High fructan wheat grains:Genetic basis and approaches
Xinguo Li, PhD. CSIRO Agriculture Flagship. Ph. 02- 6246 4848; [email protected]
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!