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Legume genomics Lotus japonicus as model
Jens Stougaard Department of Molecular Biology University of Aarhus Denmark
Wheat & barley, P&TLolium EST seguencingPotato SAGE & PMetabolomics sec metabolismArabidopsis functional genomics
Lotus japonicus as modelCharacteristics Primary plant small
Bushy plant after secondary shoot formation, and branchingPerennialPeriod from seed to flowering: 7 weeksGeneration time, seed to seed: 3-4 monthsSmall seeds: 1.2 g / 1000 seedsRegrowth from stem base/tap root
Propagation Large FlowersSelf-fertileApproximately 20 seeds per podApproximately 6000 seeds per plantNo seed scatteringHand Pollination possible
Genome Diploid, 2n=12Genome size 472 Mb
Tissue Culture Regeneration from callusAgrobacterium tumefaciens transformationRegeneration of transgenic plantsMendelian inheritance of transgene
Nodulation Fast growing Rhizobium lotiDeterminate nodules
Genetic backgroundtwo very different microsymbionts
Recognition ofRhizobium and
mycorrhiza
Symbiont or
pathogen?
Signal perception
Correct response nodule development/
mycorrhization
Regulation of developmental
changes
Reorganisation of metabolism: symbiotic life.
Reverse genetics & TILLING
Protein networks
Metabolomics
Bioimaging
Proteomics
Transcriptomics
GenomicsGenetics and gene discovery
Physiology and biochemistry
Bioinformatics
DISCIPLINES APPROACHES &
TOOLS
Lotus japonicus
BAC by BAC sequencing: Clones selected 1802
Random library phase 35Production phase 5
Phase 1 1234
Phase 3 with gene modeling & func. annotation
438 (44.9 Mb)
(486 by walking)
4089 genes have been predicteddata released
(123 Mb)
Status of Lotus genome sequencing at Kazusa
(Average gene density: 1 gene / 10.7 kb)
Number of accumulated sequence files from random sequencing:1,656,329 files Approximately 2.4-fold whole genome coverage
Amino acid biosynthesis
Biosynthesis of cofactors, prosthetic groups, and carriers
Cell envelope
Cellular processes
Central intermediary metabolism
Energy metabolism
Fatty acid, phospholipid andsterol metabolism
Photosynthesis and respiration
Purines, pyrimidines, nucleosides,and nucleotides
Regulatory functions
DNA replication, recombination,and repair
Transcription
Translation
Transport and binding proteins
Other categories
Features of the assigned protein-coding genes and the functional classification
Similar to genes of known function
Similar to Hypothetical genes
No similarity
34%
35%
BLAST cutoff of e-20
31%
Structural features of potential protein-coding genes
Features
Gene length (bp) including introns
L. japonicus A. thaliana
1,316 genes 6,451 genes*
168-21,418 (2,581) 78-17,203 (1,918)
Product length (amino acids) 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)
GC content of exons 45% 44%
GC content of Introns 33% 32%
Gene density (kb/gene) 10.7 kb 4.5 kb
*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
– 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
7 1 2 43 4 LB 66 5 1 7D 2 3 5
7 1 2 43 4 LB 66 5 1 7D 2 3 5
PrimarySecondaryIntensityR1 - C2r1 - c1 10.18R1 - C2r1 - c2 71.02R1 - C2r1 - c3 64.46R1 - C2r1 - c4103.78R1 - C2r2 - c1198.39R1 - C2r2 - c2105.58R1 - C2r2 -
c37.44
R1 - C2r2 - c4 11.14R1 - C2r3 - c1 17.26R1 - C2r3 - c2 9.98R1 - C2r3 - c3 50.78R1 - C2r3 - c4 10.14R1 - C2r4 - c1 14.26R1 - C2r4 - c2 72.93R1 - C2r4 - c3170.64R1 - C2r4 - c4 8.69
Global changes in gene expression during nodulation
Transcript levels
Nodule mean (n=8)
Root
mean
(n
=5
)
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
193.6
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
Rhizobia
Flavonoids
Nod factors (LCO)
Nod genes
Mycorrhiza
5-deoxy-strigol
?
Outline:
1. Early signalling events and the signal transduction pathway
NFR1
NFR5
Rhizobium signal(s)Nod-Factor Mycorrhiza
signal(s)
Root hair curlingNin gene activation
SYMRK
Mycorrhizapathway
Calcium spiking
Working model for the early endosymbiotic signal perception in legumes
Membrane depolarisationEarly ion fluxesRoot tip swelling
LjNup133LjCastor
LjPollux
LjCCaMK
LjCyclops
LjSym24
Plastids
Nuclear
Nin0Nup1331Nfr15Sym24Alb1LotSym73Sym35
11
Sym10221
Klavier40
Fen167
Sym8086Castor89
I
Sst13
SymRK51Sym653
Nfr561
II
Sym816
Sym10425
Sym1535
Har178
Srh84
III
Sen18
Sym10Sym6715
Sym10525
IV
Ign0
Sym720
Crinkle43Prh44Sym4350
V
Astray30
Rhl37
Pollux70
VI
Position of 35 symbiotic loci on Lotus japonicus linkage mapIdentified and published: 10. Cloned or actively worked on: >15
Sandal et al submitted
Sequenced clones located on the linkage map of L. japonicus (MG-20)http://www.kazusa.or.jp/lotus
Clones mapped by dCAPS markers:
TM0347
TM1335
TM0432
TM0793, *
TM0276, **
TM0316
TM0410
TM0060
TM0610, *
TM0159, *****
TM0035
TM0574, **
TM0059, *TM0340
TM0001, *
TM0017
TM0199
TM0070, **
TM0088, ***TM0145, *******
TM0094, ***
TM0002, ****
TM0011, *
TM0507
TM0012, **TM0010, ****TM0029, *
TM0098, ****
TM0117, **
TM0036, *
TM0101
TM0178
TM0050, *
1 2 3 4 5 6
10 cM
TM0021, **
TM0020, **
TM0018, *****
TM0013
TM0014
TM0055
TM0045, *
TM0041
TM0034, **
TM0260
TM0043, **
TM0005, *
TM0022, *
TM0049
TM0007, ***
TM0087
TM0006, **
TM0044
TM0073, **
TM0030
TM0025
TM0003, *
TM0046, *
TM0256, *
TM0062
TM0037
TM0660
TM0053TM0080
TM0048
TM0057, ***
TM0066, *****
TM0067
TM0257
TM0004
TM0042
TM0191, **
TM0058, ***
TM0063
TM0069
TM0072, *
TM0031
TM0076, ** TM0452
TM0026
TM0077, **
TM0008
TM0102, ***
TM0039, *****TM0125, ***
TM0109, *
TM0124TM0120
TM0282, *
TM0047, *TM0129, *
TM0115, *
TM0127
TM0281, *TM0247, *****
TM0093, *
TM0095
TM0096, *
TM0082
TM0134
TM0074, *
TM0081
TM0587, **
TM0133, **
TM0141, *
TM0144, *****
TM0105
TM0116, *
TM0142
TM0160
TM0135
TM0079,*
TM0140, **
TM0146
***************
TM0051
TM0111
TM0112
TM0108
TM0061, *
TM0119, **
TM0200
TM0186, *
TM0151
TM0084, *
TM0114
TM0442, *
TM0195, **TM0233
TM0121, *
TM0187
TM0675, *
TM0236, *TM0222
TM0064
TM0147
TM0153, *
TM0028, **
TM0588, *
TM0436, *
TM0292, *
TM0164
TM0136
TM0182, **
TM0157, **
TM0194TM0283
TM0175
TM0162
TM0180, *
TM0148, *****
TM0211
TM0158
TM0024
TM0118, *
TM0137TM0284
TM0215
TM0220
TM0240, ****TM0231
TM0185, **
TM0009, *
TM0318, **TM0332, *****
TM0393, *
TM0250, *
TM0225, *
TM0201, *TM0263
TM0237, ****
TM0190TM0279, **
TM0198, *TM0196
TM0203, **
TM0261, *
TM0217
TM0208, *
TM0213, *TM0246, **TM0226
TM0288
TM0265, *
TM0227TM0075, **
TM0212
TM0173TM0126TM0172TM0170
TM0244, *
TM0238
TM0307, **
TM0266
TM0214
TM0219
TM0328
TM0311
TM0299, *
TM0239
TM0302
TM0306
TM0245
TM0301
TM0314
*****TM0496, *
TM0523
TM0349
TM0154, *TM0372, *TM0487TM0334TM0670
TM0386
TM0688TM0637
TM0356
TM0033, **
TM0600TM0835, *
TM0413
TM0368
TM0490, ***
TM0254
TM0065, ****
TM0400
TM0377TM0338, *
TM0641TM0608, *
TM0374, *
TM0504, ***
TM0417
TM0370TM0621TM0512, *
TM0106, *
TM0513
TM0450
TM0538
TM0406
TM0416
TM0468
TM0649TM0616, **
TM0506, **TM0091
TM0527, *****
TM0460TM0407
TM0654, *TM0525, **
TM0542
TM0303, ***
TM0333
TM0387TM0555
TM0399, *
TM0366TM0290
TM0218TM0357, **
TM0596
TM0344TM0389
TM0138
TM0431TM0744
TM0278, *****
TM0359, **
TM0428
TM0090
TM0355
TM0553
TM0722
TM0517
TM0632
TM0317
TM0420
TM0402
TM0366
TM0582TM1240
TM1004
TM0679,*
TM0738
TM0689
TM0821
TM0778
TM0880TM0957
TM0698, *
TM0703, *
TM0953
TM0909TM0913TM0773TM0951TM0849
TM0714
TM0341
TM0494
TM0696
TM0692
TM0844
TM1208
TM0984
TM0707
TM0711, *
TM0701, *
TM0655
TM0541, ***
TM0886TM0695TM0737
TM0982
TM0800, *
TM0472
TM0817
TM1261
TM1374
BM1337TM0756
TM0508
TM0327
TM1323
TM0813
TM1496
TM1077
TM0963
TM0455
TM0096
TM0838
TM1171TM0383
TM1263
BM1206
TM0745,*
TM0590,****TM0996
TM1334, *TM1170
TM0832
TM0500
TM0597
BM1174
Clones mapped by SSR markers: 72080
Clones overlap with mapped clones:
551
Total clones located on the linkage map :1,351
Bønner, åben marked i Afrika
Genes controlling seed development and qualitypathways 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?
2D gels• Quantitative• Biased protein
content• Hard work• Identification of
one protein pr dataset makes interpretation easy
MudPIT• Not directly quantitative• Less biased protein content• Faster, but interpretation more tricky
Research projects: Pathway building
Agrawal and Rakwal, Mass Spectrom Rev. 2005
Development of legume anchor markers
for use in breeding of bean, Arachis and other crop legumes
Development of tools forgenetic mapping and comparative genomics
Anchor marker?
Model, high density of markers
Macro synteny
1
2
3
4
1
2
3
4
Crop legume
Microsynteny
Markers to correlate the dense maps of the models to the map of crop legumes
Model
Crop legume
?
A
A
B
B
orthologous regions
To be transferable, anchor markers must be derived from gene sequences
Conventional breeding
Trait of interest (yield, cold resistance, disease resistance etc)Test all offspring of breeding cross for phenotype
Unknown genetic position, unknown gene
VERY NICE TRAIT
X
intermediate phenotype?
other factors??
Present cultivar
The use of molecular markers in breeding programs
Trait of interest (yield, disease resistance etc)
Test on offspring of breeding cross
M1
M2
M3
M4
M7
M8
M9
M10
M5
M6
Select plant with desired marker combination
Multiselection possible
SYNTENY between Lotus and cropcan facilitate identification of important genes
L. japonicus
Crop, e.g. bean
Gene
M1 M2 M3 M4
TRAIT/GENE OF INTEREST
Transfer marker information to LotusIdentify candidate genes in LotusAnalyze candidate genes in bean
Lotus japonicusESTs
Lotus japonicusESTs
Mediacgotruncatula ESTs
Mediacgotruncatula ESTs
Glycine max ESTs
Evolutionary conserved
sequences (ECS)
Evolutionary conserved
sequences (ECS)
ECS with < 3 Arabidopsisorthologes
ECS with < 3 Arabidopsisorthologes
ECS with introns in Lotus genomics
sequences
ECS with introns in Lotus genomics
sequences
Multiple alignment and primer
design
Multiple alignment and primer
design
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# Size (pg)
18.000 species
Arachis and Phaseolus
Pipeline, easy to use, web-based
Primer Finder program, also web-based
Mapping parents: A. stenosperma X A. duranensis122 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
Lotus japonicus Medicago truncatula
soybean bean
peaalfalfa
Lotus corniculatus
peanut
chickpea
lentil
clover(s)
fababeans
cowpea
pigeonpea
Comparative genomics, genetics,proteomics, metabolomics ............ in legumes
lupin
Spot identificationMicromass
Maldi LR
• MALDI-TOF
• App 50 % identification
Micromass
Ultima global
• MALDI-Q-TOF
• ESI-Q-TOF
Applied Biosystems 4700
Proteomic Analyzer
• MALDI-TOF-TOF
• More than 80 % identification