MURDOCH RESEARCH REPOSITORY · 93 to produce various Chinese style end-products, which require different processing 94 quality characteristics from the Western style products. To

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Zheng, W., Peng, Y., Ma, J., Appels, R., Sun, D. and Ma, W. (2011) High frequency of abnormal high molecular weight glutenin alleles in Chinese

wheat landraces of the Yangtze-River region. Journal of Cereal Science, 54 (3). pp. 401-408.

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Accepted Manuscript

Title: High frequency of abnormal high molecular weight glutenin alleles in Chinesewheat landraces of the Yangtze-River region

Authors: Wei Zheng, Yanchun Peng, Junhong Ma, Rudi Appels, Dongfa Sun, WujunMa

PII: S0733-5210(11)00143-3

DOI: 10.1016/j.jcs.2011.08.004

Reference: YJCRS 1427

To appear in: Journal of Cereal Science

Received Date: 30 December 2010

Revised Date: 24 July 2011

Accepted Date: 2 August 2011

Please cite this article as: Zheng, W., Peng, Y., Ma, J., Appels, R., Sun, D., Ma, W. High frequencyof abnormal high molecular weight glutenin alleles in Chinese wheat landraces of the Yangtze-Riverregion, Journal of Cereal Science (2011), doi: 10.1016/j.jcs.2011.08.004

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Research Note 1

2

High frequency of abnormal high molecular weight glutenin alleles in Chinese 3

wheat landraces of the Yangtze-River region 4

Wei Zheng1, Yanchun Peng1, Junhong Ma2, Rudi Appels3, Dongfa Sun1*, Wujun Ma2,3* 5

1. Department of Agriculture, Huazhong Agriculture University. Wuhan, 6

China,430070. 7

2. Western Australian Department of Agriculture & Food. Perth, WA 6150, 8

Australia. 9

3. Centre for Comparative Genomics, Murdoch University. Perth, WA 6150, 10

Australia. 11

The first three authors contributed equally to this work; 12

*Corresponding Authors: 13

Dongfa Sun, Department of Agriculture, Huazhong Agriculture University. Wuhan, 14

China, 430070. Tel.: (86) 2787281508; Fax: (86)2787396057. email: 15

[email protected], and 16

Wujun Ma, Western Australia Department of Agriculture & Food. South Street, Perth, 17

WA 6150, Australia 18

Tel.: 61 8 93606836; fax: 61 8 93606303; email: [email protected] 19

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Abstract 20

A total of 485 common landraces of bread wheat were collected from the 21

Yangtze-River region of China. Their high molecular weight glutenin subunit 22

(HMW-GS) composition was analyzed by Matrix-assisted laser desorption/ionization 23

time-of-flight Mass Spectrometry (MALDI-TOF-MS). Among all landraces tested, 453 24

were homogeneous for HMW-GS, 32 were heterogeneous, and 37 contained abnormal 25

subunits. A total of 22 alleles were detected, including 3 at Glu-A1, 13 at Glu-B1 and 6 26

at Glu-D1, respectively. Higher variations occurred at the Glu-B1 locus compared with 27

Glu-A1 and Glu-D1. Glu-A1c (74.0%), Glu-B1b (40.4%), Glu-D1a (84.9%) appeared 28

to be the most frequent alleles at Glu-A1, Glu-B1 and Glu-D1, respectively. Two alleles 29

("null" and 1) at the Glu-A1 locus, three allele compositions (7+8, 7OE+8, 7+9) at the 30

Glu-B1 locus, and two (2 +12 and 5+10) at the Glu-D1 locus appeared to be the 31

common types in the 485 landraces. Sixteen new alleles represented by abnormal 32

subunits were identified at the Glu-B1 and the Glu-D1 locus. 33

Keywords: Yangtze-River region; High molecular weight glutenin; wheat landraces; 34

MALDI-TOF 35

Abbreviations 36

DTT: Dithiothreitol; 37

HMW-GS: High Molecular Weight Glutenin Subunits; 38

HPLC: High Performance Liquid Chromatography; 39

LMW-GS: Low Molecular Weight Glutenin Subunits; 40

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MALDI-TOF-MS: Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass 41

Spectrometry; 42

SA: Sinapinic Acid; 43

SDS-PAGE: Sodium Dodecyl Sulfate polyacrylamide Gel Electrophoresis; 44

TFA: Trifluoroacetic Acid. 45

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Introduction 46

High molecular weight glutenin subunit (HMW-GS) proteins have the ability to form a 47

gluten network, thus conferring rheological characteristics (strength and extensibility) 48

of flour dough that is essential for making bread (Bekes et al., 2001; Butow et al., 2003; 49

Ma et al., 2005). Their molecular mass ranges from ~65 to 90 kDa (Shewry and 50

Tatham, 1990; Liu et al., 2009) and are encoded by tightly-linked “x” and “y” type 51

genes at the Glu-A1, Glu-B1 and Glu-D1 loci on the long arms of chromosomes 1A, 52

1B and 1D, respectively (Payne et al., 1980). Although they are minor components in 53

terms of quantity, they are key factors in the process of bread-making due to their 54

ability to promote the formation of larger glutenin polymers (Tatham et al., 1985; 55

Shewry et al., 1992). The effect that different HMW-GS has on bread-making quality 56

has been widely studied (Bekes et al., 2001; Butow et al., 2003; Ma et al., 2005). It has 57

been shown that certain HMW-GS such as Glu-B1 i allele (17+18) and Glu-D1 d allele 58

(5+10) have a positive influence, whereas others such as Null and Glu-D1 a allele 59

(2+12) have a negative effect on dough characteristics and bread-making quality 60

(Branlard and Dardevet, 1985; Payne, 1987). Different alleles of HMW-GS have been 61

given different quality scores and are extensively used as markers for selecting 62

preferable lines in wheat breeding programs (Flæte and Uhlen, 2003). 63

The subunits 7+8, first described for bread wheat cultivar Chinese Spring, are now 64

known to be four alleles including 7+8, 7+8*, 7OE+8, and 7OE+8* (Gianibelli et al., 65

2001). It has been well-documented that the allele containing the over-expression of 66

subunit 7OE, designated Glu-B1al, has a large positive influence on bread-making 67

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quality (Marchylo et al., 1992; Lukow et al., 1992; D’Ovidio et al., 1997; Vawser and 68

Cornish, 2004). The cultivars carrying subunit 7OE formed dough with high strength as 69

indicated by increased mixing times, maximum resistance to extension, and decreased 70

resistance breakdown (Bekes et al., 2001). Dough extensibility was also increased in 71

cultivars containing subunit 7OE, although this may result from the low molecular 72

weight glutenin subunits (LMW-GS) and gliadin present in respective cultivars (Gupta 73

et al., 1994; Cornish et al., 2001). 74

SDS-PAGE and HPLC methods have been used routinely in many breeding programs 75

for selection of specific HMW and LMW subunits associated with superior quality 76

(Dworschak et al., 1998). Identification of HMW-GS using SDS-PAGE is based on 77

their electrophoretic mobility and has been considered to be relatively straight-forward 78

(Vawser and Cornish, 2004). However, some HMWGS of near identical Mr and 79

electrophoretic mobility, such as 2 and 2*, and 14+15 and 20, can cause identification 80

problems using these analytical procedures (Gianibelli et al., 2001). Recently, 81

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 82

(MALDI-TOF-MS) has become a powerful tool for characterizing wheat gluten 83

proteins (Dworschak et al., 1998; Cozzolino et al., 2001; Cunsolo et al., 2002, 2003, 84

2004; Alberghina, 2005; Muccilli et al., 2005; Chen et al., 2007; Liu et al., 2009; Gao 85

et al., 2010). It is highly accurate and sensitive, requiring only a few minutes per 86

sample to perform the measurement (Dworschak et al., 1998). 87

The history of wheat production in China has been about 4800 years (Qi, 2007). There 88

are a large number of farmer’s cultivars or landraces that have been accumulated. The 89

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Chinese National Germplasm Bank has a stock of more than ten thousand Chinese 90

wheat landrace accessions, which were collected from various wheat production 91

regions. Over the past few thousand years, these wheat accessions have been cultivated 92

to produce various Chinese style end-products, which require different processing 93

quality characteristics from the Western style products. To date, detailed genetic 94

research on these accessions is largely missing. 95

The current study aims at investigating the HMWGS compositions of Chinese wheat 96

landraces from the Yangtze-River region, where “soft” wheat is primarily produced to 97

make traditional food of the region. 98

Materials and methods 99

Plant materials 100

A total of 485 accessions of bread wheat (Triticum aestivum L.) landraces were 101

collected from the China Yangtze-River region, comprising the entire collection of the 102

past 50 years by Huazhong Agricultural University that is located in the City of Wuhan, 103

Hubei province. 104

Protein extraction 105

Protein extraction was conducted based on a procedure reported by Singh et al (1991). 106

Whole meal (20 mg) was extracted with 1.0 ml of 55% propanol-1-ol (v/v) for 5 min 107

continuous vortexing, followed by incubation (20 min at 65°C), vortexing (5 min), and 108

centrifugation (5 min at 10, 000 × g). This step was repeated three times to completely 109

remove gliadins. The HMW-GS present in the pellet was reduced with 55% 110

propanol-1-ol, 0.08 M Tris-HCl solution containing 1% dithiothreitol (DTT). For 111

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SDS-PAGE analysis, the HMW glutenins were extracted as described previously 112

(Marchylo et al., 1989). For MALDI-TOF analysis, 40% acetone was used to 113

precipitate the HMW-GS proportion followed by 80% acetone precipitation of the 114

LMW-GS proportion. The separation of HMW-GS and LMW-GS is essential since 115

different mass ranges require different MALDI-TOF working parameters, ie, 116

acceleration and grid voltages etc. 117

MALDI-TOF-MS 118

The dried mixtures of HMW-GS samples were dissolved in 60 µl acetonitrile (ACN) 119

/H2O (v/v, 50:50) containing 0.05% v/v trifluoroacetic acid (TFA) for 1 hour. Sample 120

preparation was carried out according to the dried droplet method (Kussmann et al., 121

1997), using sinapinic acid (SA) as the matrix. The matrix solution was prepared by 122

dissolving SA in ACN/H2O (50:50 v/v) with 0.05% v/v TFA at a concentration of 10 123

mg/ml. The extracted HMW-GS solution (total 60 µl) was mixed with SA solution at 124

the ratio of 1:10 (v/v) and 2 µl of this protein-SA mixture was deposited on to a 125

96-sample MALDI probe tip, and dried at room temperature. 126

MALDI-TOF mass spectrometric experiments were carried out on a Voyager DE-PRO 127

TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with 128

UV nitrogen laser (337 nm). The instrument was used with the following parameters: 129

laser intensity 2,500, mass range 50-100 kDa, acceleration voltage 25 kV, grid voltage 130

92%, guide wire 0.3%, delay time 850 ns. The Bin size was set at 20 nsec and input 131

bandwidth at 20 MHz. Spectra were obtained in positive linear ion mode and were 132

averaged from 50 laser shots to improve the S/N level. All the samples were 133

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automatically accumulated in a random pattern over the sample spot to provide the 134

final spectrum. Human transferrin (79,549 Da) was used as the external standard for 135

mass assignment. 136

Results 137

Figures 1 and 2 are typical MALDI-TOF profles of the study, while Table 1 lists all the 138

HMWGS compositions of the 485 lines. Overall, the Glu-A1 locus had three 139

predominant allele compositions including Ax1, Ax2* and Ax-null. Both Glu-B1 and 140

Glu-D1 loci showed high levels of abnormality, including double null genotype, single 141

subunit silencing, unusual subunit combination, and new subunits that have not been 142

reported in the past. 143

Glu-A1 144

MALDI-TOF analysis did not detect any abnormal allele compositions at the Glu-A1 145

locus. Out of 485 accessions, 12.4% (60) had the Glu-A1a (Ax1) allele, 13.6% (66) 146

possessed the GluA1b (Ax2*) allele, 73.8% (358) had a null allele at this locus. 147

Glu-B1 148

The Glu-B1 locus exhibited a high level of abnormality. There were 211 lines that had 149

Bx7+By8 expressed, with 196 of these lines being homogeneous and 15 lines being 150

heterogeneous at this locus. For the 15 heterogeneous lines, 11 had genotype 151

Bx7+By8/Bx14+By15; the other 4 lines had extra subunits of Bx17+By18, Bx13, 152

Bx7OE+By8�and an unusual allele with molecular weight of 78600+76800. The 153

Bx7+By9 allele appeared in 70 lines with 67 lines being homogeneous and 3 lines 154

being heterogeneous, which had extra subunits of Bx14+By15, Bx17+By18, and 155

Bx20+By20, respectively. Eighty eight lines contained the Bx7OE+By8 allele, with 156

the majority being homogeneous and only four lines had extra subunits of Bx17+By18, 157

Bx20+By20, Bx14+By15 or Bx7+By8. Subunit pair Bx7OE+By9 only appeared in 158

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one line (line 91). For allele Bx20+By20, it appeared in 38 lines with 36 being 159

homogeneous and 2 lines being heterogeneous by coupling with Bx7OE+By8 or 160

Bx7+By9. Allele Bx7b*+8 appeared in 22 lines with 20 lines being homogeneous and 161

2 lines being heterogeneous, both combining with Bx14+By15. The allele pair 162

Bx14+By15 occurred in 16 lines but none of these lines were homogeneous for this 163

locus; 12 of these lines were grouped with Bx7+By8 and the other four with 164

Bx7b*+By8�Bx7OE+By8 or Bx7+By9. Ten lines possessed a previously unreported 165

allele, Bx7 plus a By subunit with molecular weight of 73000. Sixteen lines had only 166

the By subunit expressed, and 12 lines only had the the Bx subunit expressed. When 167

Bx is silenced, the majority of the By subunit had molecular weight of about 75140, 168

which appeared as a new subunit based on this molecular weight; the only exception is 169

line 814, which had By8 subunit. When By is silenced, the majority (9 out of 12) had 170

the Bx7OE subunit, with 5 lines having only the Bx7OE and 4 lines having both 171

Bx7OE and Bx7. Lines 123 and 684 had only Bx13 and Bx20 expressed at this locus, 172

respectively; while line 227 had both Bx7 and Bx20 expressed. 173

Glu-D1 174

Overall, 84.9% (412 out of 485) lines possessed the GluD1a (Dx2+Dy12) allele, 12% 175

(58 out of 485) lines had the GluD1d (Dx5+Dy10) allele, about 1% (4 out of 485) of 176

the lines had the null allele without any Dx or Dy subunit expressed, 4 lines (about 1%) 177

possessed the GluD1b allele (Dx4+Dy12), and 2 lines had both GluD1a (Dx2+Dy12) 178

and GluD1d (Dx5+Dy10) alleles, representing heterozygotes. There was one line (No. 179

677) that had no expression of y-type, with only the Dx2 subunit expressed. Three 180

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abnormal alleles were detected. Line 163 had Dx2 plus a Dy subunit of 67,900 daltons 181

in mass, line 160 contained Dx2 plus an abnormal Dy subunit with a molecular mass of 182

69,100, and line 830 had Dx2 and another abnormal Dy type subunit of 69,900 daltons. 183

The latter two Dy subunits had significantly higher molecular weight than Dy10 and 184

12. 185

Discussion 186

In this study, the allelic variation of HMW glutenin subunits in 485 wheat accessions 187

collected from the Yangtze-River region of China was analyzed. For the Glu-A1 locus, 188

the most frequent allele was Glu-A1c (74.0%). Zhang et al. (2002), Zhu et al. (2007), 189

Liu et al. (2007) and Li et al. (2009) also reported that Glu-A1c was the predominant 190

allele in Chinese cultivars with the frequency of 91.2% among Chinese landraces, 191

89.6% among 560 local wheat landraces originating from the southwestern winter 192

wheat region in China, 81.4% among 111 accessions of Chinese common wheat 193

landraces, and 90.8% among 390 landraces of China. The most frequent alleles at the 194

Glu-B1 and Glu-D1 loci were Glu-B1b (40.4%), Glu-D1a (84.9%), respectively. Our 195

analyses of landraces in the Yangtze-River region of China are consistent with these 196

previous reports. These results indicated that these alleles displayed a similar allele 197

frequency among Chinese landraces of various sources. 198

Extensive allelic variation in HMW glutenin subunits was detected among the studied 199

landraces. A total of 453 of the accessions were homogeneous for HMW glutenin 200

subunit composition, 32 were heterogeneous and 37 accessions contained abnormal 201

subunits. A total of 22 normal alleles for the Glu-1 loci were detected, 3 belonged to 202

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Glu-A1, 13 at Glu-B1 and 6 at Glu-D1, resulting in 63 different allele combinations. 203

Wei et al. (2000) reported 5 alleles among a collection of 89 landraces from Sichuan 204

province of southwestern China. Zhang et al. (2002) detected 28 HMW glutenin 205

subunit alleles from a core collection of 3,459 Chinese landraces. Liu et al. (2007) 206

discovered 16 alleles when analyzing a collection of 111 landraces from the Hubei 207

province of China. More recently, Chen et al. (2009) reported 26 alleles among a total 208

of 493 landraces from the northwest spring wheat production region of China. Several 209

novel glutenin subunits including 1Ax5*, 1Bx6* (Dai et al., 2004), 1Bx7*, 1By8*, 210

1By8** (Liu et al., 2007), 1Dx2.6 (Cong et al., 2007), 1Bx7** (Fang et al., 2009), 211

1Dx1.5*, and 1Dy12.2* (Guo et al., 2010) have been reported in the Chinese wheat 212

landraces. Among these, the 1Dx1.5* and 1Dy12.2* genes were isolated and the 213

complete open reading frames (ORFs) were obtained. The relationship of the 1Dx1.5* 214

and 1Dy12.2* subunits with dough quality had also been studied (Guo et al., 2010). 215

These abnormal subunits encoded by special genes may play a particular role in 216

determining the viscoelastic properties of wheat flour, meeting new end-product 217

requirement (Shewry et al., 1992, 2003). In our study, apart from the 22 normal alleles, 218

twelve alleles encoding abnormal subunits with molecular weights of 69,100, 69,900, 219

73,000, 73,100, 75,140, 75,600, 76,800, 79,000, 79,100, 79,800, 83,200, 84,300 were 220

also detected. This represented a higher rate of abnormal alleles than previously 221

reported, which is likely due to the enhanced resolution in determining the molecular 222

mass of the MALDI-TOF technology (Liu et al., 2009). A more detailed study is 223

required to match these alleles to previously reported abnormal or novel alleles. 224

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It is known that genes encoding HMWGS of common wheat and emmer wheat are not 225

all expressed (Halford et al., 1989; Gianibelli et al., 2001; Sun et al., 2004). Three 226

different silencing mechanisms derived from the Glu-1 alleles have been reported. The 227

first is the insertion of transposon elements, which has been identified in the Glu-1Ay 228

allele in common wheat (Harberd et al., 1987) and tetraploid wheat (Gu et al., 2006). 229

The second is the presence of a premature stop codon within its coding region via a 230

single base substitution of C→T transition or via the deletion of base A in trinucleotide 231

CAA at the downstream of the coding sequence (Forde et al., 1985; Bustos et al., 2000; 232

Yang et al., 2006�Yuan et al., 2009). The third is caused by a deletion of 247 233

nucleotides from 17 base pairs downstream from the start sequence (Xiang et al., 2010). 234

In this study, a large number of lines had one or two HMW-GS genes silenced, 235

especially 16 lines with the 1Bx gene silenced, of which the silencing mechanism is 236

still unclear. These null alleles are valuable resources for dissecting specific allele 237

effects in wheat quality. 238

To conclude, this study studied the HMW glutenin subunit compositions of 485 wheat 239

landraces in the Yangtze-River region of China. The information obtained in this study 240

may be used by wheat breeders for breeding new cultivars meeting specific 241

end-product needs. In general, 22 HMW glutenin subunit alleles with 16 abnormal 242

subunits were identified in a collection of 485 landraces from the Yangtze-River region 243

of China. Further studies of these novel alleles are currently underway to obtain their 244

coding sequencing in order to match them with previously reported novel alleles. 245

(All accessions used in this study are maintained by Professor Dongfa Sun at the 246

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Huazhong Agriculture University. For research purpose, the collection can be obtained 247

by sending requests to [email protected]) 248

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variation and genetic diversity at Glu-1 loci in Chinese wheat (Triticum aestivum 407

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in Wheat Landraces from Southwestern Winter Wheat Region. Journal of Plant 410

Genetic Resources 8, 401–405 411

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413

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

86999.77

87109.57

86709.7668477.46

87460.11

75118.10

69286.3571219.59

65303.06 77549.8390063.16

414

Figure 1, Line 255: No Bx subunit expressed 415

Dy12 Novel By Ax1 Dx2

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416

417

60015 67015 74015 81015 88015 95015

Mass (m/z)

0

735.6

0

10

20

30

40

50

60

70

80

90

100

% In

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sity

68442.52

68359.06

86983.03

83468.54

83598.3767912.31

87675.54

65318.95 89706.1770205.73 75313.40

418

Figure 2, Line 227: No By subunit expressed. Two Bx subunits, Bx7 and Bx20 419

Dy12 Bx7 Bx20 Dx2

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420

Table 1. HMW-GS compositions of 485 lines 421

Huazhong Serial No. GluA1 GluB1 GluD1 Note

1 n 7OE+8 2+12

2 n 7+8 2+12

3 n 7+8 2+12

4 n 7b*+8 2+12

5 n 7b*+8 2+12

6 1 7+8 2+12

7 n 7+8 2+12

8 n 7+8 2+12

9 1 7+8 2+12

10 n 7+8 2+12

11 n 7b*+8 2+12

12 n 7+8/13 2+12

13 n 7b*+8 2+12

27 1 7+8 2+12

28 n 7+8 2+12

29 n 7+8/17+18 2+12

30 n 7+9 2+12

31 n 7+8/14+15 2+12

32 n 7+8 2+12

33 n 7+8 2+12

34 n 7+8 2+12

35 1 7+? 2+12 73100?

36 1 75140 2+12 no Bx

37 n 20+20 2+12

38 n 7+8 2+12

39 n 7OE+8 2+12

40 n 7OE+8/17+18 2+12

41 1 75130 2+12 no Bx

42 n 7+8 2+12

43 n 7b*+8 2+12

44 n 7+8 2+12

45 1 20+20 2+12

46 1 20+20 2+12

47 n 7+9 n

48 1 20+20 2+12

49 n 7+8 2+12

50 1 7+8 2+12

51 n 7+8 5+10

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52 2* 75150 5+10 no Bx

53 n 7+8 2+12

54 n 7+8 2+12

55 1 7+8 2+12

56 1 7+8 2+12

57 1 7+8 2+12

58 n 7+8 2+12

59 n 7OE+8 2+12

60 n 7+8 2+12

61 1 7OE+8 2+12

62 n 7+8 2+12

63 2* 7+9 2+12

64 n 7+8 2+12

65 n 7+8 2+12

66 n 7+8 2+12

67 1 7+8 2+12

68 n 7+8 2+12

69 1 7+9 2+12

70 n 7+8 2+12

71 n 7b*+8 2+12

72 n 7+8 2+12

73 n 7+8 2+12

74 n 7OE+8 2+12

75 1 7+8 2+12

76 n 7OE+8 2+12

77 n 7OE+8 2+12

78 n 20+20 2+12

79 n 7+8 2+12

80 n 7OE+8 2+12

81 n 7+8 2+12

82 n 14+15/7b*+8 2+12

83 n 7+8 2+12

84 1 7+8 2+12

85 n 7+8 2+12

86 n 7+8 2+12

87 n 7+8 2+12

88 n 7OE+8 2+12

89 n 7+8 2+12

90 1 7OE+8 2+12

91 n 7OE+9 2+12

92 1 7b*+8 2+12

93 n 7+8 2+12

94 n 7+8 2+12

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95 n 20+20 n

96 1 20+20 2+12

97 n 7+8 2+12

98 n 20+20 n

99 n 7+9 2+12

100 1 7+9 2+12

101 1 20+20 2+12

102 n 7+8 2+12

103 1 7+8 2+12

104 n 7+8 2+12

105 n 7+9 2+12

106 n 7+9 2+12

107 1 20+20 2+12

108 1 7+8 2+12

109 n 7OE+8 2+12

110 1 7+8 2+12

111 1 7+8 2+12

112 1 7+8 2+12

113 n 7+8 2+12

114 n 7+9 2+12

115 n 7+8 2+12

116 n 7OE+8 2+12

117 1 7OE+8 2+12

118 1 20+20 2+12

119 n 7+8/14+15 2+12

120 1 7+8 2+12

121 n 7+8 2+12

122 1 7+8 2+12

123 n 13 2+12 no By

124 1 7+8 2+12

125 1 7+? 2+12 73000?

126 1 7+9 2+12

127 n 7b*+8 2+12

128 n 7+8 2+12

129 n 7+8 2+12

130 n 7+8 2+12

131 n 7OE+8 2+12

132 n 7+8 2+12

133 n 7+8 2+12

134 n 7OE+8 2+12

135 n 7OE+8 2+12

136 n 7b*+8 2+12

137 2* 7b*+8 2+12

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138 n 7+8 2+12

139 n 7+8 2+12

140 n 7+8 2+12

141 1 7+9 2+12

142 n 7+8 2+12

143 n 7+8 2+12

144 1 7+8 2+12

145 n 7+8 2+12

146 n 7OE+8 2+12

147 n 7OE+8 2+12

148 n 7+9 2+12

149 n 7+8 2+12

150 n 7+8/14+15 2+12

151 n 7+8 2+12

152 n 7+8 2+12

153 1 7+8 2+12

154 1 20+20 2+12

155 1 7+8 2+12

156 n 7OE+8 2+12

157 1 7+8 2+12

158 n 7+8 2+12

159 n 7+8 2+12

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

161 n 7OE+8 2+12

162 n 7+8 2+12

163 n 7+8 2+? 67900?

164 n 7+8 2+12

165 n 7+8 2+12

166 n 7+9 2+12

167 n 7+8/14+15 2+12

168 1 7+8 2+12

169 1 7+8 2+12

170 n 20+20 2+12

171 n 7+8 2+12

172 n 7+9 2+12

173 1 7+8 2+12

174 n 7+8 2+12

175 2* 7OE+8 2+12

176 n 7+9 2+12

177 n 7OE+8 2+12

178 n 7+8 2+12

179 n 7OE+8 2+12

180 2* 7OE+8 2+12

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181 n 7OE+8 2+12

182 n 7OE+8 2+12

183 n 7+8 2+12

184 2* 7+(73000) 2+12 ?

185 2* 7+8 2+12

186 n 7+8 2+12

187 n 7+8 2+12

188 n 7+8 2+12

189 n 7+8 2+12

190 n 7+8 2+12

191 2* 7+8 2+12

192 n 7+8 2+12

193 n 7+8 2+12

194 n 7+8 4+12

195 n 20+20 2+12

196 n 20+20 2+12

197 n 7+8 2+12

198 n 7+8 2+12

199 n 20+20 2+12

200 2* 7+8 2+12

201 2* 7+8 2+12

202 n 7+8 2+12

203 n 7OE+8 2+12

204 2* 7OE+8 2+12

205 n 7+8 2+12

206 n 14+15/7+8 2+12

207 2* 7OE+8 2+12

208 n 7OE+8 2+12

209 1 7+9 2+12

210 2* 7+(73000) 2+12

211 1 7+9 2+12

212 n 20+20 2+12

213 n 7+8 2+12

214 n 17+18 2+12

215 n 14+15/7OE+8 2+12

216 n 7+8 2+12

217 2* 7+8 2+12

218 n 7+8 2+12

219 n 20+20 2+12

220 n 7+8 2+12

221 n 7+8 2+12

222 2* 7b*+8 2+12

223 n 7+9 2+12

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224 n 20+20 2+12

225 2* 7+8 2+12

226 n 7+9 5+10

227 n 7, 20 2+12 no By

228 n 7+9/14+15 2+12

229 n 7+9 5+10

230 2* 7+8 2+12

231 n 7+8 2+12

232 2* 7+8 2+12

233 n 7+8 2+12

234 n 7+8 2+12

235 n 7b*+8 2+12

236 n 7OE+8 2+12

237 n 7+8/14+15 2+12

238 2* 7+8 2+12

239 n 7b*+8 2+12

240 n 7OE+8 2+12

241 n 7+8 2+12

242 n 7+8 2+12

243 n 7+8 2+12

244 n 7OE+8 2+12

245 n 7OE+8 2+12

246 n 7+8 2+12

247 2* 7+8 2+12

248 n 7+8 2+12

249 n 7OE+8 2+12

250 n 20+20 2+12

251 n 7+8 2+12

252 2* 7+8 2+12

253 n 7b*+8 2+12

254 n 7+8 2+12

255 1 75118 2+12 no Bx

256 n 7+8 2+12

257 n 7+8 2+12

258 n 7+8 2+12

259 n 7+8 2+12

260 n 20+20 2+12

261 n 7OE+8 2+12

262 n 7+8 2+12

263 n 7+(73000) 2+12

264 n 7+8 2+12

265 n 7+8 2+12

266 n 7+(73000) 2+12

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267 n 7+8 2+12

268 n 7+8/14+15 2+12

269 n 7OE+8 2+12

270 n 7+8 2+12

271 n 7b*+8/14+15 2+12

272 n 7OE+8 2+12

273 n 7+8 2+12

274 n 7+8 2+12

275 n 7+8 2+12

276 n 7OE+8 2+12

277 n 20+20 2+12

278 n 7+8 2+12

279 n 7OE+8 2+12

280 n 7OE+8 2+12

281 n 7+8 2+12

282 n 7+8 2+12

283 n 7+9 2+12

284 n 7+9 2+12

285 n 20+20 2+12

286 n 7+8 2+12

287 n 7+8/14+15 2+12

288 n 7OE+8 2+12

289 n 7+8 2+12

290 n 7+8/14+15 2+12

291 n 7OE+8 2+12

292 n 7+8 2+12

293 n 7+8/14+15 2+12

294 n 7OE+8 2+12

295 2* 7OE+8 2+12

296 n 7b*+8 2+12

297 n 7+8 2+12

298 n 7b*+8 2+12

299 n 7OE+8 2+12

300 n 7+8 2+12

301 n 7+8 2+12

302 n 7+8 2+12

303 n 7+8/14+15 2+12

304 n 7OE+8 2+12

305 n 7OE+8/20+20 2+12

306 n 7+8 2+12

307 n 7+(73000) 2+12

308 n 7+8 2+12

309 n 7OE+8 2+12

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310 n 7+8 2+12

311 n 7+8 2+12

312 n 7+8 2+12

313 n 7OE+8 2+12

314 n 7+8 2+12

315 n 7+8 2+12

316 2* 7OE+8 2+12

317 n 7+8 2+12

615 n 7OE+8 2+12

616 n 7+8 2+12

617 n 7b*+8 2+12

618 n 7OE+8 2+12

619 n 7+9 2+12

620 n 7+(73000) 2+12

621 n 7+8 2+12

622 n 7+(73000) 2+12

623 n 7+8 2+12

624 n 7+8 2+12

625 n 7+8 2+12

626 n 20+20 2+12

627 n 20+20 2+12

628 n 20+20 2+12

629 n 7OE+8 2+12

630 n 7+8 2+12

631 n 7+9 2+12

632 n 7OE+8 2+12

633 n 7OE+8 5+10

634 n 7+8 2+12

635 n 20+20 2+12

636 n 7+8 2+12

637 n 7OE+8 2+12

638 n 7+8 2+12

639 n 7+8 2+12

640 n 7+8 2+12

641 1 7+9 2+12

642 n 7+9 2+12

643 n 20+20 2+12

644 n 7+8 2+12

645 n 7+8 2+12

646 n 7OE+8 2+12

647 n 7OE+8 2+12

648 n 7+8 2+12

649 n 20+20 2+12

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650 n 7+8 2+12

651 n 7+8/14+15 2+12

652 n 7+8 2+12

653 n 7+8 2+12

654 n 7OE+8 5+10

655 n 7+8 2+12

656 2* 7OE+8 2+12

657 n 7OE+8 2+12

658 n 7OE+8 2+12

659 n 7OE+8 2+12

660 n 7+8 2+12

661 n 7+8 2+12

662 n 7b*+8 2+12

663 n 7OE+8 2+12

664 n 7+8 2+12

665 n 7+(73000) 2+12

666 n 7+8 2+12

667 n 7+8 2+12

668 n 7+8 2+12

669 n 7b*+8 2+12

670 n 7OE+8 5+10

671 n 7+8 2+12

672 2* 7OE+8 2+12

673 2* 7OE+8 2+12

674 n 7+8 2+12

675 n 20+20 2+12

676 n 7+9 2+12

677 n 7+8 2

678 1 7+9 2+12

679 2* 7OE+8 2+12

680 n 7+8 2+12

681 n 7OE+8 2+12

682 2* 7OE 2+12 no By

683 n 20+20 2+12

684 n 20 2+12 no By

685 n 7OE+8 2+12

686 n 7+9 2+12

687 n 7OE+8 2+12

688 n 20+20 2+12

689 n 20+20 2+12

690 n 7+8 2+12/5+10

691 1 7OE+8 2+12/5+10

692 n 7+8 2+12

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693 n 7OE+8 2+12

694 n 7+8 2+12

695 n 7+8 2+12

696 1 20+20 2+12 83200

697 2* 7+9 2+12

698 n 7OE+8 2+12 83200

699 n 7+8 2+12

700 n 20+20 2+12

740 2* 7+9 5+10

741 2* 7+9 2+12

742 2* 7+9 5+10

743 2* 7+9 5+10

744 2* 7+9 2+12

745 n 17+18 5+10

746 n 17+18 2+12

747 1 75127 5+10 no Bx

748 2* 7+9 2+12

749 n 17+18 2+12

750 n 17+18 5+10

751 2* 7+8/(76800)+ (78600) 2+12

752 2* 7+9 5+10

753 n 7OE 5+10 no By

754 2* 7+9 5+10

755 n 7+9/20+20 5+10

756 2* 7+9 2+12

757 n 7+9 5+10

758 n 7OE 2+12 no By

759 n 7+9 5+10

760 2* 7OE+8/17+18 2+12

761 2* 7+9 2+12

762 n 7+9 5+10

763 n 7+9 5+10

764 n 7+9 5+10

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

770 n 7OE+8 2+12

771 2* 7+9 2+12

772 n 7+9 5+10

773 n 7+9 5+10

774 n 7+9 5+10

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775 2* 7+9 2+12

776 n 7OE+8 5+10

777 n 7OE 2+12 no By

778 1 75133 2+12 no Bx

779 2* 75140 5+10 no Bx

780 n 17+18 2+12

781 n 7OE+8 2+12

782 n 7+8 5+10

783 n 7OE+8 2+12

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

785 2* 7+9 2+12

786 2* 7+9 4+12

787 n 7+8 2+12

788 n 7+8 2+12

789 2* 17+18 2+12

790 2* 7+9 4+12

791 n 7OE 5+10 no By

792 2* 17+18 2+12

793 n 7+9 5+10

794 n 7+9 5+10

795 n 7OE+8 5+10

796 n 17+18 5+10

797 2* 75100 2+12 no Bx

798 1 7b*+8 2+12

799 n 7OE+8 5+10

800 2* 7+9 4+12

801 n 7+8 5+10

802 n 7+9 5+10

803 2* 7OE/17+18 5+10

804 2* 17+18 5+10

805 n 7+9 5+10

806 2* 75136 5+10 no Bx

807 n 17+18 2+12

808 n 17+18 5+10

809 2* 7+9/17+18 5+10

810 n 13+16 5+10

811 n 7OE/17+18 5+10

812 n 7OE/7 5+10 no By

813 2* 7+9 2+12

814 2* 8 5+10 no Bx

815 n 7+9 5+10

816 n 7OE+8 2+12

817 2* 75120 2+12 no Bx

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818 n 17+18 5+10

819 n 75118 2+12 no Bx

820 n 7+9 5+10

821 n 7+9 5+10

822 1 7OE/7 5+10 no By

823 2* 7+9 2+12

824 2* 7+9 2+12

825 2* 17+18 2+12

826 n 7OE+8 2+12

830 1 7+8 2+? 69900?

831 n 7OE+8/7+8 2+12

832 2* 7+9 2+12

833 n 7+9 5 No Dy

834 n 7+9 n

835 n 17+18 5+10

836 n 7OE+8 5+10

837 n 20+20 5+10

422

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ACCEPTED MANUSCRIPT

We have put together three points to highlight our discovery:

1. MALDI-TOF was used to analyse 485 wheat landraces from the Yangtze-river region of China;

2. High frequency of abnormal HMWGS alleles is identified; 3. A total of 37 lines contained abnormal subunits with 16 new alleles.