Identifying genes for changes in root architecture under water stress

Georgia Davis

University of Missouri

www.rootgenomics.org

Overview

Root architecture QTLs vp mutants Root transcriptome map

Qualitative Quantitative

•One gene•Discrete distribution

•Several - many genes•Continuous distribution

Quantitative Trait Mapping

Population segregating for the trait Molecular markers to create a linkage map Trait measurements Enough replication to get a good idea of

genotype vs. environmental differences

Playing the Numbers

A QTL of 15 cM contains 450 - 900 loci in maize.

QTL size is reduced by increasing recombination (ex. random intermating, larger sample of individuals) and to some degree by mapping additional genetic markers.

Molecular mapping

Compare the DNA fingerprint with trait value.

Look for bands on fingerprint associated with high value and those associated with low value.

DNA fingerprintGene 1

Mp3

13E

Va3

5

1 2 3 4 5 6

Lo Hi Int. Lo LoHi Int. Int.

Lo Hi Int. Lo LoHi Int. Int.

Gene 2

QTL Mapping

Using Intermated B73 x Mo17 (IBM) population to map root architecture under well-watered and water-stress conditions.

• Studying the IBM 94 reduced the number of candidate genes per cM to 14.3.

• The IBM genetic map is linked to the physical map and anchored sequence information allowing us to identify genes not found on the genetic map.

Mike Gerau, undergraduate

Root Architecture QTL

Measured under well-watered and water-stressed conditions: primary root length root branching root mass seminal root number shoot mass leaf relative water content

Analysis

Mean values for ww, ws and the response (ww-ws)/ww were used for QTL analysis against 643 markers spaced <10 cm apart on the genetic map.

Primary root lengthww ws resp

BranchingRoot massShoot mass

Seminal root #ww ws resp

Leaf #Leaf RWC

1 2 3 4 5 6 7 8 9 10

58 Root Architecture QTL

Candidate Genes

vp5 pds1 rt1 d10 d12 la1

hsf1 knox sod3 gst rab15 rab28

Endogenous ABA accumulation is required for root growth maintenance under water deficits

(Saab et al., 1990; 1992; Sharp et al., 1994)

vp5 (maize)

fluridone (FLU)

vp14 (maize)

phytoene

phytofluene

-carotene

neurosporene

lycopene

-,-carotene

-,-carotene

zeaxanthin

antheraxanthin

all-trans-violaxanthin

all-trans-neoxanthin

9’-cis-neoxanthin

9-cis-violaxanthin

Xanthoxin

ABA-aldehyde

ABA

*possible oxidative cleavage steps in planta; reactions catalyzed by NCED (9-cis-epoxycarotenoid dioxygenase)

Modified from Taylor et al.

(2000) J Exp Bot 51: 1563-74

*

*

ROOT TIP ABA CONTENT (ng g-1 H2O)

21 ± 5 96 ± 29118 ± 18Sharp et al. (1994) J Exp Bot 45: 1743-51

viviparous (vp) mutants have defects in carotenoid and/or ABA biosynthesis.

Six vp mutants: vp5, vp5-DR3076, vp8, vp9, vp10 and vp12

WW and WS Same measurments as QTL.

vp mutants

Ryan Dierking, undergraduate

9-cis-violaxanthin

vp14 (maize)

vp5 (maize)

fluridone (FLU)

phytoene

phytofluene

-carotene

neurosporene

lycopene

-,-carotene

-,-carotene

zeaxanthin

antheraxanthin

all-trans-violaxanthin

all-trans-neoxanthin

9’-cis-neoxanthin

xanthoxin

ABA-aldehyde

ABAModified from Taylor et al.

(2000) J Exp Bot 51: 1563-74

vp9

vp10

vp8

vp5-DR mutant

Genotype Root length (cm)

Branching Seminal Roots

Root Mass (g)

Shoot Mass (g)

Leaf No.

vp5-DR 5.973 -0.5* 0.1 0.143 0.273 0.0

wt vp5-DR 0.4 0.5 1.6 0.406 0.370 0.1

vp5 0.9 0.1 0.7 0.314 0.031 0.6

wt vp5 0.5 0.2 0.2 0.683 0.023 0.1

Mean difference between well-watered and water-stressed treatments.

* = significant at = 0.5.

vp8 mutant

Genotype Root length (cm)

Branching Seminal Roots

Root Mass (g)

Shoot Mass (g)

Leaf No.

vp8 -1.0 0.8* 0.9 1.460* 2.359* -0.5

wt vp8 0.2 0.6* 0.9 0.960* 0.125 0.0

Mean difference between well-watered and water-stressed treatments.

* = significant at = 0.5.

9-cis-violaxanthin

vp14 (maize)

vp5 (maize)

fluridone (FLU)

phytoene

phytofluene

-carotene

neurosporene

lycopene

-,-carotene

-,-carotene

zeaxanthin

antheraxanthin

all-trans-violaxanthin

all-trans-neoxanthin

9’-cis-neoxanthin

xanthoxin

ABA-aldehyde

ABAModified from Taylor et al.

(2000) J Exp Bot 51: 1563-74

vp9

vp10

vp8

Root transcriptome map

8000 root unigenes based on EST sequencing of clones from ww and ws root segments.

Goal: Use laboratory and computational methods to identify map locations.

Future: Align the map information with relevant mutant and QTL information.

Root transcriptome map

Three strategies: Wet-lab genetic mapping or physical mapping by

BAC pools. (300) E-mapping by identity with previously mapped

probe. (~1700 genes) E-mapping by sequence alignment to complete

BAC or BAC end sequence. (in progress)

Root transcriptome map

Built on IBM neighbors framework

Red are core markers Blue are newly mapped Black are prior mapped Can add kinematic

information

1L

Acknowledgements

Mike Gerau Doug Davis Theresa Musket Hector Sanchez Steve Schroeder Bill Spollen

Ryan Dierking Nicole Grweizowzciak Matt Meyer Dustin Partney Kristen Leach Dana Woodruff