High frequency of abnormal high molecular

weight glutenin alleles in Chinese wheat

landraces of the Yangtze-River region

Dongfa Sun

Wheat breeder ,Prof of Huazhong Agricultural

University, Wuhan, Hubei, 430070

1.Research background

2.Materials and methods

3.Result 4. Summary and further research

1. 1 The importance of HMW-GS Wheat glutenins are divided into high and low molecular

weight glutenin subunits (HMW-GS (65 to 90 kDa)

&LMW-GS(25 to 43 kDa ) ) based on their mobility

during SDS-PAGE. High molecular weight glutenin

subunit proteins have the ability to form gluten network

thus conferring rheological characteristics (strength and

extensibility) of flour dough that is essential for making

bread . Although they are minor components in terms of

quantity, they are key factors in the process of bread-

making due to their ability to promote the formation of

larger glutenin polymers.

1. Research background

1.2 The species of HMW-GS and their effects

● High molecular weight glutenin subunit (HMW-GS ) are

encoded by tightly-linked “x” and “y” type genes at the Glu-

A1, Glu-B1 and Glu-D1 loci on the long arms of

chromosomes 1A, 1B and 1D, respectively.

● GluB1 i allele (17+18) and GluD1 d allele (5+10) have a

positive influence on bread-making quality .

● Null and GluD1 a allele (2+12) have a negative effect

● The subunits7+8, including four alleles 7+8, 7+8*, 7OE+8,

7OE+8* , designated Glu-B1,

● 7OE+8* containing the over-expression of subunit 7OE,

has large positive influence on bread-making quality .

1. Research background

1. 3 why we do this research?

China is considered one of the secondary centres of origin for common wheat .The

history of wheat production in China has been about 4800 years . There are a large

number of landraces have been accumulated. Some novel HMW-GS alleles have

been found. in the landraces from the Yellow river

Yangtze-River region is a special place where have different ecological , multiracial

conditions ,where “soft” wheat is primarily produced to make traditional food of the

region. Over the past few thousand years, these wheat accessions have been

cultivated to produce various Chinese style end-products, which require different

processing quality characteristics from the Western style products. To date, detailed

genetic research on these accessions is largely missing. There may exist some novel

or special HMW-GS germplasms .Knowledge of the HMW-GS alleles present in

wheat landraces could help breeders to arrange crossing and analyze allelic effects

on specific quality traits. The objective of this study :

●to detect the HMW-GS compositions of Chinese wheat landraces from the

Yangtze-River region

● to explore some novel ,new or special HMW-GS germplasm

1. Research background

2.1 Plant materials

A total of 485 accessions of bread wheat

landraces were collected from the China Yangtze-

River region, comprising the entire collection of

the past 50 years by Huazhong Agricultural

University that is located in the City of Wuhan,

Hubei province.

2. Materials and methods

2.2 MALDI-TOF-MS

MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time-of-flight mass

spectrometry) has become a powerful tool for characterizing wheat gluten proteins .It

is highly accurate and sensitive, requiring only few minutes per sample to perform the

measurement. MALDI-TOF –MS experiments of this research were carried out in the

Lab of Western Australian Department of Agriculture & Food ,on a Voyager DE-PRO

TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with

UV nitrogen laser (337 nm), The dried mixtures of HMWGS samples were dissolved in 60 μl acetonitrile (ACN) /H2O (v/v, 50:50)

containing 0.05% v/v trifluoroacetic acid (TFA) for 1 hour. Sample preparation was carried out according

to the dried droplet method (Kussmann et al., 1997), using sinapinic acid (SA) as the matrix. The matrix

solution was prepared by dissolving SA in ACN/H2O (50:50 v/v) with 0.05% v/v TFA at a concentration of

10 mg/ml. The extracted HMWGS solution (total 60 μl) was mixed with SA solution at the ratio of 1:10

(v/v) and 2 μl of this protein-SA mixture was deposited on to a 96-sample MALDI probe tip, and dried at

room temperature.

The instrument was used with the following parameters: laser intensity 2,500, mass range 50-100 kDa,

acceleration voltage 25 kV, grid voltage 92%, guide wire 0.3%, delay time 850 ns. The Bin size was set at

20 nsec and input bandwidth at 20 MHz. Spectra were obtained in positive linear ion mode and were

averaged from 50 laser shots to improve the S/N level. All he samples were automatically accumulated in a

random pattern over the sample spot to provide the final spectrum. Human transferrin (79,549 Da) was

used as the external standard for mass assignment.

2.Materials and methods

3.1 Typical MALDI-TOF profiles

Figure 1 to 3 are typical MALDI-TOF profiles of the

study among 485 wheat landraces.

Overall, the GluA1 locus had three predominant

allele compositions including Ax1, Ax2* and Ax-

null.

Both GluB1 and D1 loci showed high level of

abnormality, including 4 TYPES

▼ double null genotype,

▼ single subunit silencing,

▼ unusual subunit combination,

▼ new subunits that have not been reported in the

past.

3. Result

Figure 1, Line 47: 7+8 and 17+18. GluA1 and GluD1 are not expressed

60015 67015 74015 81015 88015 95015

Mass (m /z)

0

576.1

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

Voyager Spec #1=>B C =>N R (4.00)[B P = 82905.8, 576]

82786.34

78960.62

73356.85

85845.2188393.5177188.11 92241.29

61847.14

By8 By18 Bx17

Bx7

60015 67015 74015 81015 88015 95015

Mass (m/z)

0

653.6

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Voyager Spec #1=>BC=>NR(2.00)[BP = 86992.5, 654]

86999.77

87109.57

86709.7668477.46

87460.11

75118.10

69286.35

71219.5965303.06 77549.83

90063.16

Dy12 Novel By Ax1 Dx2

Figure 2, Line 255: No Bx subunit expressed

60015 67015 74015 81015 88015 95015

Mass (m/z)

0

735.6

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Voyager Spec #1=>BC=>NR(2.00)[BP = 68447.6, 736]

68442.52

68359.06

86983.03

83468.54

83598.3767912.31

87675.54

65318.95 89706.1770205.73 75313.40

Dy12 Bx7 Bx20 Dx2

Figure 3, Line 227: No By subunit expressed. Two Bx subunits, Bx7 and Bx20

3.2 Abnormal high molecular weight glutenin allele

Table 1 lists HMW-GS compositions of some accessions

containing abnormal subunits

3.Result

Huazhong

Serial No.

GluA1 GluB1 GluD1 Note

35 1 7+? 2+12 73100?

36 1 75140 2+12 no Bx

41 1 75130 2+12 no Bx

52 2* 75150 5+10 no Bx

123 n 13 2+12 no By

125 1 7+? 2+12 73000?

160 1 7+? 2+? 69100? 75600?

163 n 7+8 2+? 67900?

255 1 75118 2+12 no Bx

263 n 7+(73000) 2+12

266 n 7+(73000) 2+12

682 2* 7OE 2+12 no By

684 n 20 2+12 no By

696 1 20+20 2+12 83200

698 n 7OE+8 2+12 83200

747 1 75127 5+10 no Bx

751 2* 7+8/(76800)+

(78600)

2+12

753 n 7OE 5+10 no By

Table 1. HMW-GS compositions of some accessions containing abnormal subunits

Huazhong

Serial No.

GluA1 GluB1 GluD1 Note

758 n 7OE 2+12 no By

765 1 75140 2+12 no Bx

766 N 7OE/7 2+12 no By

767 2* 74800 5+10 no Bx

768 2* 75100 5+10 no Bx

769 2* 75140 5+10 no Bx

777 N 7OE 2+12 no By

778 1 75133 2+12 no Bx

779 2* 75140 5+10 no Bx

784 2* 7OE/7 5+10 no By

791 n 7OE 5+10 no By

797 2* 75100 2+12 no Bx

806 2* 75136 5+10 no Bx

812 n 7OE/7 5+10 no By

814 2* 8 5+10 no Bx

817 2* 75120 2+12 no Bx

819 n 75118 2+12 no Bx

822 1 7OE/7 5+10 no By

830 1 7+8 2+? 69900?

833 n 7+9 5 No Dy

Continue

● GluA1 MALDI-TOF analysis did not detect any

abnormal allele compositions at the GluA1

locus.

60 lines had the GluA1a (Ax1) allele

66 lines possessed the GluA1b (Ax2*) allele

358 lines had a null allele at this locus.

● GluB1 The GluB1 locus exhibited a high level of abnormality.

▼Allele Bx7+By8 :expressed in 211 lines

196 lines being homogeneous

15 lines being heterogeneous at this locus. 11 had genotype

Bx7+By8/Bx14+By15; the other 4 lines had extra subunits of

Bx17+By18, Bx13, Bx7OE+By8，and an unusual allele with

molecular weight of 78600+76800.

▼ Allele Bx7+By9 : appeared in 70 lines

67 lines being homogeneous

3 lines being heterogeneous, which had extra subunits of Bx14+By15,

Bx17+By18, and Bx20+By20, respectively.

▼ allele Bx7OE+By8 :appeared in 88 lines . The majority being

homogeneous and only four lines had extra subunits of Bx17+By18,

Bx20+By20, Bx14+By15 or Bx7+By8.

▼ Subunit pair Bx7OE+By9 only appeared in one line (line 91).

▼ Allele Bx20+By20: appeared in 38 lines with 36

being homogeneous and 2 lines being heterogeneous by

coupling with Bx7OE+By8 or Bx7+By9.

▼ Allele Bx7b*+8 appeared in 22 lines with 20 lines

being homogeneous and 2 lines being heterogeneous,

both combining with Bx14+By15.

▼ The allele pair Bx14+By15 occurred in 16 lines but

none of these lines were homogeneous for this locus;12

of these lines were grouped with Bx7+By8 and the other

four with Bx7b*+By8，Bx7OE+By8 or Bx7+By9.

▼ 10 lines possessed a previously not reported allele,

Bx7 plus a By subunit with molecular weight of 73000.

● GluB1

▼ 16 lines had only the By subunit expressed, and 12

lines only had the Bx subunit expressed. When Bx is

silenced, the majority of the By subunit had molecular

weight of about 75140, which appeared as a new subunit

based on this molecular weight;

▼ The only exception is line 814, which had By8 subunit.

When By is silenced, the majority (9 out of 12) had the

Bx7OE subunit appeared, with 5 lines having only the

Bx7OE and 4 lines having both Bx7OE and Bx7 appeared.

▼ Lines 123 and 684 had only Bx13 and Bx20 expressed

at this locus, respectively;

▼ Line 227 had both Bx7 and Bx20 expressed.

● GluB1

● GluD1

▼ Dx2+Dy12 allele: 412 lines possessed the GluD1a,

▼ Dx5+Dy10 allele,58 lines had the GluD1d

▼ 4 lines had the null allele without any Dx or Dy subunit

expressed,

▼ 4 lines possessed the GluD1b allele (Dx4+Dy12),

▼ 2 lines had both GluD1a (Dx2+Dy12) and GluD1d

(Dx5+Dy10) alleles, representing hetero-zygotes.

▼ There was one line (No. 677) that had no

expression of y-type, with only the Dx2 subunit

expressed.

▼ Three abnormal alleles were detected.

Line 163 : Dx2 + Dy subunit of 67,900 dalton

line 160 : Dx2 + Dy subunit with a molecular

mass of 69,100

line 830 : Dx2 + Dy type subunit of 69,900

dalton.

The latter two Dy subunits had significantly

higher molecular weight than Dy10 and 12.

● GluD1

4. Summary and further research

● summary about abnormal subunits of this study

▼ In our study, 22 HMW glutenin subunit alleles with 16 abnormal

subunits were identified in a collection of 485 landraces from the

Yangtze-River region of China.

▼ Apart from the 22 normal alleles, twelve alleles encoding

abnormal subunits with molecular weights of 69,100, 69,900,

73,000, 73,100, 75,140, 75,600, 76,800, 79,000, 79,100, 79,800,

83,200, 84,300 were also detected

▼ In this study, a large number of lines had one or two HMW-GS

genes silenced, especially 16 lines with the 1Bx gene silenced, of

which the silencing mechanism is still unclear. These null alleles are

valuable resources for dissecting specific allele effects in wheat

quality. The information obtained in this study may be used by wheat

breeders for breeding new varieties.

● Further research

A more detailed studies of these novel

alleles are currently undergoing to obtain

their coding sequence in order to match

them with previously reported novel alleles

People contributed to this work

Prof.Dongfa Sun of Huazhong Agricultural University and his

group

Prof. Wujun Ma of Western Australian Department of

Agriculture & Food and his group

Thank you！