High fructan wheat grains:Genetic basis and approaches

Xinguo Li, PhD. CSIRO Agriculture Flagship. Ph. 02- 6246 4848; Xinguo.li@csiro.au

Outlines

Background

Development of high throughput fructan assays

Fructan variation in wheat MAGIC populations

QTLs affecting fructan content

Future perspective

• Stimulate bifidobacteria in colon, thus reduce colonic disorders (constipation, hemorrhoid) and colonic cancer

• Reduce diabetes and anaemia

• Increase nutrition absorption (calcium, iron) and strengthen bones

Fructan: a dietary fibre with health benefits

• Cell wall components (cellulose, hemicellulose, lignin)

• Arabinoxylan (AX)

• β-glucan

• Fructans: water-soluble, fructose polymers

• Resistant starch

Dietary Fibre (DF): non-digestible carbohydrates in plant food, inc. water-soluble (SF) and insoluble fibres (IF).

Glucose

fructose fructose

n: degree of polymers (DP)

DF in cereals

Potential benefits of fructans

Chicory 42%

Jerusalem artichoke 18%

Dandelion greens 14%

Garlic 13%

Leek 7%

Globe artichoke 5%

Onion 4%

Asparagus 2.5%

Wheat 2.5%

Barley 0.8%

Rye 0.7%

Banana 0.5%

Fructan content (Van Loo et al, 1995)

Fructans in flowering plants

• The 2nd richest reserve carbohydrates (next to starch) • Produced in ~15% flowering plants

Haska et al. J Cereal Sci, 2008, 48:768-774

1.4 -1.7%(white flour)

~ 0%

3.4 - 4.0%

Plant species

Fructan consumption

• Wheat is the major resource for fructan consumption, because it is a common staple food

Wheat 70%

Onion 25%

Banana, garlic and others 5%

An increase of fructan % in wheat grains (wholemeal) will significantly enhance fructan consumption.

• Fructan consumption of typical Americans from diets

3.5 g/day (Jessica Bruso, May 2015)

• Suggested daily fructan consumption for beneficial health effects

> 15 g/day (In “Diabetes Spectrum”, Feb 2013)

Total fructan intake in American diets (Moshfegh et al. 1999)

Traditional fructan assay: HPLC method

• Acid hydrolysis (hydrochloric acid, HCl) (Liu et al, 2011)

– Split extracted solution in two: acid treated vs untreated

a, Untreated: measure of water soluble sugars

b, Acid hydrolysis (fructan to glucose and fructose)

– Run both on HPLC– Then, calculate fructan content (b-a)

• Enzymatic hydrolysis– Split extracted solution into two: enzyme treated vs untreated

a, Crystalline yeast invertase (Pontis, 1966)

b, Amyloglucosidase, fructanase (Quemener et al, 1994; AOAC 997.08, 2000)

c, Amyloglucosidase, α-galactosidase, fructanase (Huynh et al, 2008)

– Run both on HPLC – Then, calculate fructan content

Fructan extraction: hot water at 80°C.Extracted solution: fructans and all other water soluble sugars

Untreated

After hydrolysis

Amyloglucosidase Galactosidase

Amyloglucosidase GalactosidaseFructanase

(Liu et al, 2011)

(Huynh et al, 2008)

HPLC

Use beaker, flask or large tubes. Require HPLC. Low throughput (~10 samples/day)

K-FRUCHK K-FRUCSpectrophotometer

Megazyme fructan kits: Spectrophotometer

Use beaker, flask or large tubes, enzyme hydrolysis and spectrophotometer; low throughput (~10 samples/day)

starch, sucrose, maltose, glucose, fructose starch, glucose, frucoseSucrase, maltase

Fructan Glucose, fructoseFructanase

Measure absorbance by Spectrophotometer

K-FRUCHK: measure absorbance

starch, sucrose, maltose, glucose, fructose glucose, fructosePullulanase, amylase, sucrase, maltase

Sodium borohydride Sorbitol, mannitol

K-FRUC: remove background

Measure absorbance

No absorbance

• Treat non-targeted sugars (background)

• Measure fructans

Flours with hot water extraction: fructans; non-targeted sugars (starch, sucrose, maltose, glucose, fructose)

Development of high throughput fructan assay Scale down the Megazyme fructan kits into plate format

Weighing flours(100-150 samples/day) Water extraction at 80 °C with shaking

(200~400 samples/day)

Hydrolysis at 40 °C with shaking(up to 90 samples/day)

Deep well plates for hydrolysis

PAHBAH colouring in 98 °C water bath Plate reader

(Microplate spectrophotometer)

SDSII Vacuum-Pump PipetteBioshake iQ

Thermo Multiskan Spectrum plate reader

Labelling and randomised design

Custom-made deep well plate clips

Recovery rate and consistency

y = 0.9439x - 0.0806R² = 0.9989

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Fru

ctan

re

cove

red

(m

g)

Pure fructan from control flour (mg)

Modified K-FRUCHKRecovery rate: 89.5%

y = 0.9551x + 0.0008R² = 0.9992

0

1

2

3

4

5

0 1 2 3 4 5

Fru

cta

n r

eco

vere

d (

mg)

Pure fructan from control flour (mg)

Modified K-FRUCRecovery rate: 95.0%

0.0

0.5

1.0

1.5

2.0

2.5

Fruc

tan

%

Modified K-FRUC

Rep1 Rep2 Rep3Rep4 Rep5 Rep6

0.0

0.5

1.0

1.5

2.0

1 5 9 13 17 21 25 29 33 37 41 45

Wheat lines

Rep1 Rep2 Rep3 Rep4

Fruc

tan

%

Modified K-FRUCHK

Comparison of the 2 high throughput fructan assays

Modified assays

Background

treatment

Background

absorbance

Absorbance after fructan

hydrolysisConsistency

between replicatesRecovery rate

(accuracy)

K-FRUCHK Measure 0.7-1.0 1.0-1.3 Lower (26% of CV) 89.5%

K-FRUC Remove ~0.06 0.2-0.5 Higher (~12% of CV) 95.0%

The modified K-FRUC assay is more suitable for fructan assay of wheat flours (containing high level of starch).

×

How high is the throughput?

• 45 samples per day (6 hrs), once wheat flours weighed, all buffers and reagents prepared

• Or 90 samples per day (7-8 hrs), once fructans of wheat flours extracted, all buffers and reagents prepared

Wheat MAGIC populationsMAGIC: Multiparent advanced generation intercross

Cavanagh et al, Curr Opin in Plant Biol, 2008, 11:215-221

AC BarrieAlsen

PastorVolcani

Xiaoyan54

WestoniaYipti

Baxter

8-way

Huang et al. Plant Biotechnology J, 2012, 10: 826-839

4-way

WestoniaYipti

Baxter

Chara

Screening wheat MAGIC populations

Population Founders Lines Lines + replicates

Yanco 2009 4-way 498768 (white flour)

1128 (wholemeal)

Narrabri 2010 4-way 727 1080 (wholemeal)

Yanco 2012 8-way 756 1260 (wholemeal)

Croppa Creek 2012 8-way 1080 1260 (wholemeal)

Yanco

Narrabri

Croppa Creek

Total: 5496 samples

Cyclone mill Brabender mill

Wholemeal White flour

Fructan variation in MAGIC population

Fruc%: 0.11-1.49

Mean: 0.77

Lines: 498

%

Yanco2009_White flour

Fruc%: 0.25~1.82

Mean:1.03

Lines: 498

fructan %

Yanco2009_Wholemeal Narrabri2010_Wholemeal

Fruc%: 0.63~2.66

Mean:1.40

Lines: 727

05

10152025303540

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Perc

ent o

f tot

al

Fructan %

Croppa Creek2012_Wholemeal

Fruc%:

0.56~2.13

Mean:1.34

Lines: 1080

05

10152025303540

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Perc

ent o

f tot

al

Fructan %

Fruc%:

0.77~2.25

Mean:1.35

Lines: 756

Yanco2012_Wholemeal

4-way

8-way

Heritability

0.634

0.8090.733

0.818 0.837

0.0

0.2

0.4

0.6

0.8

1.0He

ritab

ility

MAGIC Populations

Yanco2009-white flour Yanco2009-wholemealNarrabri2010-wholemeal Yanco2012-wholemealCroppa creek2012-wholemeal

Selection of high fructan MAGIC lines

0.0

0.4

0.8

1.2

1.6

2.0 Yanco 2009 (4-way)

Fruc

tan

%

1 3 5 7 9 11 13 15Chara

West.

..0.0

0.8

1.6

2.4 Narrabri 2010 (4-way)

Fruc

tan

%

1 3 5 7 9 11 13 15

Pastor

Yitpi

XiaoyanAlse

n0.00.40.81.21.62.02.4 Yanco 2012 (8-way)

Fruc

tan

%

1 3 5 7 9 11 13 15Pasto

r

AC Barrie

Yitpi

Westo

nia0.00.40.81.21.62.02.4 Croppa Creek 2012 (8-way)

Fruc

tan

%

QTLs affecting fructan content in wholemeal

Population Chr Marker % var PFounder effect (Fructan %)

Chara Baxter Westonia Yitpi ACBarrie Alsen Pastor Volcani Xiaoyan

Yanco 2009 (4-way)

7A 4M1 11.2 0 0.031 0.061 -0.037 -0.066

2D 4M2 16.4 0 0.073 -0.05 0.037 -0.076

2D 4M3 3.9 0 -0.043 0.043 -0.009 0.004

2B 4M4 4.1 0 -0.033 0.013 0.041 -0.026

2A 4M5 3.5 0 -0.031 -0.014 -0.002 0.044

Narrabri 2010 (4-way)

7A 4M1 9.9 0 0.031 0.061 -0.037 -0.066

2D 4M2 8.1 0 0.045 -0.005 0.025 -0.071

2D 4M3 3.4 0 -0.043 0.043 -0.009 0.004

2B 4M4 3.6 0 -0.033 0.013 0.041 -0.026

Yanco 2012 (8-way)

7A 8M1 15.5 0 0.108 -0.076 -0.09 0.09 -0.039 0.091 -0.035 -0.012

7D 8M2 6.6 0 0.071 -0.03 -0.023 -0.004 -0.055 0.055 -0.053 0.061

4A 8M3 7.4 0 -0.061 0.047 0.053 -0.043 -0.006 -0.003 0.087 -0.055

Croppa Creek 2012 (8-way)

7A 8M1 17.8 0 0.099 -0.055 -0.115 0.096 -0.056 0.146 -0.072 -0.068

7D 8M2 5.9 0 0.079 -0.019 -0.026 -0.036 -0.07 0.061 -0.057 0.056

4A 8M3 4.5 0 -0.074 0.027 0.029 -0.052 -0.006 -0.012 0.084 -0.004

Future perspective: manipulating fructan pathway

Reducing fructan degradation by gene editing or RNAi of 6&1-FEH and 6-FEH

Cimini et al, 2015. Frontiers in Plant Science

Fructan biosynthesis genes (6-SFT, 1-FFT, 1-SST, 6G-FFT)

Fructan degradation genes (1-FEH, 6-FEH, 6&1-FEH)

6&1-FEH1-FEH: break down beta-2,1 frucosyl linkage

6-FEH: break down beta-6,1 frucosyl linkage

1&6-FEH: break down beta-2,1 and -6,1 frucosyl linkage

6-FEH1-FEH

Grain filling Grain maturation & desiccation

Cell division & expansion

Project leader: Steve Jobling

Colin Cavanagh

Klara Verbyla

Zhongyi Li

Andrew Pedlar

Peggy Kooij-Liu

Ray Yuan

Acknowledgements

Jeff Rosichan

Terry Walsh

Thank you!