Light response QTL in Arabidopsis thaliana: LIGHT1 cloning

Justin BorevitzEcology & EvolutionUniversity of Chicagonaturalvariation.org

Talk Outline

• Natural Variation in Light Response

• QTL Mapping/ fine mapping

• Gene Expression candidates

• KO candidates

• Aquilegia: new model organism

• Natural Variation in Light Response

• QTL Mapping/ fine mapping

• Gene Expression candidates

• KO candidates

• Aquilegia: new model organism

Light Affects the Entire Plant Life Cycle

Light response variation can be seen under constant conditions in the labNatural Variation under selection? Test in field

Quantitative Trait Loci

SNP377

SM184

SM50

SM35

SM106

G2395

SNP65

SM40

SEQ8298

TH1

MSAT7964

MAT7787

CER45

5.50

5.87

6.34

7.01

7.30

7.44

7.60

7.79

7.96

8.13

8.29

8.65

9.32

MbMarker

Near-Isogenic Lines for LIGHT1 Ler / Cvi

mm

81N-J 17A-A/J 114 124 189Ler

6 2 4 3 3 3 Plants

Line

RVE7

GI

194

3

5.0 5.8 5.8 5.1 5.9 5.7 5.8 Phenotype

7.440

7.480

7.500

7.540

7.460

7.520

7.560

7.580

7.600

7.620

7.640

7.660

7.680

7.700

7.720

7.740

7.760

7.780

NIL189NIL194NIL114

7.540 Mb

7.675 Mb

Candidate Interval:

135 Kb34 predicted genes

OriginalMarker

Mb

LIGHT1 Fine-mapping (Ler / Cvi)

LIGHT1 (Ler / Cvi) in different light environments

0

1

2

3

4

5

6

7

8

9

White Light Blue Light Red Light

mm

Ler

189

194

114

Ler189194114 Ler189194114 Ler189194114

50 genes down regulated in NIL189 compared to Ler

5 genes up regulated in NIL189 compared to Ler

Gene Expression polymorphism between Ler and NIL189

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Ler NIL189

At1g21600

At1g21600 only gene in the LIGHT1interval that is misexpressed

7

7,5

8

8,5

9

9,5

10

0 12 24 36 48

HOURS

RE

LA

TIV

E E

XP

RE

SS

ION

Constant Light (LL)Long Day (LD)Short Day (SD)

At1g21600 is circadian-regulated

0

2

4

6

8

10

12

Salk_

0101

85

Salk_

0590

32

Salk_

0386

66

Salk_

1366

42

Salk_

0095

53

Salk_

0067

85

Salk_

0073

46

Salk_

0923

33

Salk_

0767

77

Salk_

1178

01

Salk_

1050

33

Salk_

0896

20

Salk_

0671

32

Salk_

0194

55

Salk_

1072

10

Salk_

0337

63

Salk_

0297

74

Salk_

0901

09

Salk_

0868

17

Salk_

O24

431

Salk_

5730

0

Salk_

1071

91

hypo

coty

l len

gth

(mm

)

wt.avg

mut.avg

No quantitative hypocotyl differences in 22 lines

TDNA lines in the LIGHT1 region

LIGHT1: At1g21600

Tilling: splice junction TDNA: Salk_024431

Ler: F29L

Tilling: D103N

Tilling Intron; C to T

Tilling: A30=

At1g21600 an allelic series of artificial mutations

At1g21600 is a single copy gene in multiple plant species

LF

5’ end of At1g21600 is not well conserved

At1g21600

The candidate QTN has mutated several times independantly

Natural Varition in At1g216003 nt and 1 AA change between Ler and Cvi

10 accessions sequenced for 5’ end of At1g21600Mt-0 (North Africa) shares AA change with Ler (Poland)

Sequenced 96 accessions for At1g21600, LIGHT1 candidate geneMagnus Nordborg

At1g21600 AA change is associated with Hypocotyl length and Flowering time

Hypocotyl data: P-valueRed light + PhyB 2 0.01828Red light + PhyB 7 0.01627

White light + PhyB 7 0.04189

Blue light + PhyB 1 0.03489

Flowering time data:LD True Leaf Number + FRI 0.01482LD Days to Flowering + FRI 0.004912

LIGHT1 (Ler / Cvi) : future projects

Further fine-mapping: Screen 4000 NIL189 Het plants

Phenotype KO mutants (25 of the 34 candidate genes)

Sequence some candidate SNPs across accessions

Transgenics (complementation / functional analysis)

Seasons in the Growth Chamber

• Changing Day length• Cycle Light Intensity• Cycle Light Colors• Cycle Temperature

Day Length

0:00

2:00

4:00

6:00

8:00

10:00

12:00

14:00

16:00

18:00

20:00

22:00

sep

oct

nov

dec

jan

feb

mar

apr

may jun jul

aug

month

hour

s

Sweden

Spain

standard

standard

Light Intensity

0

200

400

600

800

1000

1200

1400se

p

oct

nov

dec

jan

feb

mar

apr

may jun jul

aug

month

W/m

2

Sweden

Spain

standard

Temperature

-10

-5

0

5

10

15

20

25

30

35

sep

oct

nov

dec

jan

feb

mar

apr

may jun jul

aug

monthde

gree

s C

Spain High

Spain Low

Sweden High

Sweden Low

standard

Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome

> 20k dbEST 11/14/2003

Animal lineage: good coverage

Plant lineage: crop plant coverage

NSF Genome Complexity

• 35,000 ESTs 5’ and 3’• 350 arrays, RNA and genotyping

– High density SFP Genetic Map

• Physical Map (BAC tiling path)– Physical assignment of ESTs

• QTL for pollinator preference – ~400 RILs, map abiotic stress

– QTL fine mapping/ LD mapping

• Develop transformation techniques

Scott Hodges (UCSB)

Elena Kramer (Harvard)

Magnus Nordborg (USC)

Justin Borevitz (U Chicago)

Jeff Tompkins (Clemson)

NaturalVariation.orgNaturalVariation.orgSalkTodd MichaelOlivier LoudetJoanne ChoryDetlef WeigelJoseph Ecker

ScrippsSam Hazen

University of Chicago

Xu ZhangEvadne Smith

UC DavisJulin Maloof

University of Chicago

Xu ZhangEvadne Smith

UC DavisJulin Maloof

SalkTodd MichaelOlivier LoudetJoanne ChoryDetlef WeigelJoseph Ecker

ScrippsSam Hazen