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Ind J Plant Physiol. (January–March 2015) 20(1):97–102 DOI 10.1007/s40502-015-0141-z

SHORT COMMUNICATION

Allelic variations of functional markers for high molecular weight glutenin genes in Indian wheat (Triticum aestivum L.) cultivars and their correlation with bread loaf volume Umesh Goutam • Ratan Tiwari • R. K. Gupta Sarvjeet Kukreja • Ashok Chaudhury

•

Received: 26 May 2014 / Accepted: 1 February 2015 / Published online: 12 February 2015 Ó Indian Society for Plant Physiology 2015

Abstract Glutenins, the polymeric proteins are composed of high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits. High molecular weight glutenin subunit (HMW-GS) plays a key role in determining bread making quality. The HMW-GS encoded by Glu-1 loci (GluA1, GluB1 and GluD1) are highly polymorphic. Thus it is very important to identify the alleles at Glu-1 loci for all three genomes for wheat quality improvement. In the present study 53 Indian wheat genotypes were evaluated for HMW-GS composition using PCRbased DNA markers. In total, two alleles (Ax2*, Ax1 or Axnull) at Glu-A1, four allele/allelic pairs (Bx7, Bx7 ? By8, Bx7 ? By9, Bx17 ? By18) at Glu-B1 and two allelic pairs (Dx2 ? Dy12 and Dx5 ? Dy10) at Glu-D1 were identified using diagnostic DNA markers. Allele or allelic pair Ax2* (62.3 %) at Glu-A1, Bx7 ? By8 (64 %) at Glu-B1 and Dx2 ? Dy12 (58.5 %) at Glu-D1 were present most frequently in wheat genotypes taken for study. Moreover, the allelic combination Ax2*, Bx17 ? By18 and Dx5 ? Dy10 exhibited highest bread loaf volume in comparison to other allelic combinations studied. The set of markers used here thus represents a useful alternative for

U. Goutam R. Tiwari R. K. Gupta Directorate of Wheat Research, Post Box 158, Karnal 132001, India U. Goutam A. Chaudhury Department of Bio and Nanotech, Guru Jambheshwar Univeristy of Science & Technology, Hisar, India U. Goutam (&) S. Kukreja Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India e-mail: [email protected]

screening of HMW-glutenin allele (s) for improvement of bread making quality in Indian wheat genotypes. Keywords Triticum aestivum L. HMW glutenin Functional marker Bread loaf volume Among the cereals, wheat is unique because its flour alone has the ability to form dough that exhibits the rheological properties required for the wider diversity of food preparations. In dough, most of the proteins are converted into gluten complex. The gluten proteins consist of glutenin, responsible for elasticity of dough and gliadins, for providing extensibility to dough (Gianibelli et al. 2001; Maucher et al. 2009). Glutenins are composed of high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (GS) based on their mobility on SDS-PAGE. The HMW-GS are encoded by genes at the Glu-1 loci, present on the long arms of the homeologus group 1 chromosome (Payne and Lawrence 1983). At each Glu-1 locus (Glu A-1, Glu B-1 and Glu-D1), there are two tightly linked HMW-GS genes, which encode two types of subunits, the larger one termed as x-type and the smaller one as y-type (Harberd et al. 1986). Allelic variation existing at each of the HMW glutenin loci demonstrated that dough strength and elasticity is largely influenced by the alleles present at the Glu-D1 locus. The HMW glutenin subunits Dx5 ? Dy10 have been associated with high dough strength and good bread making quality as compared to allelic subunits Dx2 ? Dy12 (Sramkova et al. 2010). In the past, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) has been routinely used to analyze large number of HMW-GS alleles in Indian wheat cultivars (Ram 2003; Singh et al. 2007). However, study by (Gianibelli et al. 2002) has revealed that SDSPAGE analysis of wheat glutenin sometimes results in

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incorrect interpretation of different HMW-GS alleles. The method is time consuming and is not well suited to the high throughput system (Ma et al. 2003). In an effort to overcome these constraints, the availability of PCR-based DNA markers serves as a valuable tool for selection of desired trait with relative ease. The use of PCR-based DNA markers significantly supplements the alternative to SDSPAGE. In last two decades, number of PCR-based DNA markers has been used to differentiate between different HMW-GS alleles (Goutam et al. 2013). The current study thus focuses on the use of identified DNA markers to differentiate between HMW-GS alleles in the bread wheat genotypes grown in different agroclimatic zones of India. Fifty three spring wheat cultivars from five wheat agroclimatic zones of India, viz, North Western Plains Zone (NWPZ), Northern Hills Zone (NHZ), North Eastern Plains Zone (NEPZ), Central Zone (CZ) and Peninsular Zone (PZ) were included in this study. All fifty three wheat cultivars were grown over two crop seasons (2008–2009 and 2009–2010). The experimental material represents the cultivars released during 1965–2011 (Table 1). DNA isolation and PCR analysis was done in Plant Molecular Biology laboratory, Directorate of Wheat Research (DWR), Karnal, India. Genomic DNA was extracted from one-week-old seedlings by SDS method (Dellaporta et al. 1983). The primer sequences, expected product size and annealing temperatures of amplification products are shown in supplementary data. Polymerase chain reaction (PCR) amplifications were carried out in 25 ll volume with STS markers for HMW-GS genes (Ahmad 2000; Ma et al. 2003 and Lei et al. 2006). Bread was baked in Quality and Basic Sciences laboratory, DWR, Karnal, India according to the standard AACC method 26-10 (2000) with slight modifications and loaf volume was measured by the rapeseed displacement method (Dhingra and Jood 2004). A total of 53 bread wheat genotypes were characterized for allelic combinations of HMW-GS allelic composition. Several methods based on biochemical and molecular techniques are available to detect the presence of HMWGS composition (Goutam et al. 2013) in wheat. In the present study, we evaluated the utility of six PCR-based DNA markers for detecting the presence of HMW-GS alleles. All markers were able to amplify the bands that distinguished between wheat genotypes for different HMW glutenin alleles at Glu-1 loci. Using Ax2* allele-specific PCR primers (Ahmad 2000), a band of 1.319 kb was obtained in 33 genotypes, whereas, amplicon was absent in 20 Ax1 or Axnull genotypes. Out of 53 wheat genotypes, 33 genotypes showed the expected product size of 1.319 kb, indicating the presence of Ax2*

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Ind J Plant Physiol. (January–March 2015) 20(1):97–102

allele, whereas absence of a PCR product in remaining twenty genotypes indicated presence of either Ax1 or Axnull allele (Fig. 1a). Four alleles (Bx7, Bx17, By8 and By9) of Glu-B1 locus were amplified by PCR-based markers developed by Ma et al. (2003) and Lei et al. (2006). PCR analysis using Bx primers (Bx7/Bx17) produced single DNA fragment (669 bp) in genotypes containing the Bx17 allele and two fragments (630 and 766 bp) in genotypes for Bx7 allele/ allelic pair (Fig. 1b). Among the 53 Indian wheat genotypes screened with this marker, thirty four genotypes were found positive for Bx7 allele/allelic pairs (Bx7 ? By8, Bx7 ? By9) and 15 were found positive for Bx17 allele (Table 1). Remaining four genotypes, viz, C 306, HI 1500, HD 2888 and HW 2004 did not show PCR amplicon for Bx7 or Bx17 allele. The genotypes containing By8 allele produced a 527 bp (Fig. 1c) fragment, while non-By8 genotypes were negative for amplicon. Sixteen genotypes showed presence of By8 allele (Table 1). The allele specific primers for identification of different HMW-glutenin allele were used to amplify By9 gene segments. Using By9 AS-PCR primers, a band of 707 bp (genotypes carrying By9 allele) was obtained in 14 genotypes, whereas, amplicon of 662 bp (genotypes carrying non By9 allele) was present in 39 wheat genotypes (Fig. 1d, Table 1). Ma et al. (2003) studied 50 Australian wheat genotypes and reported 39.3 % genotypes with Bx17 allele whereas 36.4 % genotypes were found positive for Bx7 allele. When result of all four AS-PCR (Table 1) primers was collectively analyzed it was found that 64 % genotypes showed presence of Bx7allele/allelic pairs (Bx7, Bx7 ? By8, Bx7 ? By9) while remaining 28 % genotypes showed presence of Bx17 allele (Table 1). This data indicates that Bx7 allelic pair is more prominent in Indian wheat genotypes as compared to Bx17 allele. A multiplex PCR assay for fifty three wheat genotypes was carried out which permitted the discrimination of the major HMW glutenins in a single PCR reaction and agarose gel assay. Primer combinations (Dx5 and Dy10/12) were used to establish a simple multiplex PCR assay to distinguish both alleles of HMW glutenin, 1Dx and 1Dy, respectively (Fig. 1e). In the present study, 22 genotypes amplified 450 and 576 bp fragments, indicating the presence of Dx5 and Dy10 alleles, respectively. Remaining 31 genotypes amplified 612 bp fragment associated with Dy12 allele (Table 1), whereas absence of Dx5 allele indicating presence of Dx2 allele. The overall frequencies of allelic pairs Dx5 ? Dy10 and Dx2 ? Dy12 were 41.5 and 58.5 %, respectively. It shows that Dx2 ? Dy12 allelic pair is predominantly present in Indian wheat genotypes. Table 1 shows the range of variation of bread loaf volume (BLV) of 53 bread wheat genotypes studied. The BLV varied from 498 ml (HI 1500) to 606 ml (HI 977) with an

HS 490

HS 507

Sonalika

32

33

34

HI 1563

22

VL 892

HD 2967

21

HS 420

MACS 6145 HD 2888

19 20

31

K 0307

18

30

NW 1014

17

HS 295

HUW 234

16

VL 829

K 8027

15

29

NW 2036

14

28

DBW 14

13

VL 829

HD 2733

12

28

K 9107

11

HS 277

C 306

10

27

HI 1544

9

VL 616

HW 2004

8

VL 804

HI 1500

7

26

Raj 4037

6

25

GW 173

5

HS 240

MP 4010 DL 788-2

3 4

VL 738

GW 322

2

23

Lok 1

1

24

Cultivar

S.no.

II54-388/AN/3/YT54/N10B/LR 64

KAUZ/MYNA/VUL/BUC/FLK/4/MILAN

HS 364/HPW 114//HS 240/HS 346

WH 542/PBW 226

LAJ 3302//CMH 73A-497/3*CNO 79

(OT-AZ/IA 555-ALD’’S‘‘/ALD’’S‘‘-NATN) PJN’’S‘‘-PEL ’’S‘‘.

BWSN/CPAN2099

BWSN/CPAN2099

KVZ/CGN

SONALIKA/CPAN 1507

CPAN 3018/CPAN 3004//PBW 65

NS12.07/LIRA’’S’’//VEE ,,S’’

AU/KAL-BB//WOP’’S‘‘/PAVON’’S‘‘

MACS 2496*2/MC 10

ALD/COC//URES/HD2160 M/HD2278

C306 ? LR28 C 306/T. sphaerococcum//HW 2004

K 8321/UP 2003

HAHN ,,S‘‘

HUW 12* 2/CPAN 1666//HUW 12

NP875/4/N10B/Y53//Y50/3/KT54B/5/2*K852

BOW/CROW/BUC/PVN

RAJ 3765/PBW 343

ATTILA/3/TUI/CARC//CHEN/CHTO/4/ATTILA

K 8101/K 68

RGN/CSK3//2*C591/3/C217/N14//C281

HINDI 62/BOBWHITE/CPAN 2099

C 306 *7//TR 380-14 #7/3 AG14

HW2002*2//STREMPALLI/PNC 5

DL 788-2/RAJ 3717

TW275/7/6/1/LOK-1

ANGOSTURA 88 K7537/HD2160/HD2278//L24/K4.14

GW 173/GW 196

S308/S331

Pedigree

NWPZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NHZ

NEPZ

NEPZ

NEPZ NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

NEPZ

CZ

CZ

CZ

CZ

CZ

CZ CZ

CZ

CZ

Zone

1965

2010

2007

2007

2002

1992

2002

2002

1992

1986

2002

1996

1989

2010

2011

2002 2005

2006

1997

1985

1984

2002

2002

2001

1995

1965

2007

1995

2002

2003

1994

2002 1996

2002

1981

Year of release

?

–

?

?

?

?

–

–

–

?

?

–

–

?

?

? –

?

?

?

?

–

?

?

?

–

–

–

?

?

?

? –

?

?

Ax2*

?

?

?

?

?

–

?

?

?

?

?

?

?

?

–

– –

–

?

?

–

?

?

?

–

–

?

–

–

–

?

– –

?

–

BX7

–

–

–

–

–

?

–

–

–

–

–

–

–

–

?

? –

?

–

–

?

–

–

–

?

–

–

–

–

?

–

? ?

–

?

BX17

–

?

?

?

?

–

–

–

–

–

–

?

–

?

–

– –

–

?

?

–

?

?

–

–

–

?

–

–

–

?

– –

?

–

BY8

?

–

–

–

–

–

?

?

?

?

?

–

?

–

–

– –

–

–

–

–

–

–

?

–

–

–

–

–

–

–

– –

–

–

BY9

?

–

?

?

?

?

?

?

–

?

?

–

–

?

–

– ?

?

?

?

?

–

?

–

–

?

?

?

?

?

?

? ?

?

–

Dx2 ? Dy12

Table 1 Allelic variation of HMW glutenin genes at Glu-A1, Glu-B1, Glu-D1 loci and mean bread loaf volume (BLV) of 53 Indian wheat genotypes

–

?

–

–

–

–

–

–

?

–

–

?

?

–

?

? –

–

–

–

–

?

–

?

?

–

–

–

–

–

–

– –

–

?

Dx5 ? Dy10

531

557

517

532

534.5

515.3

520

520

521.8

512

523.3

558

549.3

555

568

502.8 521.5

603

538

547

538.8

598

543.8

576.3

599.5

503.3

538

519.5

498

573

567.7

560 564

535.3

573.5

BLV (ml)

Ind J Plant Physiol. (January–March 2015) 20(1):97–102 99

123

123

UP 2425

PBW 175

PBW 396

DBW 17

PBW 502 PBW 550

PBW 590

HD 2329

NI 5439

Raj 4083

HD 2189

40

41

42

43

44 45

46

47

48

49

50

HD 2932

Raj 3765

39

53

PBW 373

38

NIAW 34

PBW 343

37

HI 977

HD 2687

36

51

WH 542

35

52

Cultivar

S.no.

Table 1 continued

KAUZ/STAR//HD 2643

GLL/AUST 61.157/CNO/NO 66/3/Y50E/3/KAL

CNO 79/PRL ‘‘S’’

HD 1963/HD 1931

PBW 343/UP 2442//WR 258/UP 2425

REMP 80/3* NP 710

HD 1962/E 4870/3/K 65/5/HD1553/4/UP262

WH 594/RAJ3814//W 485

W 485/PBW 343//RAJ 1482 WH 594/RAJ 3858//W 485

CMH79A.95/3*CNO 79//RAJ3777

CNO67/MFD//MON’’S’’/3/SERI

HD 2160/WG 1025

HD 2320/UP 2263

HD 2402/VL639

ND/VG9144//KAL/BB/3/YCO’’S‘‘/4/VEE#5 ’’S‘‘

ND/VG9144//KAL/BB/3/YCO’’S‘‘/4/VEE#S ’’S‘‘

CPAN2009/HD 2329

JUP/BJY’’S’’//URES

Pedigree

PZ

PZ

PZ

PZ

PZ

PZ

NWPZ

NWPZ

NWPZ NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

NWPZ

Zone

2007

1985

1995

1979

2006

1973

1982

2008

2003 2007

2006

1999

1989

1999

1995

1996

1995

1999

1992

Year of release

?

?

–

?

–

–

?

?

– ?

?

–

?

–

–

–

–

?

?

Ax2*

–

–

?

–

?

–

?

?

? ?

?

?

?

–

?

?

?

?

?

BX7

?

?

–

?

–

?

–

–

– –

–

–

–

?

–

–

–

–

–

BX17

–

–

–

–

?

–

–

–

– –

–

–

?

–

?

–

–

–

–

BY8

–

–

?

–

–

–

?

?

– ?

–

?

–

–

–

–

–

?

?

BY9

?

–

–

?

–

?

?

–

– –

–

–

?

–

?

–

–

?

–

Dx2 ? Dy12

–

?

?

–

?

–

–

?

? ?

?

?

–

?

–

?

?

–

?

Dx5 ? Dy10

585

606.5

535.3

577.5

590.3

592

500

562

550.3 544

564.5

549.3

512.8

560

542

557

544.5

526.5

542.8

BLV (ml)

100 Ind J Plant Physiol. (January–March 2015) 20(1):97–102


Fig. 1 a PCR amplification of HMW glutenin Ax2* gene with allelespecific PCR marker in bread wheat genotypes. 1 DBW 14, 2 DBW 17, 3 GW 173, 4 GW 322, 5 HD 2189, 6 HD 2733, 7 VL 616, 8 HS 490, 9 HI 977, 10 HS 420, 11 HW 2004, 12 K 0307, 13 K 8027, 14 1-Kb DNA ladder. b PCR amplification of HMW glutenin alleles Bx7/Bx17 in bread wheat genotypes with allele specific-PCR marker related to bread quality. 1 DL 788-2, 2 GW 173, 3 HD 2189, 4 HD 2687, 5 HS 295, 6 HS 277, 7 C 306, 8 HS 420, 9 K 0307, 10 NIAW 34, 11 NI 5439, 12 PBW 175, 13 100–1,200 bp DNA ladder. c PCR amplification of HMW glutenin By8 gene with allele-specific PCR marker in bread wheat genotypes. 1 HI 977, 2 HD 2329, 3 C 306, 4 DBW 17, 5 DBW 14, 6 DL 788-2, 7 HD 2189, 8 GW 173, 9 HD 2687, 10 HD 2733, 11 HI 1500, 12 HS 240, 13 HS 277, 14 GW 322, 15 HS 295, 16 HW 2004, 17 K 0307, 18 K 8027, 19 HS 420, 20 100 bp DNA ladder. d PCR amplification of HMW glutenin By9 gene with allelespecific PCR marker in bread wheat genotypes. 1 HI 977, 2 HD 2329, 3 HS 295, 4 K 0307, 5 HD 2733, 6 K 8027, 7 NIAW 914, 8 1.2 Kb DNA ladder. e Multiplex PCR for amplification of HMW glutenin 1DX5 and 1Dy10/1Dy12 alleles with 450 and 576/612 bp DNA fragments, respectively from bread wheat genotypes. 1 100 bp DNA ladder, 2 PBW 343, 3 HI 977, 4 PBW 373, 5 NIAW 34, 6 HW 2004, 7 HI 1500, 8 MACS 6145, 9 RAJ 3765

average value of 547.8 ml. Higher BLV (more than 575 ml) was found only in nine genotypes (K0 307, HD 2189, HD 2932, NI 5439, NW 2036, K 9107, RAJ 4083, HD 2733 and HI 977), 14 genotypes were found to possess

101

moderate BLV (550–575 ml), whereas, 30 genotypes showed poor (525–550 ml) and inferior values of BLV (\525 ml). Out of 33 genotypes (carrying Ax2* allele), only six genotypes viz., HI 977, K 9107, K 0307, HD 2189, HD 2932 and HD 2733 showed higher BLV (Table 1). Inspite of the presence of Ax2* allele in remaining twenty seven genotypes, BLV was moderate to low. It might be because of the fact that out of 27 genotypes, 20 genotypes were also carrying the Dx2 and Dy12 alleles responsible for poor bread making quality (Payne and Lawrence 1983) whereas, remaining seven genotypes showed moderate value of BLV with presence of favorable alleles Dx5 and Dy10. At Glu-B1 locus, 34 genotypes carrying Bx7 ? By8 and Bx7 ? By9 allele/allelic pairs, 31 genotypes showed moderate to lower values of BLV (Table 1). Out of these 31 genotypes, 18 genotypes showed poor BLV and 13 genotypes showed moderate value with presence of allelic pair Dx2/Dy12 and Dx5/Dx10, respectively (Payne and Lawrence 1983). Out of 15 genotypes carrying Bx17 ? By18 allelic pair, only three genotypes, viz, HS 295, K 8027 and MACS 6145 was found with poor BLV (Table 1). Two genotypes, viz, HS 295 and K 8027 were also carrying poor bread making alleles Dx2 and Dy12. Lawrence et al. (1987) could not find any difference in dough quality between Bx7 ? By8 and Bx17 ? By18 subunits but they reported that Bx17 ? By18 provided greater loaf volume and was as effective as Dx5 ? Dy10 for improving dough and baking quality (Lawrence et al. 1987). At Glu-D1 locus, out of 22 genotypes carrying Dx5 ? Dy10 allelic pair, only two genotypes, viz, MACS 6145 and HS 277 showed inferior value of BLV, whereas six genotypes, viz., HS 240, WH 542, PBW 343, PBW 396, PBW 550 and NIAW 34 were found with reasonably poor values of BLV. Out of these eight genotypes having poor BLV, seven genotypes were not carrying favorable alleles at Glu-B1 loci (Bx17 ? By18) and one genotype MACS 6145 exceptionally showed poor BLV, inspite of having all the favorable alleles. Out of 31 genotypes (carrying Dx2 ? Dy12 allelic pair), only four genotypes namely, K 0307, NI 5439, HD 2932 and HD 2189 showed higher values of BLV (Table 1). It might be because of the fact that these genotypes also have other favorable allelic pair Bx17 ? By18 at Glu-B1 locus, which contributed positively as Dx5 ? Dy10 allelic pairs for bread quality (Lawrence et al. 1988). The effect of interaction of HMW-glutenin allelic combination on BLV was also analyzed. The relationship between different combination of HMW-glutenin subunits and bread loaf volume is shown in Fig. 2. The highest mean value was observed for HMW-GS combination Ax2*, Bx17 ? By18 and Dx5 ? Dy10, whereas Ax2*,

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Bread loaf volume (ml)

600 580 560 540 520 500 17+18, 5+10 17+18, 2+12

7+9, 5+10

7+8, 2+12

7+9, 2+12

HMW glutenin allelic combination

Fig. 2 Relationship between different combination of HMW-glutenin subunits and bread loaf volume of 53 bread wheat genotypes

Bx7 ? By9 and Dx2 ? Dy12 subunits combination showed lowest mean value. Out of 53 genotypes, only five genotypes, viz, HI 977, K 9107, HD 2967, LOK 1 and MACS 6145 showed favorable allelic combination (Ax2*, Bx17 ? By18 and Dx5 ? Dy10) in Indian wheat genotypes. This indicates that HMW-GS Dx5 ? Dy10 are superior to Dx2 ? Dy12 for good bread making properties (Payne and Lawrence 1983; Gupta and MacRitchie 1991; Luo et al. 2001).

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Goutam, U., Kukreja, S., Tiwari, R., Chaudhury, A., Gupta, R. K., Dholakia, B. B., & Yadav, R. (2013). Biotechnological approaches for grain quality improvement in wheat: Present status and future possibilities. Australian Journal of Crop Science, 7, 469–483. Gupta, R. B., & MacRitchie, F. (1991). A rapid one-step onedimensional SDSPAGE procedure for analysis of subunit composition of glutenin in wheat. Journal of Cereal Science, 14, 105–109. Harberd, N. P., Bartels, D., & Thompson, R. D. (1986). DNA restriction fragment variation in the gene family encoding highmolecular-weight (HMW) glutenin subunits of wheat. Biochemical Genetics, 24, 579–596. Lawrence, G. J., MacRitchie, F., & Wrigley, C. W. (1988). Dough and baking quality of wheat lines deficient in glutenin subunits controlled by the Glu-Al, Gut-B1 and Glu-D1loci. Journal of Cereal Science, 7, 109–112. Lawrence, G. J., Moss, H. J., Shepherd, K. W., & Wrigley, C. W. (1987). Dough quality of biotypes of eleven Australian wheat cultivars that differ in high-molecular-weight glutenin subunit composition. Journal of Cereal Science, 6, 99–101. Lei, Z. S., Gale, K. R., He, Z. H., Gianibelli, C., Larroque, O., Xia, X. C., et al. (2006). Y-type gene specific markers for enhanced discrimination of high-molecular weight glutenin alleles at the Glu-B1 locus in hexaploid wheat. Journal of Cereal Science, 43, 94–101. Luo, C., Griffin, W. B., Branlard, G., & McNeil, D. L. (2001). Comparison of low and high molecular weight wheat glutenin allele effects on flour quality. Theoretical Applied Genetics, 102, 1088–1098. Ma, W., Zhang, W., & Gale, K. R. (2003). Multiplex-PCR typing of high molecular weight glutenin alleles in wheat. Euphytica, 134, 51–60. Maucher, T., Figueroa, J. D. C., Reule, W., & Pena, R. J. (2009). Influence of low molecular weight glutenins on viscoelastic properties of intact wheat kernels and their relation to functional properties of wheat dough. Cereal Chemistry Journal, 86, 372–375. Payne, P. I., & Lawrence, G. J. (1983). Catalogue of alleles for the complex gene loci, Glu-A1, Glu-B1, and Glu-D1 which code for high-molecular weight subunits of glutenin in hexaploid wheat. Cereal Research Communications, 11, 29–35. Ram, S. (2003). High molecular weight glutenin subunit composition of Indian wheats and their relationships with dough strength. Journal of Plant Biochemistry and Biotechnology, 12, 151–155. Singh, A. M., Devaeshwar, J. J., Ahlawat, A. K., & Singh, B. B. (2007). Identification of novel variants of high molecular weight glutenin subunits in Indian bread wheat landraces. Cereal Research Communications, 35, 99–108. Sramkova, Z., Gregova, Z., Slikova, S., & Studike, E. (2010). Wheat varieties released in Slovakia and their bread-making quality. Cereal Research Communications, 38, 386–394.