Institute of Crop Science/National Wheat Improvement Centre, CAAS

Email: zhangyongzhy@263.net

Bio-fortification for high micronutrients

in wheat breeding program in China

Yong Zhang and Zhonghu He

The 11th International Gluten Workshop. 12-15, 2012, Beijing

Outline

General information

Cultivar screening for high micronutrients

Fe/Zn consistency over locations

Nitrogen application on Fe/Zn transportation

Effect of milling on micronutrient contents

Fe bioavailability of wheat samples

Basic information for wheat in China

Largest producer and consumer in

the world

Main food for 50% of the

population, especially for northern

China’s less developed area

Area: 24 million ha; Yield: 4.8 t/ha

Production: 115 million tons in 2012,

with 78% for food

General information

Challenges for wheat production

Yield improvement, with wide adaptability

Resistance to bio- and abiotic stresses

Processing and nutrition qualities

Basic information for malnutrition HarvestPlus (IFPRI): Fe, Zn, Vitamin deficiency throughout

the world, with population over 2 billions, in undeveloped area

HEALTHGRAIN (EU): Phytochemicals, especially phenolics

for chronic diseases and metabolic syndrome, cardiovascular

incidence in urban China is 18%, doubled in the last 10 years

Immense economic and social costs, biofortification is a best way to solve the problem

Objectives of HarvestPlus China what program

To select high Fe/Zn varieties with good agronomic performance and broad adaptation - acceptable to farmers

To improve nutrient malnutrition, prevent or reduce Fe/Zn deficiency and relative diseases - beneficial to poor consumers

Strategy

Variety testing and screening, then breeding

Combination of various disciplines, i.e.,

breeding, plant nutrition, and human

nutrition

Link with HarvestPlus wheat

program through Dr. Ivan

Monasterio from CIMMYT

HarvestPlus Wheat Annual Meeting

Collaborative institutes

CAAS: germplasm screening, multi-location field

trial, effect of milling and processing on nutrients

CAU: Fe and Zn analysis, and their transportation

efficacy from root to shoot, and to grain

Sichuan University: bio-availability analysis

Eight lines with high Fe/Zn in Chinese wheat

In the 8 high Fe genotypes, 5 had high Zn

Euphytica, 2010, 174:303-313

Genotype Fe Genotype Zn

Jimai 26 60.2 Henong 326 58.2

Henong 326 58.1 Jimai 26 51.9

Han 3475 52.7 Nongda 3197 47.8

Jingdong 8 51.8 Jingdong 8 46.1

Zhoumai 3 49.4 Zhongmai 175 45.8

Jimai 3 49.4 Nongda 179 45.6

Nongda 3197 47.1 Lumai 23 45.5

Nongda 179 46.4 Weimai 8 42.4

265 genotypes were sown at Anyang in Henan province in two seasons

Cultivar screening

Cultivar screening for high micronutrients

Three cultivars Henong 326, Jimai 26 and Jingdong 8 have

high Fe/Zn, among which Jingdong 8 is the leading cultivar

as check in Northern China Plain before 2010

There is significant and positively correlations among Fe and

Zn, it is possible to combine high Fe/Zn in wheat breeding

Zhongmai 175 with high yield potential, released in Northern

Plain and Northern Yellow and Huai Valleys Drought and

Fertile Region, has high Zn, extend 0.2 million Ha in 2012

Six lines with high free and bound phenolic acids

Season

Cultivar

Free phenolic (mg/kg)

Cultivar

Bound phenolic (mg/kg)

2008-2009 Jishi 02-1 45 Kaimai 18 756

Taishan 23 39 Linyou 145 725

Xiaoyan 6 21 Zhongmai 895 684

Liangxing 66 20 Shaanyou 225 683

Xinong 979 20 Liangxing 66 674

Zhongmai 895 19 Zhangmai 349 666

2009-2010 Jimai 19 25 Kaimai 18 951

Liangxing 66 25 Jimai 19 923

Shaanyou 225 23 Xiaoyan 6 844

Zhongmai 895 19 Zhongmai 155 815

Zhongyu 5 16 Zhongmai 895 783

Zhongmai 875 16 Liangxing 66 777

Liangxing 66 and Zhongmai 895 perform high and more consistent for

both free and bound phenolic acids

Journal of Cereal Science, 2012, in press

Consistency of Fe/Zn over locations and years

Source df Fe Zn P TKW Protein

Genotype (G) 23 9.7 2.8 7.7 50.6 37.1

Environment (E) 13 66.0 89.5 75.8 28.8 33.6

G×E 299 24.3 7.7 16.5 20.6 29.3

E/G 6.8 32.0 9.8 0.6 0.9

Euphytica, 2010, 174:303-313

Fe/Zn consistency

Fe of Zhongyou 9507 at 7 locations over 2 seasons

Zn of Zhongyou 9507 at 7 locations over 2 seasons

Cultivar performance over locations

Significant effects of genotype, environment, and G × E on Fe/Zn were observed

Significant variation for Fe/Zn in the same genotype was observed in 7 locations over years, however, the variation of Fe is much lower than Zn

QTL with Xwmc312 on chromosome 1A QTL with Xgwm251 on chromosome 4B

QTL Closest Marker Position AE LOD PVE (%) Total PVE (%)

QZinc.caas-1A Xwmc312 2.0 1.61 3.1 14.0 29.2

QZinc.caas-4B Xgwm251 6.5 -1.78 2.7 15.2

72 DH lines were sown at Anyang in Henan province in two seasons

QTL mapping of Zn

Effect of N on Fe in root and shoot

Optimized N application significantly increase Fe uptake of shoot and plant,

63%-82% of Fe is kept in root

Trait Check 174 kg/ha

Root

Fe concentration (mg/kg) 1654a 1431a

Fe uptake (mg/m2) 98a 137.0a

Shoot

Fe concentration (mg/kg) 65.3a 86.6a

Fe uptake (mg/m2) 21.3b 80.8a

Shoot/Root ratio of Fe concentration 0.04 0.06

Shoot/Root ratio of Fe uptake 21.77 58.99

Shoot/(Root+Shoot) ratio of Fe uptake 18% 37%

Total (Root+Shoot)

Fe concentration (mg/kg) 338.8a 309.1a

Fe uptake (mg/m2) 129.0b 217.8a

Liangxing 99 was sown at Quzhou in Hebei province in 2010-2011

Effect of nitrogen application

Effect of N on Zn in root and shoot

Optimized N application significantly increase Zn uptake in root, and Zn

concentration and uptake of shoot and plant. 16%-42% of Zn is kept in root

Trait Check 174 kg/ha

Root

Zn concentration (mg/kg) 44.8a 45.4a

Zn uptake (mg/m2) 2.7b 4.4a

Shoot

Zn concentration (mg/kg) 11.6b 24.3a

Zn uptake (mg/m2) 3.7b 22.6a

Shoot/Root ratio of Zn concentration 0.26 0.54

Shoot/Root ratio of Zn uptake 1.4 5.2

Shoot/(Root+Shoot) ratio of Zn uptake 58% 84%

Total (Root+Shoot)

Zn concentration (mg/kg) 17.0b 26.3a

Zn uptake (mg/m2) 6.4b 26.9a

Effect of N on Zn/Fe distribution in wheats

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0kg/ha 174kg/haZ

n D

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Grain Stem+Leaves Husk Root

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0kg/ha 174kg/ha

Fe

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Grain Stem+Leaves Husk Root

N application decrease root Fe and Zn by 9% and 6%, increase grain Fe

and Zn by 2% and 3% than control, significantly increase the efficacy of

Fe/Zn transportation from root to shoot and grain, but the efficiency for Zn

was much better than that for Fe

Source df Fe Zn P PAP Phytase

Milling 7 3324*** 2506*** 367306*** 67006*** 10134***

Genotype 42 20*** 28*** 1564** 126** 1157***

Error 294 62 65 6060 508 2956

There were significant effect of milling and genotype on all micronutrient

concentrations, with milling being the predominant

43 cultivars sown in Jinan, Shandong province in 2005-2006

ANOVA of milling on micronutrients

Effect of milling and processing

Fraction Fe Zn

Mean Range Mean Range

Break 1 6.9edf 4.6-10.9 7.3d 4.1-14.2

Break 2 7.9ed 3.6-12.1 7.9d 1.8-12.1

Break 3 15.7c 10.4-27.7 12.7c 9.4-17.7

Reduction 1 5.5f 3.2-13 7.8d 4.9-13.8

Reduction 2 6.5ef 2.5-10.4 8.9d 4.8-16

Reduction 3 8.9d 5-12.8 11.4c 6.9-17

Shorts 43.5b 30.8-61.3 49.1b 35.4-74.2

Bran 100.5a 77.4-124.4 86.3a 69.2-128.2

Fe/Zn contents of streams from Buhler mill

Fraction P PAP Phytase

Mean Range Mean Range Mean Range

Break 1 844de 555-1097 548d 445-649 233e 45-587

Break 2 878de 80-1224 558d 479-690 259e 107-762

Break 3 1314c 985-1715 684c 578-832 617c 221-936

Reduction 1 777e 321-978 524d 437-606 209e 8-437

Reduction 2 1033de 458-8199 574d 484-697 337de 74-452

Reduction 3 1072dc 461-1382 579d 510-667 403d 112-594

Shorts 3860b 886-4999 1262b 967-1684 1449b 580-3061

Bran 10819a 8541-12960 4839a 4166-5579 1683a 630-3726

Phosphorus contents of streams from Buhler mill

Effect of milling on micronutrients

In the same flour stream, at least 2-3 times difference on Fe/Zn among cultivars was observed

Very good (B1, B2, R1 and R2) and fine quality (B1, B2, B3, R1, R2 and R3) flour has low Fe/Zn

Micronutrients concentrate in short and bran fraction

Relationship between nutrient contents and flour yield

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55 60 65 70 75 80 85 90 95 100

Fe

Zn

Total P

PAP

Phytase

Flour yield

% o

f to

tal

in g

rain

Journal of Cereal Science, 2008, 48:821-828

Total phosphorus decreased the most with decreasing flour yield

Phytic acid (PAP) and phytase activity decreased the least

Relationship of phenolic acids and flour yield

Flour yield

mg

/kg

Journal of Cereal Science, 2012, in review

Ferulic was the predominant, and Caffeic and P-coumaric

was important in phenolic acids

Phenolic acids concentrate in short and bran fraction

0

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200

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400

500

600

700

60% 70% 80% 90% 100%

Ferulic

Caffeic

Chlorogenic

Syringic

Gentisic

P-coumaric

Phenolics

Effect of processing method on micronutrients

Process Fe Zn P PAP

Flour 6.7c 8.4c 857a 565 a

Bread 8.4b 9.2b 860a 1.5 b

Steamed bread 8.2b 9.6a 867a 1.6 b

Noodle 9.5a 9.7a 855a 1.7 b

Processing method significantly increase concentration of

Fe/Zn, but decrease phytic acid P very deeply at the same

Sample information for in vitro digestion/Caco-2 cell model

Biomedical and Environmental Sciences, 2012, in press

Sample Flour yield (%) Fe (mg/kg) PAP

Yumai 18 78 15.8 0.78

Jingdong 8 78 20.8 0.82

Zhongmai 9 78 22.7 0.87

Zhongmai 175 78 20.9 0.79

Yumai 2 78 16.0 0.68

Zhongyou 9507 78 29.2 0.91

Fe bioavailability

Bioavailability in In-Vitro Digestion/Caco-2 cell model

d

b

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cd

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Yumai 18 Jingdong 8 Zhongmai 9 Zhongmai 175 Yumai 2 Zhongyou 9507

BV

& B

VP

G (

% o

f co

ntr

ol)

Fe BVPG (% of control) Fe BV (% of control)

Zhongyou 9507 and Jingdong 8 have high Fe bioavailability

Phytate is an important Fe BV inhibitors, as reductions in phytate

content in cereals lead to strong increases in Fe BV

Conclusions

Jingdong 8 and Zhongyou 9507 have high Fe/Zn. Liangxing

66 and Zhongmai 895 have high and consistent phenolic acids

Fe, Zn and PAP are significantly influenced by G, E, milling,

and processing

Two QTLs on 1A and 4B contribute 29.2% of the PVE of Zn

Nitrogen can significantly increase Fe/Zn transportation

efficacy from soil to shoot and grain, but the efficiency for Zn

is much better than that for Fe

Zhongyou 9507 and Jingdong 8 have high Fe bioavailability

based on in-Vitro digestion/Caco-2 cell model

Perspective

More genotypes need to be screened and their stability across

environments need to be investigated

QTL s on Zn should be confirmed, molecular marker will be

validated for breeding program. How about Fe?

Effects of other fertilizer and their interaction on Fe/Zn

transportation efficacy will be done next

Effects of milling and processing on bioavailability will be

done next, and how about animal and human trial?

Need to meet efforts to combine high yield potential and

major disease resistance with nutrition quality, farmer’s need

Acknowledgements

Prof. Yan Zhang, Mr. Jianwei

Tang, and Xiaoyong Shen, Lan

Wang, CAAS

Drs. Rongli Shi and Qichao Song,

CAU

Drs. Lin Bai and Dr. Ji Lei, SCU

Dr. Raymond P. Glahn, Cornell

University

Funding from HPC and Chinese Ministry of Agriculture