Genomic and Metabolomic Approaches for …imbgl.cropsci.illinois.edu/school/2011/Jay_Boddu.pdfQTL Microarrays and MPSS Kernel culture Illumina platform Deep sequencing mQTL Asparagine

Genomic and Metabolomic Approaches for Improving Nitrogen Use Efficiency (NUE) in Corn

Jay Boddu

Genomic and Metabolomic Approaches for Studying Nitrogen Use Efficiency (NUE) in Corn

PAST & PRESENT

QTL identification • NUE, NutE, NupE, Grain yield, Biomass etc.

Long-term selection • Illinois Protein and Oil lines

Development of functional markers • Expression QTL (eQTL), metabolic QTL (mQTL)s and SNPs

Evaluation of source-sink relationships • N uptake, utilization and remobilization

Identification of pathways involved in N metabolism • Microarrays, MPSS, Kernel culture and Deep sequencing

Effect of the fixed alleles on select phenotypes • Allelic fixation and heterotic vigor

PAST & PRESENT

QTL identification • NUE, NutE, NupE, Grain yield, Biomass etc.

Long-term selection • Illinois Protein and Oil lines

Development of functional markers • Expression QTL (eQTL), metabolic QTL (mQTL)s and SNPs

Evaluation of source-sink relationships • N uptake, utilization and remobilization

Identification of pathways involved in N metabolism • Microarrays, MPSS, Kernel culture and Deep sequencing

Effect of the fixed alleles on select phenotypes • Allelic fixation and heterotic vigor

QTL

Microarrays and MPSS

Kernel culture

Illumina platform Deep sequencing

mQTL

Asparagine Cycling Pathway

eQTL

Asparaginase, Asparagine synthetase and their regulation

Work flow of the NUE project management

Genetics

Genomics

Functional Genomics

Metabolomics

Historical Increases in Maize N Utilization

Year

Gra

in y

ield

(M

g h

a-1

)

1965 1970 1975 1980 1985 1990 1995 2000 2005

4

5

6

7

8

9

10

Grain yield

Fe

rtilize

r N ra

te (k

g h

a-1)100

120

140

160

180

Fertilizer use

Data USDA-NASS, 2006

UNIQUE RESOURCES AT UNIVERSITY OF ILLINOIS

Soybean Maize

IBMRIL X IHP1

Experiment 2006

IHP

ILP

Reverse Selections

cycle 48

IRLP

IRHP

Nitrogen Responsive Nursery Illinois Protein Lines

Nitrogen Use Efficiency (NUE) :

• Grain yield increase per unit fertilizer applied

Nitrogen Utilization Efficiency (NUtE) :

• Ratio of grain yield to the total plant N

Nitrogen Uptake Efficiency (NUpE) :

• Percentage of fertilizer-applied N found in the plant

3. The Illinois protein lines differ in the N utilization efficiency

Genetic material predominantly used in Nitrogenes project

1. IBMRIL population for linkage analyses

2. Nested Association Mapping (NAM) founder lines Tzi8 Tropical Ky21 Temperate

CML333 Tropical MS71 Temperate

CML277 Tropical Oh43 Temperate

Ki11 Tropical Oh7B Temperate

NC358 Tropical M162W Temperate

CML247 Tropical B97 Temperate

CML52 Tropical HP301 Sweet & Pop

CML228 Tropical IL14H Sweet & Pop

Ki3 Tropical P39 Sweet & Pop

NC350 Tropical

CML69 Tropical

CML103 Tropical

CML322 Tropical

Mo18W Mixed

M37W Mixed

Tx303 Mixed

Photoperiod Insensitive

Photoperiod Sensitive

0

1

2

4

5

6

Grain

Stover

R1 R1R6 R6

IHP ILP

0

3

Grain

Stover

g N/plant

681018Total Shank AAs

146Alanine

247Serine

13< 110Arginine

35611Asparagine

62213Aspartate

23%43%36%Glutamine

15

ILP

5

B73

4Glutamate

IHP


146Alanine

247Serine

13< 110Arginine

35611Asparagine

62213Aspartate

23%43%36%Glutamine

15

ILP

5

B73

4Glutamate

IHPA B C

0

5

10

15

20

25

30

35

40

(k

g g

ra

in

/ k

g p

la

nt N

)

FR1064 x IHP1

FR1064 x ILP1

N Utilization Efficiency

0 50 100 150 200 250

Nitrogen Rate (kg/ha)

0

1

2

4

5

6

Grain

Stover

R1 R1R6 R6

IHP ILP

0

3

Grain

Stover

g N/plant


146Alanine

247Serine

13< 110Arginine

35611Asparagine

62213Aspartate

23%43%36%Glutamine

15

ILP

5

B73

4Glutamate

IHP


146Alanine

247Serine

13< 110Arginine

35611Asparagine

62213Aspartate

23%43%36%Glutamine

15

ILP

5

B73

4Glutamate

IHPA B C

0

5

10

15

20

25

30

35

40

(k

g g

ra

in

/ k

g p

la

nt N

)

FR1064 x IHP1

FR1064 x ILP1


0 50 100 150 200 250


Genetic Variation for N Utilization in Maize Hybrids

0

50

100

150

200

250

300

N u

tiliza

tio

n (

g/p

lt)

Grain Stover

0

50

100

150

200

250

300

N u

tiliza

tio

n (

g/p

lt)

Grain Stover Soil N (~75 kg/ha)

US Elite Hybrids

US Elite Hybrids

B73 x Temperate NAM

B73 x Temperate NAM

B73 x Tropical NAM

B73 x Tropical NAM

Soil N + 200 kg/ha (~275 kg/ha)

NUE-related QTL in maize

Coque et al., 2006

Hirel et al., 2001

Gallais and Hirel., 2004

Genetic Utilization (GU), NUE, NUtE and NUpE QTL in maize

Genetic map of IBMRIL population showing QTL detected for genetic utilization (red boxes), nitrogen (N) use efficiency (green boxes), N uptake efficiency (dark blue boxes), and N utilization efficiency (light blue boxes). B = B73 allele increases trait value, M = Mo17 increases trait value. Open boxes are QTL at 0 N, filled boxes are QTL at high N. The markers flanking the interval for each QTL or cluster of QTL are indicated

Grain composition QTL

Genetic map of IBMRIL population showing QTL detected for kernel protein concentration (red boxes), starch concentration (green boxes), and oil concentration (dark blue boxes). B = B73 allele increases trait value, M = Mo17 increases trait value. Open boxes are QTL at 0 N, filled boxes are QTL at high N. The flanking markers for each QTL or cluster of QTL are indicated

Functional genomics to identify genes behind the observed QTL

QTL

Functional genomics to identify genes behind the observed QTL

QTL

Genes

NITROGEN RESPONSIVE TRANSCRIPTOME ANALYSIS USING MICROARRAYS

Question Tissue types Genotypes Stages N treatment

N Uptake Root B73

Seedling No IHP

Remobilization Leaves

B73

V10 & V16 No IHP

ILP

Mo17

Source behavior Leaves

B73

V8 Yes IHP

ILP

IRHP

IRLP

Sink development Seeds

B73

8 DAP & 16 DAP No IHP

ILP

IRHP

IRLP

Sink development Earshoot

B73

Earshoot at anthesis Yes B73 X Mo17

High yielding IBMRIs

Low yielding IBMRIs

Tropical genotypes

Chorismate

Anthranilate

N-(5-Phospho-β-D-ribosyl)-

anthranilate

1-(2-Carboxy-Phenylamino)-

1-deoxy-D-ribulose 5-phosphate

(3-Indolyl)-Glycerol

phosphate

L-Tryptophan

Tryptamine

AS

PAT

PAI

InGPS

TSA

Indole

TSB

TDC

24W 24Fg 48W 48Fg 72W 72Fg 96W 96Fg 144W144Fg

Expression value1 10 102 103 104

Tryptophan decarboxylase

Tryptophan decarboxylase

Anthranilate synthase beta subunit

Phosphoribosyl anthranilate isomerase

Tryptophan synthase-beta chain


Tryptophan synthase-alpha chain

Indole 3-glycerolphosphate synthase

Phosphoribosyl anthranilate transferase

Anthranilate synthase alpha subunit


Indole 3-glycerolphosphate synthase

HY07PO2u_at

Contig8200_at

Contig5883_s_at

Contig11064_at

Contig6407_at

Contig10115_at

Contig5542_at

Contig17478_at

Contig3904_at

HK05P12r_s_at

Contig23667_at

Contig11623_at

0

500

1000

1500

2000

2500

3000

3500

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

24 48 72 96 144

Time points (h)

Ex

pre

ss

ion

va

lue

W

Fg

0.0

100.0

200.0

300.0

400.0

500.0

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

0.0

500.0

1000.0

1500.0

2000.0

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

0.0

500.0

1000.0

1500.0

2000.0

2500.0

24 48 72 96 144

Time points (h)

Ex

pre

ss

ion

va

lue

W

Fg

0.0500.0

1000.0

1500.02000.02500.03000.0

3500.04000.0

24 48 72 96 144

Time points (h)

Exp

ressio

n v

alu

e

W

Fg

Boddu et al., MPMI, 2006

Can we observe something like this ?

IHP 921

ILP 469

IHP 726

IRHP 647

ILP 1114

IRLP 1394

BMO (High N) 1087

B73 (Low N) 725

BMO (Low N) 1146

B73 (Low N) 961

IBMHY (High N) 20

IBMHY (Low N) 17

IBMLY (High N) 52

IBMLY (Low N) 24

ILP 1757

IHP 1554

ILP 309

IRHP 284

ILP 256

IRLP 191

B73 957

IHP 888

B73 1014

ILP 813

B73 954

IHP 698

B73 916

ILP 667

B73 (High N) 1123

IHP (High N) 715

B73 (High N) 1085

IHP (Low N) 854

High N 320

Low N 221

High N 537

Low N 170

B73 667

IHP 506ROOT

IHP vs IRHP

ILP vs IRLP

BMO (High N) vs

B73 (Low N)

BMO (Low N) vs

B73 (Low N)

IBMHY (High N) vs

IBMHY (Low N)

IBMLY (High N) vs

IBMLY (Low N)

Ear shoot

8 DAP Leaf

B73 vs IHP

ILP vs IHP

ILP vs IRHP

16 DAP seeds

Up- regulated inComparison

ILP vs IRLP

IHP vs ILP

Plant organ/Tissue

B73 (High N) vs

IHP(High N)

B73 (High N) vs

IHP(Low N)

B73 (High N) vs

B73(Low N)

IHP (High N) vs

IHP(Low N)

B73 vs IHP

B73 vs ILP

B73 vs IHP

B73 vs ILP

V16 Leaf

V10 Leaf

V8 Leaf

Genotype, Tissue, Development and N treatment specific

differentially accumulating maize transcriptome

Important conclusions from microarray experiments

A baseline dataset was derived Genotype and organ/tissue specific patterns were observed Distinct nitrogen-specific responses were recorded in IBMRILs that differed in their yield response Photosynthesis specific transcriptional machinery was induced by high N condition in juvenile earshoot Amino acid metabolism transcripts (eg. AS, ASNase, GS, AspAT etc.) that participate in asparagine cycling pathway showed prominent N-response Coordinated expression of asparagine synthetase and asparaginase was found in protein lines

The Kernel Culture: A Controlled Environment Approach

1.31-0.632-0.77Grain protein concentration

1.320.510.86Harvest index

1.820.200.38Total plant N concentration

1.330.31-0.10Total biomass

1.840.260.61R1 stover N concentration

1.12-0.14-0.53R1 stover biomass

1.15-0.31-0.24V8 stover N concentration

1.000.030.04V8 stover biomass

0.530.25N utilization efficiency

0.590.04N uptake efficiency

0.320.31N use efficiency

1.07-0.330.19Kernel weight

1.580.900.93Kernel number

1.88Grain yield (GY)

N

Response

GYmax

correlation

GY0

correlationTrait

1.31-0.632-0.77Grain protein concentration

1.320.510.86Harvest index

1.820.200.38Total plant N concentration

1.330.31-0.10Total biomass

1.840.260.61R1 stover N concentration

1.12-0.14-0.53R1 stover biomass

1.15-0.31-0.24V8 stover N concentration

1.000.030.04V8 stover biomass

0.530.25N utilization efficiency

0.590.04N uptake efficiency

0.320.31N use efficiency

1.07-0.330.19Kernel weight

1.580.900.93Kernel number

1.88Grain yield (GY)

N

Response

GYmax

correlation

GY0

correlationTrait

unpollinated

ovules

pollinated but

aborted kernels

Kernel abortion Kernel Culture

Amino Acid Metabolism in Maize Earshoots. Implications for Assimilate Preconditioning and Nitrogen Signaling

Seebauer et al., 2004. Plant Physiol.

Agilent 1200 series rapid resolution HPLC system Illumina Genome Analyser

Transcriptomics Metabolomics

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0 84 168 252 336

Supplemental N Rate (kg/ha)A

sn

:Gln

Rati

o

B73 x IHP1

B73 x ILP1

B73 x Mo17

10

15

20

25

30

0 84 168 252 336

Supplemental N Rate (kg/ha)

To

tal A

min

o A

cid

s (

mg

/g d

ry w

eig

ht)

B73 x IHP1

B73 x ILP1

B73 x Mo17

A B

Genotype specific free amino acid concentrations

• High N utilization is associated with low leaf Asn.

• Greater Asn accumulation in IHP hybrid is correlated with high grain protein and low N utilization in IHP.

N Utilization Differences in B73 x IPS Hybrids

0

5

10

15

20

25

30

35

40

45

0 50 100 150 200 250

N Rate (kg/ha)

Mg

grain/k

g plant

N


B73 x IHP1

B73 x ILP1

B73 x Mo17


0

2

4

6

8

0 84 168 252 336

Leaf [Asparagine]

B73 x Mo17

B73 x IHP1

B73 x ILP1

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

0 84 168 252

Supplemental N (kg/ha)

Gra

in Y

ield

(M

g/h

a)

B73 x MS71

B73 x Oh7B

B73 x Mo17

Mean 25 hybrids

Grain yield responses to N of 25 Diversity hybrids

Genotypes selected for kernel culture experiment

B73 X MS71: Lowest N response B73 X Mo17: Medium N response B73 X Oh7B: Highest N response B73 X IHP1 B73 X ILP1

Physiological extremes for N response

Genotype dependent grain filling in kernel culture as inferred by kernel weights

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

B73 X IHP1

B73 X ILP1

B73 X Mo17

B73 X MS71

B73 X Oh7B

B73 X IHP1

B73 X ILP1

B73 X Mo17

B73 X MS71

B73 X Oh7B

B73 X IHP1

B73 X ILP1

B73 X Mo17

B73 X MS71

B73 X Oh7B

B73 X IHP1

B73 X ILP1

B73 X Mo17

B73 X MS71

B73 X Oh7B

HIGH LOW HIGH LOW

HIGH LOW

2 DIC

5 DIC

PM

Culture N

Field N

High Low High Low

High Low

Nsufficient

Ndeficient

Nrecovery

N signalcarryover

B73 X Oh7B showed highest N-recovery from 2 DIC to 5 DIC Indicating its high N-utilization efficiency

DIC: Days In Culture

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

0 N 25 N 0 N 25 N 0 N 25 N 0 N 25 N

High Low High Low

Cob Kernel

Low High Low High Low High Low HighHigh Low High Low

Cob Kernel

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

B73 X

IHP1

B73 X

ILP1

B73 X

Mo1

7

B73 X

MS7

1

B73

X O

h7B

B73 X

IHP1

B73 X

ILP1

B73 X

Mo1

7

B73 X

MS7

1

B73

X O

h7B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73

X O

h7B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73

X O

h7B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73

X O

h7B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73 X

Oh7

B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73 X

Oh7

B

B73 X

IHP1

B73

X IL

P1

B73 X

Mo1

7

B73 X

MS7

1

B73 X

Oh7

B

0 N 25 N 0 N 25 N 0 N 25 N 0 N 25 N

High Low High Low

Cob Kernel

Asparagine

Glutamine


Cob Kernel

PM

Culture N

Field N

Tissue

0

5000

10000

15000

20000

25000

30000

35000

40000

B73

X IL

P1

B73

x O

h7B

B73

X IH

P1

B73

X M

S71

B73

X M

o17

B73

X IL

P1

B73

x O

h7B

B73

X IH

P1

B73

X M

S71

B73

X M

o17

B73

X IL

P1

B73

x O

h7B

B73

X IH

P1

B73

X M

o17

B73

X M

S71

B73

X IL

P1

B73

x O

h7B

B73

X IH

P1

B73

X M

o17

B73

X M

S71

Low N High N Low N High N

High N Low N

Kernel

2 DIC

N sufficient

N deficient

N recovery

N signal carryover

Genotype and N-treatment specific asparagine accumulation in cultured kernels at 2 days in culture

Culture N

Field N

0

5000

10000

15000

20000

25000

30000

B73

x O

h7B

B73

X IL

P1

B73

X IH

P1

B73

X M

o17

B73

X M

S71

B73

x O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

S71

B73

X M

o17

B73

x O

h7B

B73

X IL

P1

B73

X M

o17

B73

X IH

P1

B73

X M

S71

B73

x O

h7B

B73

X M

o17

B73

X IH

P1

B73

X IL

P1

B73

X M

S71


High N Low N

Kernel

5 DIC

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

0 N 25 N 0 N 25 N 0 N 25 N 0 N 25 N

High Low High Low

Cob Kernel


Cob Kernel

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

B73

X IH

P1

B73

X IL

P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

0 N 25 N 0 N 25 N 0 N 25 N 0 N 25 N

High Low High Low

Cob Kernel

Asparagine

Glutamine


Cob Kernel

PM

Culture N

Field N

TissueN

sufficient N

deficient N

recovery N signal carryover

Genotype and N-treatment specific asparagine accumulation in cultured kernels at 5 days in culture

Culture N

Field N

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o17

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Cob Kernel


Cob Kernel

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Mo1

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MS7

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P1

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P1

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Mo1

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MS7

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Mo1

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MS7

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o17

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MS7

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o17

B73 X

MS7

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B73

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B73

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P1

B73

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P1

B73

X M

o17

B73

X M

S71

B73

X O

h7B

0 N 25 N 0 N 25 N 0 N 25 N 0 N 25 N

High Low High Low

Cob Kernel

Asparagine

Glutamine


Cob Kernel

PM

Culture N

Field N

TissueN

sufficient N

deficient N

recovery N signal carryover

Genotype and N-treatment specific asparagine accumulation in cultured kernels at physiological maturity

Culture N

Field N

In majority of the tested parameters such as N-deficiency, N-sufficiency and N-recovery, the cultured kernels of B73 X Oh7B hybrid showed lowest asparagine levels in agreement with the previously observed fact that the asparagine levels negatively correlates with N-utilization

Diverse genotypes to test correlations between select agronomic traits with immature earshoot free amino acids

DIVERSITY

PHOTOPERIOD

SENSITIVE

DIVERSITY

PHOTOPERIOD

INSENSITIVE

EXPIRED PATENT

VARETY PROTECTION

LINES

OLD INBRED

LINES

CML69 B97 LH1 IHP

CML228 CML103 LH123 ILP

CML247 HP301 LH82 D1

CML277 IL14H Mo17 D3

CML322 Ky21 PH207 D2

CML333 Ms71 PHG35 D9

Ki3 Oh43 PHG39 D4

Ki11 Oh7B PHG47 FR1064

M37W P39 PHG84 D5

Mo18W PHJ40 M162W

Tx303 PHZ51 Va85

Tzi8

These inbreds were crossed to IHP and ILP and were grown at High and Low N conditions

Hybrids InbredsAlanine Arginine Asparagine

Aspartic

acid

Glutamic

acidGlutamine Lysine Threonine Alanine Arginine Asparagine

Aspartic

acid

Glutamic

acidGlutamine Lysine Threonine

Grain Weight 0.00 0.00 -0.13 0.02 0.01 -0.06 0.00 -0.08 Grain Weight -0.50 -0.39 -0.32 0.01 -0.07 -0.32 -0.13 -0.50

Cob Weight -0.15 -0.09 -0.19 -0.02 -0.11 -0.04 -0.05 -0.22 Cob Weight -0.32 -0.10 -0.34 0.04 0.09 -0.31 -0.38 -0.28

Stover Biomass -0.13 -0.01 -0.05 -0.15 -0.10 -0.14 -0.14 -0.07 Stover Biomass 0.31 0.22 0.17 0.12 0.28 -0.13 -0.31 0.51

Total N -0.13 -0.07 -0.12 -0.18 -0.09 -0.21 -0.17 -0.14 Total N 0.26 0.30 0.13 0.12 0.30 0.00 -0.16 0.49

Grain Moisture 0.01 -0.20 -0.22 -0.19 -0.10 -0.18 -0.08 -0.21 Grain Moisture 0.08 -0.04 -0.02 -0.23 -0.09 -0.06 0.12 -0.17

Starch 0.03 -0.21 -0.31 -0.18 -0.01 -0.29 -0.10 -0.30 Starch -0.45 -0.55 -0.28 0.03 -0.16 -0.45 -0.05 -0.44

Oil -0.04 0.06 0.18 0.30 0.18 0.30 0.27 0.15 Oil -0.01 0.01 0.05 0.15 0.00 0.25 0.15 0.20

Protein -0.03 0.20 0.31 0.16 0.00 0.25 0.07 0.30 Protein 0.44 0.58 0.26 -0.03 0.20 0.34 -0.10 0.46

Hybrids InbredsAlanine Arginine Asparagine

Aspartic

acid

Glutamic

acidGlutamine Lysine Threonine Alanine Arginine Asparagine

Aspartic

acid

Glutamic

acidGlutamine Lysine Threonine

Grain Weight 0.30 0.03 0.01 0.27 0.41 -0.05 0.11 0.42 Grain Weight 0.33 0.34 0.38 -0.23 -0.43 0.33 0.42 -0.21

Cob Weight 0.06 -0.01 0.02 0.02 0.07 -0.24 -0.11 0.06 Cob Weight 0.02 -0.01 -0.04 0.05 -0.15 0.05 0.03 -0.07

Stover Biomass 0.00 0.06 -0.03 -0.12 -0.17 0.02 -0.06 -0.15 Stover Biomass -0.17 -0.27 -0.49 0.66 0.65 -0.16 -0.57 0.21

Total N -0.02 0.07 -0.08 -0.14 -0.10 -0.09 -0.05 -0.12 Total N -0.15 -0.25 -0.35 0.48 0.46 -0.19 -0.44 0.22

Grain Moisture 0.13 -0.15 -0.22 -0.01 0.15 -0.09 0.19 0.19 Grain Moisture -0.09 0.33 0.11 0.01 -0.02 0.23 0.22 0.05

Starch 0.16 -0.03 -0.14 0.08 0.28 -0.23 0.14 0.17 Starch 0.15 0.46 0.28 -0.39 -0.52 0.26 0.50 -0.38

Oil -0.15 0.17 0.08 -0.09 -0.17 0.17 -0.25 -0.19 Oil 0.04 -0.05 0.12 -0.12 -0.25 -0.24 0.12 0.09

Protein -0.11 0.08 0.15 -0.02 -0.20 0.21 -0.10 -0.12 Protein -0.18 -0.53 -0.38 0.47 0.65 -0.25 -0.61 0.43

High N

Low N

-1 to -.4

-.39 to -.2

.2 to .39

.4 to 1

Hybridization of diverse genotypes with IHP/ILP alleles decreased the predictive ability of earshoot free amino acids For some amino acids especially Asn and Gln the correlations reversed with N treatment

Correlations between NUE related traits with earshoot free amino acids in diverse genoypes

trait starch oil protein biomass yield HI Knum stovN NHI

ala 0.16 -0.03 -0.15 -0.18 0.09 0.2 0.09 -0.25 0.24

arg 0.03 -0.13 0 -0.21 -0.16 0.07 -0.11 -0.28 0.19

Asparagine 0.13 -0.02 -0.12 -0.29 -0.06 0.19 -0.04 -0.33 0.24asp -0.15 0 0.16 0.04 -0.28 -0.19 -0.21 -0.12 0.04

glu -0.07 0.1 0.06 -0.06 -0.2* -0.06 -0.13 -0.23 0.15

Glutamine 0.08 -0.05 -0.07 -0.21 -0.11 0.1 -0.05 -0.27 0.18

Correlations between NUE related traits with earshoot free amino acids in IBMRIL population

High N

Low N

Trait Starch Protein RS Biomass Yield HI Knum StovN NHI

ala 0.26 -0.26 0.22 -0.3 0.25 0.29 0.21 -0.22 0.23

arg 0.27 -0.26 0.27 -0.41 0.3 0.37 0.3 -0.31 0.29

Asparagine 0.3 -0.32 0.34 -0.46 0.37 0.44 0.32 -0.35 0.36asp -0.1 0.08 0.04 -0.04 -0.05 -0.02 0.04 0.12 -0.11

glu -0.05 0.02 0.05 -0.09 -0.01 0.03 0.05 0.15 -0.12

Glutamine 0.14 -0.15 0.16 -0.33 0.16 0.25 0.16 -0.2 0.18

Chr. 1 2 3 4 5 6 7 8 9 10

Metabolite QTL (mQTL) identification using earshoot asparagine and glutamine concentrations

Asn

Gln

Aspartate + Glutamine Asparagine + Glutamate

NO3-

Glutaminesynthetase 2

(GS2)

Nitratereductase

(NR)

Asparaginesynthetase

(AS)

Asparaginase(ASNase)

ATP AMP + PPi

NH4+

Nitritereductase

(NiR)

Aspartateaminotransferase

(AspAT)

S1-ClassbZIP

The Asparagine Cycling Pathway

Genes selected for expression QTL (eQTL) analysis

Gene Forward Primer Sequence Reverse Primer Sequence Clone Bin

Tubulin Beta 7 AGCAGATGTGGGACTCCAAGAACAT GTTCTTGTTCTGCACGTTCAGCATT NA NA

Glutamine Synthetase 2 CGCCCTACACCGACAAGGTCAT GCTTCTCCTGTGCTAGATCCATCGTA NA 10.07

Nitrite Reductase 2 CCCTACACCAACCTTCTCTCCTCCTA GCATGTACGCGAGGTCGTTGAT AC183914.2 4.07

Nitrite Reductase 6 GGCCATCCATTCTTGAGCGTGTA GATGAAGCTTGTGACAAAGGCGAA AC193463.3 6.02

Malate Dehydrogenase CTTCTGCACCAGGTACGACCTC TCTTGTCCGAAAACCTCACCA AC206527.1 1.08

Aspartate Aminotransferase TTGGAGGAGTTAACCAGGCGAGAA AGGCCTCAGAAACCCCAAGAATT AC196774.3 5.08

Asparagine Synthetase 2 CGGAGCCCTCCCCGGACAGCACCATCTTCACG GTCGTCGGCCTCGAGGACGGCATCGACGCGAT AC208436.1 1.03

Asparaginase CCCGCCATCTAAACACAAATCAAA CAATCTCCATGCAATCGCTACTACTAGTG AC206980.1 2.02

PEP Carboxylase CCTTGAGCCACTTGAGCTGTGCTA GGATGTCCAGCTTCACCAGGGA NA 9.03

Expression QTL (eQTL) identification for selected genes using V8 leaf of IBMRI population

Gene Chromosome Position (cM) Flanking Markers cis/trans LOD R2 Additive Effect

GS2 6 106.6-127.6 rz242a-psb108 trans 6.78 0.25 0.49

8 258.2-265.3 php20714-umc1460 trans 3.75 0.11 0.26

10 120.6-130.7 umc130-lim2 trans 3.29 0.10 -0.23

NiR2 6 108.6-132.4 rz242a-umc1595 trans 3.95 0.13 0.19

6 270.4-288.5 mmp62-umc1020 trans 5.04 0.17 0.22

NiR6 1 573.5-585.7 csu374b-bnlg1556 trans 2.89 0.09 -0.57

3 304.8-310.6 umc26a-csu636 trans 2.77 0.08 0.56

5 112.2-125.6 rz630f-psr922a trans 3.57 0.1 0.6

7 285.1-315.4 npi394-umc56 trans 10.8 0.26 -1.28

MDH 2 266.7-273.2 bnlg108-umc1861 trans 3.02 0.1 0.45

4 325.4-332.5 umc1945-mmp97 trans 2.67 0.08 0.39

6 246.6-256.4 umc1352-umc2141 trans 25.87 0.55 3.24

AspAT 5 580.9-586.7 umc1225-umc104b cis 2.69 0.09 -0.21

6 94.6-119.4 mmp10-mmp51 trans 6.89 0.22 0.3

10 115.9-123.8 isu085b-npi105a trans 4.17 0.14 0.27

10 193.2-196.7 umc64a-umc1246 trans 2.56 0.08 -0.2

AS2 5 5.1-15.3 csu1087-umc1445 trans 3.54 0.13 -0.33

6 107.0-129.2 rz242a-psb108 trans 4.99 0.16 0.37

8 95.9-104.8 umc1483-bnlg1194 trans 2.98 0.12 0.31

8 218.5-225.1 mmp195f-umc1415 trans 3.88 0.11 -0.33

10 170.9-173.9 umc1239-umc155 trans 2.91 0.08 0.25

Asnase 2 43.1-71.3 umc1165-umc1265 cis 19.94 0.56 1.26

2 189.0-203.1 umc1776-mmp42 trans 3.69 0.07 0.51

5 117.3-137.1 rz630f-mmp130 trans 3.62 0.06 -0.43

5 407.5-412.6 mmp104-mmc0481 trans 2.54 0.05 -0.42

9 262.4-284.4 bnlg1209-npi580a trans 3.48 0.06 -0.43

PEPC 1 54.3-75.8 cdo1081b-bnlg1014 trans 3.86 0.12 -0.17

2 270.6-275.1 umc1259-umc2088 trans 2.79 0.09 0.14

6 139.1-169.6 csu923-npi223a trans 7.92 0.29 0.26

0

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Leaf developmental stage

bZIP

ILP IHP B73

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ILP IHP B73

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ILP IHP B73

V10 V12 V14Leaf developmental stage

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ASNase

Cycle 65

Cycle 105

Cycle 65

Cycle 105

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ASNase

IHP ILP

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ASNase

ZmbZIP1

Cycle 65 Cycle 105

IHP ILP IHO IHP ILP IHOR

ela

tive

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B.

Coordinated expression of Asparaginase and Asparagine synthetase genes

0

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bZIP

ILP IHP B73

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ILP IHP B73

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ILP IHP B73

V10 V12 V14Leaf developmental stage

Re

lati

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lati

ve

ex

pre

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lati

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ex

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ZmbZIP1

ZmASN1

ASNase

Cycle 65

Cycle 105

Cycle 65

Cycle 105

AS

ASNase

IHP ILP

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ZmASN1

ASNase

ZmbZIP1

Cycle 65 Cycle 105

IHP ILP IHO IHP ILP IHOR

ela

tive

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tive

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tive

exp

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B.

Coordinated expression of Asparaginase and Asparagine synthetase genes

QTL controlling RNA expression of Asn pathway cycling genes, free amino acids, and agronomic N utilization (NutE) in the IBM population

B.

AspAT(mRNA)

ZmbZ

IP1

ctg270

AC205337 AC191608AC186399 AC205882

ctg271

Bin 6.02

A.

ctg69

AC204017 AC186420

Bin 2.02

What next…

Find gene groups using the improved coverage obtained from analysis of kernel culture mRNAseq data Improve confidence on the identified gene groups by comparing overlaps between mRNAseq data and pre-existing profiling data Obtain genomic locations of differentially accumulating transcrips and connect the dots between the NUE QTL and the genes Design reciprocal crosses using diverse genotypes with IHP, ILP B73 and Mo17 to improve and reaffirm the predictability of NUE using earshoot free amino acid profiles

AND

Try to improve N U E…

… as much as WE CAN

Christine

Dominique

Evan Han

Michael

Farag Wei Ravikarn

Yuhe

Jay

Abe Wess

Jeff Church Devin Nichols

Fred Below Collaborator

& His Lab

Ed Buckler Collaborator

& His Lab

The Stephen Moose Corn Functional

Genomics Lab

at Illinois

Matt Hudson Collaborator

& His Lab

Documents

Genomic and Metabolomic Approaches for …imbgl.cropsci.illinois.edu/school/2011/Jay_Boddu.pdfQTL Microarrays and MPSS Kernel culture Illumina platform Deep sequencing mQTL Asparagine