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European Networking Summer School(ENSS)

Plant Genomics & Bioinformatics

Standing Committee for Life, Earth and EnvironmentalSciences (LESC)

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Supported by:Austria Fonds zur Förderung der wissenschaftlichen Forschung (FWF)

Belgium: Fonds voor Wetenschappelijk Onderzoek (FWO)

Finland: Academy of Finland - Research Council fo Biosciences and Environment

Ireland: Irish Research Council for Science Engineering and Technology (IRCSET)

Italy: Consiglio Nazionale delle Ricerche (CNR) - Dipartimento Agroalimentare

Netherlands: Nederlandse Wetenschappelijk voon Onderzoek (NWO)

Norway: The Research Council of Norway

Poland: The Polish Academy of Science

Romania: Ministry of Education and Research

United Kingdom: Biotechnology and Biological Sciences Research Council (BBSRC)

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AIMS

• Support plant genome research networks based bytraining of young investigators

• Summer courses with theoretic and practical training

• Access to technologies, resources, skills and know-how

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ENSS 2009 Plant Bioinformatics, Systems and Synthetic Biology

27-31 July 2009University of Nottingham, UK

Natalio Krasnogor, Jaume Bacardit, Malcolm Bennett

ENSS 2010 Plant Epigenetics

September 2010Leibniz Institute of Plant Genetics and Crop

Plant Research (IPK) in Gatersleben, GermanyMichael Florian Mette

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Comparative and Functional Genomics

Functional genomics is the understanding of the function of genes and other parts of the genome

Comparative genomics involves the use of computer programs to line up multiple genomes/genes for the identification of similarities

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What is needed to do comparative and functional genomics?

model organism

Why are model organisms important?

Criteria for a good model organism?

Relationship of the model to important crop plants?

How many genes are the same?

Why using knock out/down mutants?

How will they help us determine gene function?

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What will you hear?

Background on annotating gene function using comparativegenomic tools

Example for the use comparing genomes/genes from individuals between populations to determine their function

Example to show how these tools can be employed to get a glimps on the function of a yet unknown gene

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All Starts With Genome Sequencing Projects

http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi

http://www.ensembl.org/info/about/species.html

How many plant genomes have been sequenced?

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Plant Genome Sequencing Projects

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Improvements in the rate of DNA sequencing overthe past 30 years and into the future

Stratton et al., 2009

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Quote from Joe Ecker IARC2009Capillary sequencing – 500 people – 7 years – 70.000.000 $

Perlegen sequencing – 50 people – 1 year – 70.000 $

Next generation sequencing – 2 people –7 days – 7.000 $ - 50 x coverage

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Paradigm Change

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After genome sequencing still many questions remain – example Arabidopsis

MASC 2007

MASC 2009

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Sequenced genomes – the basis to address questions on

Function of all genes

Role of single nucleotide polymorphisms (SNPs, natural variation)

Where? - Localization (organs, tissues, cellular, sub-cellular)

Functional redundancies/diversification of gene families

Role of noncoding regions and repeats in the genome

Biological role(s)

When? – Regulation (transcriptional, post-transcriptional, post-translational,..)

Interacting partners - Networks

Role of alternative splicing variants

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Functional Genomic ToolsSequences genome, full-length cDNA clones

Gene knock-outs, knock-downs (T-DNA, transposon, amiRNA, tilling, gene targeting, collection of natural variants, ....)

Methods for studying functions of nonprotein-coding sequences

Comprehensive analysis of gene expression (microarray, deepsequencing, cell sorting, laser dissection, reporter constructs, … )

Large-scale protein analyses (proteomics, protein arrays, large scaleY2H, interactomes-networks, 3D structures)

Metabolomics

-omics

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Comparative genomics between species

comparison of genomes from different taxa

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Comparative genomics within a speciescomparison of genomes from different individuals between populationsthat might be differentially adapted to particular environments ….

Weigel Lab

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What to compare? on the structural level:

sequence similarity (nucleic acid, protein, domains)gene location (synteny)gene structure (length, number of exons) amount of noncoding DNA highly conserved regions (fundamental/essential genes)highly/less polymorphic regions (indication of adaptation,

selection)

on the functional level: expression pattern epigenetic regulation post-transcriptional translational regulation subcellular localization interactionspost-translational regulation/modification

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How to compare?Search tools for homologies: BLAST

FASTA

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How to compare?http://www.expasy.org/

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Example

1. BLAST:

Proteome analysis in Poplar result a peptide of MILSALLTSVGINLGLC

UniGene

2. UniGene

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G – Entrez Gene

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G – Entrez Gene

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Domains – Function - Localization?

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Example

1. BLAST:

Proteome analysis in Poplar result a peptide of MILSALLTSVGINLGLC

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G – Entrez Gene

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G – Entrez Gene

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alternatively spliced

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GBrowser

gene family 13 members

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Synteny SearchCan the annotation of one member of the gene family in any plant species

guide to the function?

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Search for HYP1

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Common Function?

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Other Synteny Tools

http://chibba.agtec.uga.edu/duplication/

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Plant Genome Duplication Database (PGDD)

http://chibba.agtec.uga.edu/duplication/

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Intra-genome Dotblot analysis at PGDD

duplication of chr 3 and chr 2

non-synonymous substitution (Ka)synonyoumous substitution (Ks)

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Cross-genome Dotblot analysis at PGDD

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PGDD

microsynteny

Vitis vinifera

Medicagotrunculata

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Back to TAIR

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SUBA-Database

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novel putative function

HYP1

http://aramemnon.botanik.uni-koeln.de/

15 related proteins

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What is AtGFS10?http://www.ncbi.nlm.nih.gov/sites/gquery

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Topology Prediction

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Strongly predicted to be in the secretroypathway

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Expression Analyses

Poplar homologs

http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi

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Expression Analyses

highest in male catkins

highest in leaves

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Upon water stressand

day/night cycles

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co-expressed gene in Arabidopsis

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Expression of the Arabidopsis homolog uponosmotic stress

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Still no entry in Proteins Wiki

http://proteins.wikia.com/wiki/

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Summary Peptide/Protein AnnotationMILSALLTSVGINLGLC

• belongs to gene family –• members HYP1 (hypothetical protein 1)• RXW8 (name of cDNA) • ERD4 (early responsive to dehydration)• AtGFS10 (protein involved in vacuolar sorting fo storage proteins,

green fluorescent seed, gfs mutant)

Quote: „no closely related homologs of GFS10 in the Arabidopsis genome“but topology similar – Aramemnon and 39% amino acid homology

• integral membrane protein

• plasma membrane, secretory pathway

• induced during water stress

wild-type

secrete vacuole-targeted GFP out of the seed cells

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What next?

Poplar gene with homolog in Arabidopsis (81%) First functional tests in Arabidopsis: knock outs

over-/ectopic-/ inducible expressionin vivo localization – XFP, immunointeraction with other proteins

Design experiments for functional characterization

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QTL-Analysis or Association Mapping

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Understanding functional consequences of natural variation: trichome patterning in

Arabidopsis

Example for the comparison of genomes/gene and their function from individuals between populations

Julia Hilscher, Christian Schlötterer, Marie-Theres Hauser

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Trichomes in Arabidopsis• Single cell structure• Present on leaves, stem, petioles, sepals• 32C->polyploid• Model for cell fate specification

A B

DC

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Trichome function• Arabidopsis

– Protection against herbivoryMauricio & Rausher (1997), Handley, Ekbom & Ågren (2005)

• A. lyrata– Protection against herbivory

Kivimäki, Kärkkäinen, Gaudeul, Løe & Ågren (2007)

• Other plants– Decrease of water loss– Increased light reflection– Freezing tolerance– Ca++ homeostasis– Heavy metal storage– Metabolite production and storage – Cotton fiber development

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Cross-species function of Trichomeregulators

Cotton fiber development

Trichome development of Arabidopsis 35S::GaMYB2 in Arabidopsis

„hairs on seeds“

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Trichome density differs in natural populations

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Gr-

1Bl

h-1

Bur

-0/1

Ba-

1B

uckh

orn

Pas

sLe

rN

ok-0

Ga-

0Lz

-0W

t-5O

y-0

Di-0

Van

-0B

rest

-1C

t-1/4

No-

0A

n-1

Wa-

1/3

83-3

Tsu-

0Ki

n-0

Pi-0

Pa-

1/1

T-20

-IA

g-0/

4T-

12-I

I-33

N7

Lip-

0/1

RLD

-1S

toc

5T-

29-I

Mh-

0M

t-0G

e-0

Tsch

a-1

Hi-0

Kar

a7-2

Sf-2

Pa-

3D

ül/4

Nd-

0Es

t-1Yo

-0S

t-0 Col

Aa-

0S

haG

rivo-

1B

ayU

k-3

Es-0

9481

Te-0

Ws-

0Te

in-1

Bla-

1K

as-1

Ms-

0R

yb2

Mas

/2Ita

-0C

ond

Per

-1/5

Lim

384

-1Al

c-0/

1Ts

ar/1

Edi

-0/2

Gy-

0P

og-0

/2B

as-2

/320

-13

Rub

-1/2

Can

-0

Can-0Gr-1

1.5 mm

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QTL mapping

x

F1x F1

F2

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Composite Interval Mapping

• 266 F2 individuals

• 24 microsatellites

• A single QTL on chromosome 2 explains 33% of the variation in trichome number

0

20

40

60

80

100

120

0 10 20 30 40 50 60

LR0ng

a63

Ath

ZFP

G

T27K

12-S

p6

T2K

10

0

20

40

60

80

100

120

0 10 20 30 40 50

LR0

[cM]

CIW

2

F19G

14A

nga2

235

ngaT

3B23

nga3

61ng

a347

0ng

a369

2

ngaT

2P4

Chr I Chr II

0

20

40

60

80

100

120

0 10 20 30 40 50 60

LR0

nga1

62

3g28

3 7

F1P

2TG

F

nga6

0

20

40

60

80

100

120

0 10 20 30 40

LR0

[cM]

JV30

/31

nga8

CIW

7

nga1

139

Chr III Chr IV

0

20

40

60

80

100

120

0 10 20 30 40

LR0

[cM]

nga1

51 ng

a13

9 AtS

0191

nga1

29

Chr V Trichome numberTrichome density

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Components of trichomedevelopment

Patterning involves positive and negative regulators with redundant functions

WTtry cpc

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Model of epidermal patterning

Phenotypic class of mutants

Gene Action

no trichomes“glabrous”

GL1 Positive regulators

TTG1

decreased trichome #

GL3/EGL3

GL2

nests of trichomes

TRY Negative regulators

single-repeat R3 MYB genes,

act non-cell autonomous

increasedtrichome #

CPC

GL3 GL3

TTG1

GL1GL2, …

CPC, TRY, ETC1, ETC2, TCL1, TCL2,ETC3

modified from Larkin et al., 2003

All of the genes or their paralogs are also involved in root hair patterning

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Composite Interval Mapping

TCL2

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Fine mapping on chromosome II• Selective genotyping

– 465 F2 with extreme phenotype– 10 additional markers

• 66 recombinants in the initial interval -> expected mapping resolution of 40kb

• 288 kb mapping interval with remainig 87 genes

ngaT

3B23

nga3

000

nga3

023

HD

UP

3038

Sm

all M

ybs

nga3

073

nga3

098

nga3

61ng

a310

82ng

a311

9

nga3

168

nga3

341

% re

com

bina

ntch

rom

osom

es

0

1

2

3

4

5

6

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3 candidate genes

Three single-repeat R3 MYB genes are located in the fine-mapping interval

At2g30440

TCL1 ETC2

KIS

TCL2

At2g30430

1kb

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Association with trichome number

CPC

84-I

Gr

Can

Oy

Ler

Col

84-I

CPC

Gr

Can

Oy

Ler

Col

Ws

20-13

84-I

CPC

Gr

Can

Oy

Ler

Col

Ws

20-13

low trichome number accessions

high trichome number accessions

Small MYB_A

Small MYB_B

Small MYB_C

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Complementation test

mutants are complemented with the wildtype alleles from high and low trichome number accessions

smal

lmyb

Col

Sm

allM

ybG

r-1

smal

lMY

BLe

r

smal

lMY

BC

an-0

Sm

allM

YB

20-1

3

# of

tric

hom

es/le

af

0

100

200

300

400

F1 cross to

low trichome numberaccession

high trichome numberaccession

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Identification of QTN

Many SNPs show association with trichome phenotype

Screen many high/low trichome accessions for recombinants

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Identification of QTN: 2 candidates-53 +55

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Identification of QTN

Gr-1

Can-0

A

C A

G-53 bp +55 bpposition

Gr-1

Can-0

C

C G

G

Gr-1

Can-0

A

A A

A

allele background

SNP status

wt

+55 Gr-1

-53 Can-0

+55 Can-0

-53 Gr-1

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Identification of QTNTransgenic complementation

0

50

100

150

200

250

300#

of tr

icho

mes

/leaf

+55Gr-1 +55Can-0

Gr-1Can-0

- 53Can-0 - 53Gr-1

allele background

ANOVA

SNP state: p=0.001

allele background: p=0.35

Mean ± s.e.m. of n>21 T1 lines each, 3 leaf positions

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QTN is highly conserved among family members

+55 mutation leads to an amino acid replacement: Lysine (K) to Glutamate (E)

K: ancestral, yet unknown importance

Alpha Helices 1-3 constituting R3 MYB domain with conserved W residues forming cluster

Predicted bHLH interaction motif: [DE]Lx2[RK]x3Lx6Lx3R (Zimmermann et al., Plant J 2004)

Required for CPC movement (Kurata et al., Development 2005)

H1 H2 H3

CPL3

TCL1

CPL3

TCL1

K19E

TCL2

TCL2TCL1

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Competition between activators and repressors

TTG1

GL3/EGL3

TTG1

GL3/EGL3smallmyb

target genes

GL1

target genes

smallMyb

GL1

TTG1

GL3/EGL3

competition

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3 possible factors for trichome patterning

Binding strength to GL3 or the regulatory region

Movement rate to neighboring cells

Stability

Lysine modification by: methylationN-glycosylationubiquitylationsumoylationacetylation

Glutamate modification by: methylation

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Next steps

Biochemistry

Cell Biology

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Sumoylation? Use ExPASy

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Take Home Message

Functional genetics & natural variation – powerful tool

Strong QTL or Association

33% of natural variation in trichome number was explainable by a single aa replacement

Classical genetic studies failed to identify the major modifier of trichome number

Typical accession used for functional tests (Ws, Col) have intermediate-high trichome number and the weak suppressor allele

Requirements for success

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Association Mapping

trichome density

In future

– phenotyping ecotypes/accession- look up into the association map- identify the gene with the variable SNPs- functional confirmation

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More sequences * More functions * More to compare

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THANKS

QUESTIONS?