Legume genomicsLotus japonicus as model

Wheat & barley, P&TLolium EST seguencingPotato SAGE & PMetabolomics sec metabolismArabidopsis functional genomics

Jens Stougaard Department of Molecular Biology University of Aarhus Denmark

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

Signal perception

Recognition ofRhizobium and

mycorrhiza

Correct response nodule development/

mycorrhization

Symbiontor

pathogen?

Regulation of developmental

changes

Reorganisation of metabolism:symbiotic life.

DISCIPLINESAPPROACHES &

TOOLSGenetics and gene discovery Genomics

Reverse genetics & TILLING Bioimaging

Transcriptomics Physiology and biochemistry

MetabolomicsProteomics Lotus japonicus

BioinformaticsProtein networks

Status of Lotus genome sequencing at KazusaNumber of accumulated sequence files from random sequencing:1,656,329 files Approximately 2.4-fold whole genome coverage

BAC by BAC sequencing:Clones selected 1802 (486 by walking)

Random library phase 35Production phase 5

Phase 1 1234

Phase 3 with gene modeling & func. annotation

438 (44.9 Mb)

4089 genes have been predicted

(123 Mb)

data released(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

Similar to genes of known function

Similar to Hypothetical genes

No similarity

34%

35%

31%

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

BLAST cutoff of e-20 Translation

Transport and binding proteins

Other categories

Structural features of potential protein-coding genes

L. japonicus A. thalianaFeatures 1,316 genes 6,451 genes*

Gene length (bp) including introns 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 geneexpression during nodulationTranscript levels

Root

mea

n(n

=5)

870 genes induced >2-foldin nodules (P<0.05)

Metabolism Transport

Signalling Transcription

Protein synthesis Cell Division

Cell Biogenesis Intracellular transport

Unknown Function No homology

Nodule mean (n=8)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 fromdifferent 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

Nod factors (LCO)

Rhizobia

Flavonoids

Nod genes

Mycorrhiza

5-deoxy-strigol

?

Working model for the early endosymbiotic signal perception in legumes

NFR1

NFR5

Mycorrhizasignal(s)

Rhizobium signal(s)Nod-Factor

Root hair curlingNin gene activation

SYMRK

Mycorrhizapathway

Calcium spiking

Membrane depolarisationEarly ion fluxesRoot tip swelling

LjNup133LjCastorLjPollux

LjCCaMK

LjCyclops

LjSym24

Plastids

Nuclear

Position of 35 symbiotic loci on Lotus japonicus linkage mapIdentified and published: 10. Cloned or actively worked on: >15

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

Sandal et al submitted

Sequenced clones located on the linkage map of L. japonicus (MG-20)http://www.kazusa.or.jp/lotus

TM0347TM1335

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, *

TM0101TM0178

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, **TM0233TM0121, *

TM0187TM0675, *

TM0236, *TM0222TM0064

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

TM0302TM0306

TM0245

TM0301

TM0314

*****TM0496, *

TM0523TM0349

TM0154, *TM0372, *TM0487TM0334TM0670

TM0386

TM0688TM0637

TM0356

TM0033, **

TM0600TM0835, *

TM0413TM0368

TM0490, ***TM0254

TM0065, ****

TM0400

TM0377TM0338, *

TM0641TM0608, *

TM0374, *

TM0504, ***

TM0417

TM0370TM0621TM0512, *

TM0106, *

TM0513

TM0450

TM0538

TM0406TM0416

TM0468

TM0649TM0616, **TM0506, **TM0091TM0527, *****

TM0460TM0407

TM0654, *TM0525, **

TM0542

TM0303, ***TM0333TM0387TM0555

TM0399, *

TM0366TM0290

TM0218TM0357, **TM0596TM0344TM0389

TM0138

TM0431TM0744

TM0278, *****TM0359, **

TM0428

TM0090

TM0355

TM0553

TM0722TM0517

TM0632

TM0317

TM0420

TM0402

TM0366

TM0582TM1240

TM1004

TM0679,*

TM0738

TM0689

TM0821

TM0778TM0880TM0957

TM0698, *

TM0703, *

TM0953

TM0909TM0913TM0773TM0951TM0849

TM0714

TM0341TM0494

TM0696

TM0692

TM0844

TM1208

TM0984

TM0707

TM0711, *

TM0701, *

TM0655

TM0541, ***TM0886TM0695TM0737

TM0982

TM0800, *

TM0472

TM0817

TM1261

TM1374

BM1337TM0756

TM0508

TM0327

TM1323

TM0813TM1496TM1077

TM0963

TM0455

TM0096

TM0838

TM1171TM0383

TM1263

BM1206

TM0745,*

TM0590,****TM0996

TM1334, *TM1170

TM0832

TM0500

TM0597

BM1174

Total clones located on the linkage map : 1,351

Clones mapped by dCAPS markers:Clones mapped by SSR markers: 720

80Clones overlap with mapped clones: 551

Bønner, åben marked i Afrika

Genes controlling seed development and qualitypathways for protein, carbohydrate, oil, metabolite biosynthesisand 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 datasetmakesinterpretation easy

MudPIT• 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 forgenetic mapping and comparative genomics

Anchor marker?Markers to correlate the dense maps of the models to the map of crop legumes

MicrosyntenyMacro synteny

Model, high density of markers ModelCrop legume Crop legume1

2

3

4

1

2

34

?A

orthologousregions B

A

B

To be transferable, anchor markersmust be derived from gene sequences

Conventional breeding

Unknown genetic position,unknown gene

VERY NICE TRAITTrait of interest (yield, cold resistance, disease resistance etc)

Test all offspring of breeding cross for phenotype

X

Presentcultivar intermediate

phenotype?

other factors??

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 desiredmarker combination

Multiselection possible

SYNTENY between Lotus and cropcan facilitate identification of important genes

L. japonicus

Crop, e.g. beanM1 M2 M3 M4

TRAIT/GENE OF INTEREST

GeneTransfer 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

INTRONINTRON

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 PhaseolusPipeline, 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 bp1 Indel / 1600 bp

Mapping parents: Phaseolus vulgaris BAT93 X JALO EEP558117 primer pairs tested and 74 markers developed

Comparative genomics, genetics,proteomics, metabolomics ............ in legumes

chickpea

peanut lentil

Lotus japonicus Medicago truncatula

soybean bean

peaalfalfa

Lotus corniculatus

lupin clover(s)

pigeonpea fababeans

cowpea

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