Understanding the effects of HMW-GS and LMW-GS on processing quality using near-isogenic lines and development of functional markers for LMW-GS in wheat

Xianchun Xia, Zhonghu He

National Wheat Improvement Center,

Chinese Academy of Agricultural Sciences

[email protected]

Understanding the effects of HMW-GS and LMW-

GS on processing quality using near-isogenic lines

and development of functional markers for LMW-

GS in wheat

Plant materials

Quality parameters

Flour yield, energy, extensograph maximum resistance,

farinograph stability time, and noodle viscoelasticity

Chinese steamed bread, fresh white Chinese noodle, pan

bread quality

glutenin macropolymers, dough properties

A3b A3d A3e A3f B3a B3c B3d B3f B3g B3h B3i D3a D3b D3d D3f

Glu-A3 Glu-B3 Glu-D3

B3-688 B3-691 A3-620

D3-525

B3-570

D3-578

B3-510 D3-432

D3-575

B3-621

A3-620

D3-525

D3-575

D3-578

B3-530 D3-442

B3-607

a b B3i D3a

Fig. 1 Separation of HMW-GS and LMW-GS on SDS-PAGE

HMW-GS

D-group

B-group

C-group

643

565 568 662

640

402

624

601

544

548

548

548

548

548

700

700

700 800

700

688

688

688 813

688 691 570 510

441

441

441

583

586

525

525

370

650 650

& 621 620

646 700

600

593

600

LMWGS3c LMWGS3b

LMWGS3c

LMWGS3b

LMWGS3b

LMWGS3b

LMWGS3b

LMWGS3b

LMWGS3b

Aroona

A3c/B3b/D3c

Aroona-A3b

Aroona-A3d

Aroona-A3e

Aroona-A3f

Aroona-B3a

Aroona-B3c

Aroona-B3d

Aroona-B3f

Aroona-B3g

Aroona-B3h

Aroona-B3i

Aroona-D3a

Aroona-D3b

Aroona-D3d

Aroona-D3f

Fig. 2 Separation of

LMW-GS genes in

Aroona NILs using

the LMW-GS gene

molecular marker

system.

Identification of LMW-GS genes in 16 Aroona NILs by

using the LMW-GS gene marker system

A3b A3c A3d A3e A3f B3a B3b B3c B3d B3f B3g B3h B3i D3a D3b D3c D3d D3f

The effects of glutenin subunits on dough properties

Different letters indicate the significant difference at 5% probability level

The effects of glutenin subunits on pan bread quality


The effects of glutenin subunits on quantity of

gluten protein fractions


The effects of glutenin subunits on fresh white Chinese

noodle quality


The effects of glutenin subunits on northern style Chinese

steamed bread quality


Trait Combination

ZSV Large 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c

1/17+18/2+12/Glu-A3c/Glu-B3b/Glu-D3c

Small 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c

ST Long 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c

1/7+9/2+12/Glu-A3f/Glu-B3b/Glu-D3c

Short 1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c

1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

Ext Good 1/7+9/2+12/Glu-A3c/Glu-B3i/Glu-D3c

1/7+9/2.2+12/Glu-A3c/Glu-B3b/Glu-D3c

Bad 1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c


The effects of combination of HMW-GS and LMW-GS on

dough properties

Trait Combination

Gli/Glu Low 1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c

1/7+9/3+12/Glu-A3c/Glu-B3b/Glu-D3c

High 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

1/7+9/2+12/Glu-A3c/Glu-B3c/Glu-D3c

%UPP High 1/7+9/5+10/Glu-A3c/Glu-B3b/Glu-D3c

1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3d

Low 2*/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3c



quantity of gluten protein fractions

Trait Combination

PBTS High 1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3f

1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c

Low 1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

N/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3c

Noodle

viscoelasticity

Good 1/7+9/2+12/Glu-A3c/Glu-B3d/Glu-D3c

1/7+9/2+12/Glu-A3c/Glu-B3f/Glu-D3c

Bad 1/7*+8/2+12/Glu-A3c/Glu-B3b/Glu-D3c

1/7+9/2+12/Glu-A3b/Glu-B3b/Glu-D3c

SBTS High 1/7+9/2+12/Glu-A3c/Glu-B3b/Glu-D3f

1/7+9/2+12/Glu-A3c/Glu-B3a/Glu-D3c

Low 1/7+9/2+12/Glu-A3c/Glu-B3g/Glu-D3c

1/7+9/2+12/Glu-A3c/Glu-B3d/Glu-D3c


processing quality

Cloning of Glu-B3 genes and

development of functional markers

Primers used for cloning Glu-B3 genes

Target

gene

Primer Sequence (5＇→3＇) Reference

gene

Primer

locationa

Ann temp

(oC)

GluB3-1 LB1F GCACAAGCATCAAAACCAAGA AB262661 -13b 58

LB1R GACACTTTATTTGTCACCGCTG AB262661 1129

GluB3-2 LB2F AACCTAACGCATTGTACCAAAAATC AY542898 208 61

LB2R GGCGGGTCACACATGACA AY542898 1499

GluB3-3 LB3F CATCACAAGCACAAGCATCAA Y14104 472 58

LB3R CATATCCATCGACTAAACAAA AB119006 709

GluB3-4 LB4F CACCCTATACAAGGTTCCAAAAT Y14104 77 60

LB4R TATTTCCATAATTTAAACTAGTTTGT AB062852 +1346 b

Primers localization for cloning Glu-B3

Fig.1 Electrophoresis of the PCR products of the four gene-specific primer sets

on agarose gel. a GluB3-1, GluB3-4 and GluB3-2 genes, 1 Chinese Spring, 2

N1AT1B, 3 N1BT1A, 4 N1BT1D and 5 N1DT1B. b GluB3-3 gene, 1, 2, 5, 6, 7

1BL.1RS lines, 3 Aroona-B3c, 4 Aroona-B3d, 8 Aroona-B3h, 9 Aroona-B3i, 10

N1AT1B, 11 N1BT1A, 12 N1BT1D and 13 N1DT1B. M DNA Ladder 2000

(100, 250, 500, 750, 1,000 and 2,000 bp)

a. LB1F-LB1R LB4F-LB4R LB2F-LB2R

b. LB3F-LB3R

PCR amplification

Amplified the isogenic lines with different Glu-B3 protein alleles.

Cloned 4 genes at Glu-B3 locus,

with 17 allelic variants.

Glu-B3 allelic variants

Gene

Allelic

variants Allele Size

Accession No.

GluB3-1 GluB3-11 a 1213 EU369699 GluB3-12 b 1228 EU369700 GluB3-13 e 1225 EU369701 GluB3-14 f 1222 EU369702 GluB3-15 g 1162 EU369703

GluB3-2 GluB3-21 a 1396 EU369704

GluB3-22 bef 1393 EU369721 GluB3-23 g 1393 EU369705

GluB3-3 GluB3-31 c 1290 EU369715 GluB3-32 d 1290 EU369716 GluB3-33 h 1290 EU369717 GluB3-34 i 1290 EU369718

GluB3-4 GluB3-41 acd 1636 EU369724 GluB3-42 b 1636 EU369719 GluB3-43 eh 1636 EU369727 GluB3-44 fg 1636 EU369729 GluB3-45 i 1615 EU369720

Relationship between Glu-B3 protein

alleles and gene haplotypes Lines Allele GluB3

-11

GluB3

-12

GluB3-

13

GluB3-

14

GluB3-

15

GluB3-

21

GluB3-

22

GluB3-

23

Aroona-B3a a + +

Aroona b + +

Aroona-B3c c

Aroona-B3d d

Cheyenne e + +

Aroona-B3f f + +

Aroona-B3g g + +

Aroona-B3h h

Aroona-B3i i

Lines Allele GluB3

-31

GluB3

-32

GluB3-

33

GluB3-

34

GluB3-

41

GluB3-

42

GluB3-

43

GluB3-

44

GluB3-

45

Aroona-B3a a +

Aroona b +

Aroona-B3c c + +

Aroona-B3d d + +

Cheyenne e +

Aroona-B3f f +

Aroona-B3g g +

Aroona-B3h h + +

Aroona-B3i i + +

Sequence alignment of allelic variants

GLUB3-11 CAACCACAACAATTTCCACAACAGCAACCATGTTCACAACAACAACAACAACAACAACAACAACAACAAC 280





GLUB3-11 AACAACAACAACAACAACAACAACAACAACAACAACAA............CCATTATCGCAGCAACAACA 338

GLUB3-12 AACAACAACAACAACAACAACAACAACAACAACAACAACaacaacaacaaCCATTATCGCAGCAACAACA 350

GLUB3-13 AACAACAACAACAACAACAACAACAACAACAACAACAACaacaacaa...CCATTATCGCAGCAACAACA 347

GLUB3-14 AACAACAACAACAACAACAACAACAACAACAACAACAACaacaa......CCATTATCGCAGCAACAACA 344

GLUB3-15 AACAACAACAA........................................................... 292

GLUB3-11 ACCACCATTTTCACAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 408




GLUB3-15 ....CCATTaTCgCAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 357

GLUB3-11 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 478





GLUB3-11 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAA...CC 545

GLUB3-12 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAAcaaCC 560



GLUB3-15 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAA...CC 494

Allele-specific PCR markers for the discrimination

of different Glu-B3 protein alleles Marker

name

Primer

set

Sequence (5＇→3＇) Target

allele

Fragment

size

PCR condition

gluB3a SB1F GCACAAGCATCAAAACCAAGA a 1095 94℃/35s-55℃/35s-72℃/90s

SB1R TGGCACACTAGTGGTGGTC

gluB3b SB2F ATCAGGTGTAAAAGTGATAG b 1570 94℃/35s-56℃/35s-72℃/90s

SB2R TGCTACATCGACATATCCA

gluB3c SB3F CAAATGTTGCAGCAGAGA c 472 94℃/35s-56℃/35s-72℃/90s

SB3R CATATCCATCGACTAAACAAA

gluB3d SB4F CACCATGAAGACCTTCCTCA d 662 94℃/35s-58℃/35s-72℃/90s

SB4R GTTGTTGCAGTAGAACTGGA

gluB3e SB5F GACCTTCCTCATCTTCGCAa e 669 94℃/35s-58℃/50s-72℃/90s

SB5R GCAAGACTTTGTGGCATT

gluB3fg SB6F TATAGCTAGTGCAACCTACCAT fg 812 94℃/35s-62℃/35s-72℃/90s

SB6R CAACTACTCTGCCACAACG

gluB3g SB7F CCAAGAAATACTAGTTAACACTAGT

C

g 853 94℃/35s-60℃/35s-72℃/90s

SB7R GTTGGGGTTGGGAAACA

gluB3h SB8F CCACCACAACAAACATTAA h 1022 94℃/35s-60℃/35s-72℃/90s

SB8R GTGGTGGTTCTATACAACGA

gluB3i SB9F As SB6F i 621 94℃/35s-58℃/35s-72℃/90s

SB9R TGGTTGTTGCGGTATAATTT

gluB3bef SB10F GCATCAACAACAAATAGTACTAGAA bef 750 94℃/35s-60℃/35s-72℃/90s

SB10R GGCGGGTCACACATGACA

STS markers

gluB3a

1095bp

gluB3b

1615bp

gluB3c

472bp

a b c d e f g h I

a b c d e f g h I

gluB3d

662bp

gluB3e

669bp

gluB3bef

750bp

STS markers

a b c d e f g h I

gluB3i

621bp

gluB3h

1022bp

gluB3g

853bp

gluB3fg

812bp

STS markers

Validation of molecular markers

Twenty wheat cultivars and 141 CIMMYT advanced

lines were tested with the markers. The results were

consistent with those detected by SDS-PAGE, except

for 4 lines.

Wang et al. 2009, Theor Appl Genet 118:525–539

Allele-specific PCR markers for the discrimination

of different Glu-A3 protein alleles

Marker

name

Primer

set

Sequence (5＇→3＇) a Target

allele

Fragmen

t

size

PCR condition

GluA3a LA1F AAACAGAATTATTAAAGCCGG a 529 94℃/35s-60℃/35s-72℃/90s

SA1R GGTTGTTGTTGTTGCAGCA

GluA3b LA3F TTCAGATGCAGCCAAACAA b 894 94℃/35s-60℃/35s-72℃/90s

SA2R GCTGTGCTTGGATGATACTCTA

GluA3ac LA1F AAACAGAATTATTAAAGCCGG acb 573 94℃/35s-60℃/35s-72℃/90s

SA3R GTGGCTGTTGTGAAAACGA

GluA3d LA3F TTCAGATGCAGCCAAACAA d 967 94℃/35s-60℃/35s-72℃/90s

SA4R TGGGGTTGGGAGACACATA

GluA3e LA1F AAACAGAATTATTAAAGCCGG e 158 94℃/35s-60℃/35s-72℃/90s

SA5R GGCACAGACGAGGAAGGTT

GluA3f LA1F AAACAGAATTATTAAAGCCGG f 552 94℃/35s-60℃/35s-72℃/90s

SA6R GCTGCTGCTGCTGTGTAAA

GluA3g LA1F AAACAGAATTATTAAAGCCGG g 1345 94℃/35s-60℃/35s-72℃/90s

SA7R AAACAACGGTGATCCAACTAA

Glu-A3 marker

AF33-A3A3R

GluA3a

AF14-A3AC2R

GluA3ac

AF63-A6B3R

GluA3b

A3a A3b A3c A3d A3e A3f A3g

Wang et al. 2010, J. Cereal Sci 51：305-312

Glu-A3 marker

AF63-A6D2R

GluA3d

Af522-A5d1r

GluA3d

AF14-A3E3R

GluA3e




A3F1F-A3F3R

GluA3f

AF33-A1G1R

GluA3g

Glu-A3 marker


Glu-A3 multiplex PCR

GluA3be GluA3df

GluA3bf GluA3dg

Glu-A3 multiplex PCR

GluA3be + GluA3df

GluA3bf + GluA3dg

a

b

1345bp

967bp

894bp

552bp

GluA3b GluA3d

GluA3f

GluA3g

967bp

894bp

552bp

158bp

GluA3e

GluA3f

GluA3b GluA3d

Wang et al. 2010, J Cereal Science 51：305-312

Acknowledgement

Ken Shepherd, SARDI Grain Quality Research Laboratory

Marie Appelbee, SARDI Grain Quality Research Laboratory

Rudi Appels, University of Murdoch

Wujun Ma, University of Murdoch

Roberto Peña, CIMMYT

Aimin Zhang, Institute of Genetics, CAS

Peiyuan Mu and Zheru Fan for their assistance in the field trials

Xianlin Zhao

Linhai Wang

Xiaofei Zhang

Hui Jin

These studies were supported by

the National Basic Research Program (2009CB118300),

National Natural Science Foundation of China (30830072),

International Collaboration Project from Ministry of

Agriculture (2011-G3)

Thank you ！

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Understanding the effects of HMW-GS and LMW-GS on processing quality using near-isogenic lines and development of functional markers for LMW-GS in wheat