International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. 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
xiaxianchun@caas.net.cn

2. Plant materials

3. 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

4. a b
Glu-A3 Glu-B3 Glu-D3
A3b A3d A3e A3f B3a B3c B3d B3f B3g B3h B3i D3a D3b D3d D3f B3i D3a
HMW-GS
D-group
B3-688 A3-620
B3-691
B-group A3-620 B3-621
B3-570 D3-575
D3-575 B3-607
D3-578 D3-578
D3-525 D3-525
C-group B3-530
B3-510
D3-432 D3-442
Fig. 1 Separation of HMW-GS and LMW-GS on SDS-PAGE

5. LMWGS3c 650 650 LMWGS3b Aroona
620 & 621 A3c/B3b/D3c
Aroona-A3b
643
Aroona-A3d
370 402
565 568 662
700
LMWGS3c
Aroona-A3e
646
Aroona-A3f
640
600 LMWGS3b Aroona-B3a
548 593
624
700 688 LMWGS3b
Aroona-B3c
548
700 688 LMWGS3b Aroona-B3d
548
Aroona-B3f
548 601
Aroona-B3g
544
700 LMWGS3b 800
Aroona-B3h
548 688 813
700 688 Aroona-B3i
510 570 691 LMWGS3b
Fig. 2 Separation of
Aroona-D3a
441 525 LMW-GS genes in
441
Aroona-D3b Aroona NILs using
583
the LMW-GS gene
441 Aroona-D3d
586 molecular marker
525
600 LMWGS3b Aroona-D3f system.

6. Identification of LMW-GS genes in 16 Aroona NILs by
using the LMW-GS gene marker system

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

8. The effects of glutenin subunits on dough properties
Different letters indicate the significant difference at 5% probability level

9. The effects of glutenin subunits on pan bread quality
Different letters indicate the significant difference at 5% probability level

10. The effects of glutenin subunits on quantity of
gluten protein fractions
Different letters indicate the significant difference at 5% probability level

11. The effects of glutenin subunits on fresh white Chinese
noodle quality
Different letters indicate the significant difference at 5% probability level

12. The effects of glutenin subunits on northern style Chinese
steamed bread quality
Different letters indicate the significant difference at 5% probability level

13. The effects of combination of HMW-GS and LMW-GS on
dough properties
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
1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

14. The effects of combination of HMW-GS and LMW-GS on
quantity of gluten protein fractions
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
1/7+9/2+12/Glu-A3e/Glu-B3b/Glu-D3c

15. The effects of combination of HMW-GS and LMW-GS on
processing quality
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 Good 1/7+9/2+12/Glu-A3c/Glu-B3d/Glu-D3c
1/7+9/2+12/Glu-A3c/Glu-B3f/Glu-D3c
viscoelasticity
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

16. Cloning of Glu-B3 genes and
development of functional markers

17. Primers used for cloning Glu-B3 genes
Target Primer Sequence (5＇→3＇) Reference Primer Ann temp
gene gene locationa (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

18. Primers localization for cloning Glu-B3
a. LB1F-LB1R LB4F-LB4R LB2F-LB2R
b. LB3F-LB3R
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)

19. PCR amplification
 Amplified the isogenic lines with
different Glu-B3 protein alleles.
 Cloned 4 genes at Glu-B3 locus,
with 17 allelic variants.

20. Glu-B3 allelic variants
Allelic Accession No.
Gene variants Allele Size
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

21. Relationship between Glu-B3 protein
alleles and gene haplotypes
Lines Allele GluB3 GluB3 GluB3- GluB3- GluB3- GluB3- GluB3- GluB3-
-11 -12 13 14 15 21 22 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 GluB3 GluB3- GluB3- GluB3- GluB3- GluB3- GluB3- GluB3-
-31 -32 33 34 41 42 43 44 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 + +

22. Sequence alignment of allelic variants
GLUB3-11 CAACCACAACAATTTCCACAACAGCAACCATGTTCACAACAACAACAACAACAACAACAACAACAACAAC 280
GLUB3-12 CAACCACAACAATTTCCACAACAGCAACCATGTTCACAACAACAACAACAACAACAACAACAACAACAAC 280
GLUB3-13 CAACCACAACAATTTCCACAACAGCAACCATGTTCACAACAACAACAACAACAACAACAACAACAACAAC 280
GLUB3-14 CAACCACAACAATTTCCACAACAGCAACCATGTTCACAACAACAACAACAACAACAACAACAACAACAAC 280
GLUB3-15 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
GLUB3-12 ACCACCATTTTCACAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 420
GLUB3-13 ACCACCATTTTCACAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 417
GLUB3-14 ACCACCATTTTCACAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 414
GLUB3-15 ....CCATTaTCgCAGCAACAACAACCACCATTTTCACAGCAACAACCACCATTTTCGCAGCAACAACAA 357
GLUB3-11 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 478
GLUB3-12 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 490
GLUB3-13 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 487
GLUB3-14 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 484
GLUB3-15 CCAGTTCTACCGCAACAGCCATCATTTTCGCAGCAACAACTACCACCATTTTCACAACAACAACCACCGT 427
GLUB3-11 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAA...CC 545
GLUB3-12 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAAcaaCC 560
GLUB3-13 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAAcaaCC 557
GLUB3-14 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAAcaaCC 554
GLUB3-15 TTTCACAACAACAACAACCAGTACTACCGCAACAACCACCATTTTCGCAACAACAACAACAACAA...CC 494

23. Allele-specific PCR markers for the discrimination
of different Glu-B3 protein alleles
Marker Primer Sequence (5＇→3＇) Target Fragment PCR condition
name set allele size
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 g 853 94℃/35s-60℃/35s-72℃/90s
C
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

24. STS markers
a b c d e f g h I
gluB3a
1095bp
gluB3b
1615bp
gluB3c
472bp

25. STS markers
a b c d e f g h I
gluB3d
662bp
gluB3e
669bp
gluB3bef
750bp

26. STS markers
a b c d e f g h I
gluB3fg
812bp
gluB3g
853bp
gluB3h
1022bp
gluB3i
621bp

27. 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

28. Allele-specific PCR markers for the discrimination
of different Glu-A3 protein alleles
Marker Primer Sequence (5＇→3＇) a Target Fragmen PCR condition
name set allele t
size
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

29. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
AF33-A3A3R
GluA3a
AF63-A6B3R
GluA3b
AF14-A3AC2R
GluA3ac
Wang et al. 2010, J. Cereal Sci 51：305-312

30. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
AF63-A6D2R
GluA3d
Af522-A5d1r
GluA3d
AF14-A3E3R
GluA3e
Wang et al. 2010, J. Cereal Sci 51：305-312

31. Glu-A3 marker
A3a A3b A3c A3d A3e A3f A3g
A3F1F-A3F3R
GluA3f
AF33-A1G1R
GluA3g
Wang et al. 2010, J. Cereal Sci 51：305-312

32. Glu-A3 multiplex PCR
GluA3be GluA3df
GluA3bf GluA3dg

33. Glu-A3 multiplex PCR
a
GluA3b GluA3d
GluA3be + GluA3df
967bp
894bp
GluA3f
552bp
GluA3e
158bp
b
GluA3bf + GluA3dg GluA3g
GluA3b GluA3d 1345bp
967bp
GluA3f
894bp
552bp
Wang et al. 2010, J Cereal Science 51：305-312

34. 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 Peñ CIMMYT
a,
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

35. 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)

36. Thank you ！