM0292, *
TM0452
TM0142
TM0226
TM0246, **
TM0213, *
TM0406
TM0208, *
TM0416
TM0115, *
TM0049
TM0711, *
TM0160
TM0468
TM0203, **
TM0164
TM0701, *
TM0649
TM0616, **
TM0136
TM0506, **
TM0091
TM0217
TM0527, *****
TM0112
TM0135
TM0460
TM0407
TM0127
TM0261, *
4
TM0525, **
TM0654, *
TM0288
TM0026
TM1334, *
TM0256, *
TM1170
TM0265, *
TM0007, ***
TM0182, **
TM0832
TM0692
TM0432
TM0079,*
TM0227
TM0075, **
TM0347
TM0157, **
TM1335
TM0194
TM0283
TM0212
TM0119, **
TM0247, *****
TM0281, *
TM0173
TM0126
TM0172
TM0170
TM0087
TM0093, *
TM0500
TM0003, *
TM0030
TM0219
TM0542
TM0061, *
TM0244, *
TM0303, ***
TM0006, **
TM0333
TM0238
TM0387
TM0555
TM0044
TM0108
TM0399, *
TM0162
TM0046, *
TM0307, **
TM0597
TM0844
TM0175
TM0266
BM1174
TM0025
TM0004
TM0073, **
TM0214
TM0069
TM0042
5
TM0260
TM0180, *
TM0698, *
TM0366
TM0290
TM0327
TM0328
TM0703, *
TM0200
TM0218
TM0357, **
TM0953
TM0596
TM0148, *****
TM0344
TM0389
TM0095
TM0311
TM0138
TM0909
TM0913
TM0773
TM0951
TM0849
TM0239
TM0158
TM0714
TM0048
TM0299, *
TM0211
TM0062
TM1323
TM0186, *
TM0431
TM0744
TM0341
TM0151
TM0494
TM0024
TM0813
TM0278, *****
TM1496
TM0359, **
TM1077
TM0696
TM0428
TM0072, *
TM0963
TM0043, **
TM0090
TM0455
TM0034, **
TM0096, *
TM0096
TM0355
TM0077, **
TM0146
Total clones located on the linkage map : 1,351
Clones mapped by SSR markers:
Clones mapped by dCAPS markers:
Clones overlap with mapped clones:
720
80
551
6
TM0082
TM0553
TM1004
TM0472
TM0722
TM0679,*
TM0517
TM0014
TM0738
TM0302
TM0817
TM0306
TM0689
TM1261
TM0084, *
TM0245
TM0632
TM0821
TM0057, ***
TM1374
TM0041
TM0037
TM0317
TM0140, **
TM0013
TM0420
TM0137
TM0778
TM0045, *
TM0880
TM0957
TM0066, *****
TM0402
BM1337
TM0756
TM0118, *
TM0301
TM0114
TM0366
TM0055
TM0314
TM0582
TM1240
TM0508
10 cM
Bønner, åben marked i Afrika
Genes controlling seed development and quality
pathways for protein, carbohydrate, oil, metabolite biosynthesis
and micronutrient deposition
1D PAGE of seed proteins at
diffferent developmental stages
MultiDimensional Protein
Identification Technology (MudPIT)
MultiDimensional Protein
Identification Technology (MudPIT)
• Two LC MS/MS runs performed
on 40 gel slices of 1D PAGE
separated proteins
• 2578 peptide MS/MS queries
• App 400 protein hits
• Analysis ongoing
2D-reference maps and proteome database
2D electrophoresis
•
•
•
•
PDquest analysis ~1200 spots pr gel
Spots excised
In-gel tryptic digestion
Identification of proteins by MS
Identifications
• App 400 proteins identified by MudPIT
• App 100 proteins identified from 2D gels
Why do we use both techniques?
MudPIT
2D gels
• Quantitative
• Biased protein
content
• Hard work
• Identification of one
protein pr dataset
makes
interpretation easy
• Not directly quantitative
• Less biased protein content
• Faster, but interpretation more tricky
Research projects: Pathway
building
Agrawal and Rakwal, Mass Spectrom Rev. 2
Development of
legume anchor markers
for use in breeding of bean, Arachis and other crop legumes
Development of tools for
genetic mapping and comparative genomics
Anchor marker?
Markers to correlate the dense maps of the models to the map of crop legumes
Macro synteny
Microsynteny
Model, high density of markers
Model
Crop legume
1
Crop legume
A
2
?
orthologous
regions
1
2
A
3
3
4
B
4
To be transferable, anchor markers
must be derived from gene sequences
B
Conventional breeding
Unknown genetic position,
unknown gene
VERY NICE TRAIT
Trait of interest (yield, cold resistance, disease resistance etc)
Test all offspring of breeding cross for phenotype
Present
cultivar
X
intermediate
phenotype?
other factors??
The use of molecular markers in breeding programs
Multiselection possible
M5
M6
M7
M8
M3
M1
M4
Trait of interest (yield, disease resistance etc)
M9
M10
M2
Test on offspring of breeding cross
Select plant with desired
marker combination
SYNTENY between Lotus and crop
can facilitate identification of important genes
TRAIT/GENE OF INTEREST
Crop, e.g. bean
M1 M2
M3
L. japonicus
Gene
Transfer marker information to Lotus
Identify candidate genes in Lotus
Analyze candidate genes in bean
M4
Lotus japonicus
ESTs
Mediacgo
truncatula ESTs
Evolutionary
conserved
sequences (ECS)
ECS with < 3
Arabidopsis
orthologes
ECS with introns in
Lotus genomics
sequences
Multiple
alignment
and primer
design
Glycine max ESTs
Multiple sequence
alignment
INTRON
INTRON
PriFi-Using Multiple Alignment of Related Sequences to Find Primers
for Amplification of Homologs/Orthologs
http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main
Legume Phylogeny
Chromosome#
18.000 species
Size (pg)
Arachis and Phaseolus
Pipeline, easy to use, web-based
Primer Finder program, also web-based
Mapping parents: A. stenosperma X A. duranensis
122 primer pairs tested and 90 markers developed
1 SNP / 90 bp
1 Indel / 1600 bp
Mapping parents: Phaseolus vulgaris BAT93 X JALO EEP558
117 primer pairs tested and 74 markers developed
Comparative genomics, genetics,proteomics, metabolomics ............ in legumes
chickpea
peanut
lentil
soybean
bean
Lotus japonicus
Medicago truncatula
lupin
clover(s)
pea
alfalfa
Lotus corniculatus
pigeonpea
fababeans
cowpea
Spot identification
Micromass
Micromass
Maldi LR
• MALDI-TOF
Ultima
global
• App 50 %
identification
• MALDI-QTOF
• ESI-Q-TOF
Applied Biosystems
4700
Proteomic Analyzer
• MALDI-TOF-TOF
• More than 80 %
identification