J. Genet. & Breed. 56: 245-250 (2002)
Polymorphism of gluten proteins in Indian dicoccum wheat (Triticum turgidum ssp. dicoccum) revealed by SDS and Acid-PAGE M.D. Oak, S.A. Tamhankar, V.S. Rao* and S.B. Bhosale Agharkar Research Institute, Pune, 411 004, India. Fax: +91-20-5651542 E-mail:
[email protected] Received October 17, 2001 ABSTRACT Analysis of glutenin and gliadin profiles in 42 dicoccum wheats, comprising 37 local genotypes, two exotic genotypes and three released varieties, revealed five different patterns for high molecular weight (HMW) glutenin subunits, resulting from the combination of two alleles at Glu-Al locus and three alleles at the Glu-Bl locus. Glu-All and Glu-Blh were the most frequent alleles detected. Most of the dicoccurs also had a protein product encoded by the Glu-Al locus, which is generally not seen in durum wheats. Three different g-gliadin fractions coded by Gli-Bl locus were detected. Five different LMW-B glutenin patterns were detected at Glu-3 loci. At GH-A2 only one variety showed a - 1 allele and rest of them showed a - 2 allele. Only one commercial dicoccum variety DDK1001 showed the presence of ggliadin 45 in A-PAGE and also the presence of Glu-B3 allele, associated with good pasta quality. Key Words: Electrophoresis, Gliadins, Glutenins, Quality.
INTRODUCTION Wheat is the world's most important food grain, consumed by humans as whole grains, cracked grain particles either semolina or flour, breakfast cereals and several leavened products. The end use quality of wheat largely depends on the quality and quantity of gluten proteins (PAYNE, 1987; CARRILLO et al, 1990a; POGNA et at, 1990). Early studies (DAMIDAUX et al, 1978; PAYNE, 1984) demonstrated the usefulness of gliadin g - 4 5 and g - 4 2 , encoded at Gli-Bl locus (JOPPA et al, 1983), as biochemical markers of good and poor pasta quality, respectively. Further studies (POGNA et al, 1988; 1990; Ruiz and CARRILLO, 1995) showed that the low molecular weight (LMW) glutenin subunits, encoded at the Glu-3 loci and tightly linked to the Gli-1 loci (SINGH and SHEPHERD, 1988) were responsible for differences in quality. Recent studies have reported the effect of few allelic variants at the Glu-A3, Glu-B3 and Glu-B2 loci on pasta making quality (Ruiz and CARRILLO, 1995; VAZQUEZ et al, 1996; NIETO-TALADRIZ et al, 1997). The hulled wheats are becoming increasing* Corresponding author.
ly popular as eco-friendly cereal crops for the production of special traditional nutritious, attractive foods such as extruded, cooked products and breakfast cereals (CORBELLINI et al, 1999; MARCONI et al, 1999). Triticum dicoccum is one such primitive, hulled, allotetraploid emmer species. This wheat has high protein content, high lysine and fiber (GALTERIO et al, 1994). Its consumption reduces the risk of colon cancer and heart diseases and hence dicoccum wheatbased foods are being promoted as health food (ICARDA, 1991). India is one of the few countries in the world where dicoccum wheat (locally known as Khapli or Popatiya) is under cultivation (50,000 ha area) in some parts of Karnataka, Maharashtra and Gujarat states. This hard and highly vitreous dicoccum wheat is preferred to produce several types of salty and sweet traditional products (MISRA, 1998). The evaluation of emmer (T. dicoccum) wheat for characteristics of technological importance, such as protein content, gluten strength and pasta making, is only in its initial stage. Earlier reports on electrophoretic analysis of storage proteins in T. dicoccum have revealed
246 TABLE 1 Glutenin and gliadins in Indian dicoccum wheat genotypes Glu-Bl
Glu-Al
Serial Name
Glu-3
Gli-Bl
No.
Allele 1 2 3 4 5 6 7 8 9 10 11 12 13 14
15 16 17 18
19 20 21 22 23
24 25 26 27 28 29 30 31 32 33
34 35 36
37 38 39 40 41 42
Arbhavi Local Azar DDK1001 DDK1009 Ex - 33 Ex - 7 Farrum K-6413 HW- 19 HW- 24 HW- 27 HW- 28 HW- 65 HW- 66 HW- 67 HW- 68 HW- 70 HW- 71 HW- 72 HW- 75 HW-2 HW-63
I I C
KDH
I I
Khapli - 53 Khapli 2-9-8 Khapli pink
Khapli-1 Khapli-2 Khapli-3 Khapli-4 MACS 2574 N - 4914 NP - 200 NP - 201 NP -202 Popatiya RL 4045 RL 5045 Rufrum II 2701 Sangali 2-2 TTC
Ugar khapli Yellow Khapli
I I I I I I I I I I I
I I I I I I
I
I I I I I I
c
I I I I I I
I I
I I I I
Band
Allele
Band
I I N I I I I I 1 I I I I I I I I I I I I I I I I
h h b b
14+15 14+15
I
IV
I I I N I I I I I I I I I I I I
h h h h h
considerable allelic variation at the Glu-1 and Glu-3/Gli-l loci coding for glutenins and gliadins (VALLEGA and WAINES, 1987; GALTERIO et al., 1994;
LIU and SHEPHERD, 1996, PFLUGER et al., 2001).
However, relatively little work has been done to exploit this genetic variability for practical purposes.
IV IV
h h
h h
h h h h h IV IV
h h IV
h h h h h
IV IV IV
h h h h IV
h h h
7+8 7+8
23+18 23 + 18 14+15 14+15 14+15 14+15 14+15 14+15 14+15 14+15 14+15 23 + 18 23 + 18 14+15 14+15 23 + 18 14+15 14+15 14+15 14+15 14+15 23 + 18 14+15 14+15 14+15 14+15 14+15 23 + 18 23 + 18 23+18 14+15 14+15 14+15 14+15 23+18 14+15 14+15 14+15
LMW-5 LMW-5 LMW-3 LMW-5 LMW-5 LMW-2 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-1 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-4 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-5 LMW-1
44 44
45 44 44 47 44 44 44 44 44 44 44 44
44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44
44 44 44 44 44 44
This study was undertaken to broaden the present knowledge of gluten protein profiles of Indian dicoccum genotypes in relation to their end use quality. The present paper describes the variability of dicoccum gluten protein profiles, mainly g-gliadins and low molecular weight glutenins (LMW B subunits).
247 MATERIALS AND METHODS
TABLE 2 Varieties used as standards for the Glu-1 and Gli-Bl loci
Plant material
Locus
Forty-two dicoccum wheat (T. dicoccum) genotypes analysed in the present study are listed in Table 1. These were selected from the dicoccum. wheat collection at Agharkar Research Institute, Pune, India. Among them, 37 are local genotypes, 2 are exotic and 3 released varieties. Seeds of the varieties used as standards for glutenin and gliadin subunits were procured from Directorate of Wheat Research, Karnal, India and Dr. Branlard, Station d'Amelioration des Plantes, Clermont-Ferrand, France (Table 2).
Glu-Al
Glu-Bl
Extraction and electrohoresis High and low molecular weight glutenins and gliadins were extracted from single seed of each genotype. At least six seeds per genotype were analyzed in order to verify their protein homogeneity. Glutenins were extracted following sequential extraction procedure of SINGH et al (1991) with slight modifications. First, 100ml 70% alcohol was added to powdered seed, incubated at 37C° for 20 min and then centrifuged at 9000 x g for 5 min. Supernatant was used for gliadin electrophoresis, which was carried out according to the procedure described by KHAN et al. (1985). Protein bands were numbered according to BUSHUK and ZILLMAN (1978) using Marquis as standard cultivar. Residue was extracted three times (30 min) with 50% (v/v) propan-1-ol and discarded to remove gliadins. Glutenins were extracted from the residue, using buffer containing 50% propan-1-ol, 0.8 M Tris-HCI, (pH 8.0), reduced using 1% DTT and alkylated using 1.4% of 4-vinylpyridine. Electrophoresis was carried out as described by PAYNE et al. (1980) on 8% acrylamide gel (0.75 mm thick) using Bio-Rad Mini-Protean-II unit. For resolution of certain HMW glutenin subunits gels were run for longer time. The HMW bands were numbered according to PAYNE and LAWRENCE (1983), VALLEGA and WAINES (1987) and BRANLARD et al. (1989). The relative mobility of these subunits was determined by comparison with standard genotypes listed in Table 2. Low molecular weight glutenin subunit patterns have been temporarily designated as LMW-1 to LMW-5.
RESULTS
Gli-Bl
Alleles
Subunits
a
1
b c
2* Null
b d e f h i IV
7+8 6+8 20 13 + 16 14+15 17+18 23 + 18
Chinese spring Edmore Aric 581/1 Durtal Anatolien 6523 Kalyansona Kyperounda-2
g-42 g-45 g-43.5 g-44
Langdon Edmore Bijapur 370-4 Vijay
Standard variety
Ensat 398 MACS-2496 Durtal Ensat 387
Almost all dicoccums showed the presence of allele Glu-All except two recent varieties (DDK1001 and MACS-2574). At Glu-Bl locus, Glu-Blh allele is more frequent (Fig. la, lane D). Allelic variation at Gli-Bl loci The Acid-PAGE analysis showed four classes of gliadins namely alpha, beta, gamma and omega (Fig. 2). We were specifically interested in analyzing the gamma gliadins, encoded by Gli-Bl locus, which are used as biochemical markers to determine the pasta making quality. At Gli-Bl three alleles are detected (Table 1, Fig. 2, lanes C, D and E). Only one variety DDK 1001 showed the presence of gamma gliadin 45 and linked 35-omega gliadin (Fig. 2, lane D) and one exotic line EX-7 showed the presence of gamma gliadin 47 (Fig. 2, lane C). Rest of the genotypes showed the presence of gamma gliadin 44 (Fig. 2, lane E). Only one genotype MACS-2574 showed Gli-Al coded band y-51 (Fig. 2, lane F).
Allelic variation at Glu-1 loci
Allelic variation at Glu-3 loci
The dicoccum genotypes analyzed showed five different HMW glutenin banding patterns (Table 1, Fig. la) resulting from the combination of two alleles (/ and c) at Glu-Al locus (Fig. lb, lanes C and E) and three alleles at Glu-Bl locus (h, IV and b) (Fig. 1 a, lanes A, B and C).
Low molecular weight glutenins are important because the variation in LMW-B glutenins decides the end use quality. SDS-PAGE profiles of LMW glutenins showed five different LMW glutenin subunit banding patterns considering only B subunits at Glu-3 loci (Table 1, Fig. la).
248
Each genotype had four to six LMW glutenin B subunits (Fig. la, lanes B and C), coded by GluA3, Glu-B3 and Glu-B2 loci. The five variant LMW B glutenin patterns are temporarily designated as LMW-1 to LMW-5 (Fig. la). The differences between the patterns were for mobility of bands. The LMW-5 is most frequent type and the rest of them are present only in one genotype. At Gli-A2 locus two types of alleles are detected (Fig. 2, lanes E and C), allele a-2 being more common. DISCUSSION
Fig. 1a
Fig. 1b a. Allelic variation at Glu-1 and Glu-3 loci. A: KDH F: Edmore B: EX-7 G: Langdon C: DDK-1001 H: HD-4502 D: MACS-2574 I: MACS-2496 E: DDK-1009 b. Allelic variation at Glu-Al loci. D: Yellow Khapli A: Kalyansona B: MACS-2496 E: MACS-2574 C: KDH F: DDK-1001 Different HMW glutenin subunits are numbered according to Payne and Lawrence (1983), Vallega and Waines (1987) and BRANLARD et ah (1989). Different LMW B glutenin patterns are numbered below the lanes. Standard varieties Edmore, Langdon and HD-4502 are durums, while MACS-2496 and Kalyansona are aestivums.
1 - SDS-PAGE profiles of glutenin subunits from Indian dicoccums. FIGURE
Allelic frequencies in dicoccum and its comparison with durum wheat Gluten proteins of Indian dicoccum wheat did not show much variation at Glu-1, Gli-Bl and Glu-3 loci. This is in contrast with the earlier reports by VALLEGA and WAINES (1987), Liu and SHEPHERD (1996) and PFLUGER et at. (2001). These authors have reported more variation at these loci. This may be due to the fact that the material analyzed by these authors was from diverse germplasm sources. The limited variation observed in the present analysis may be due to several reasons. Since these seeds were originally collected directly from farmers' fields, the same genotype may be known by different names in different regions. The limited area of cultvation may also result in less diversity. Very low polymorphism was also observed earlier, when a part of this germplasm was analyzed using DNA based markers (PUJAR et ah, 1999; 2002). Nearly all the dicoccums showed the protein band coded by Glu-Al locus, in contrast to a null allele at this locus in most of the durums. Since most of the dicoccums had Glu-AlI allele, we used pair of primers (D'OVIDIO et ah, 1995), which are able to amplify complete coding region of Glu-Alx type genes. A band larger in size than that of Glu-Ala type allele was observed (data not shown). At Glu-Bl locus total three alleles were observed. Allele Glu-Blh was the most common, while Glu-Blb was found only in two cultivars (DDK1001 and DDK1009). This differs from the earlier reports by VALLEGA and WAINES (1987) and PFLUGER et al. (2001) where Glu-Blb was the most common allele observed at Glu-Bl
249
Fig. 2 A: Marquis C: EX-7 E: DDK-1009 G: Langdon I: Bijapur 370-4
B: Edmore D: DDK-1001 F: MACS-2574 H: Vijay J: Marquis.
Gliadin bands are numbered according to BUSHUK and ZILLMAN (1978). Thick arrows show g gliadins 47 in lane C, 45 in lane D and 44 in lane E. Thin arrow show w gliadin 35 band in lane D. Arrow head shows the presence of g gliadin 51 in lane F. Reference g gliadin 50 is numbered in lane J. Marquis (lanes A and J), Edmore (lane B), Langdon (lane G), Vijay (lane H) and Bijapur 370-4 (lane 1) are used as standards. Square brackets show the a - 1 and a-2 alleles in lanes E and C respectively.
2 - Acid-PAGE profiles of gliadins from Indian dicoccums. FIGURE
locus. Alleles Glu-Blc (7 + 9) and Glu-Bli (17 + 18) were not observed, which are very common in bread wheat. These results are in agreement with VALLEGA and WAINES (1987), BRANLARD et at (1989) and CIAFFI et at. (1993). These results also support the hypothesis put forth by BRANLARD and coworkers (1989) that these glutenin subunits may have been derived from mutations, which occurred only in hexaploid wheats. At GliBl only three alleles are detected, most of them showed the presence of gamma gliadin 44, but the gamma gliadin 44 containing genotypes did not show the presence of linked omega gliadins 34-37, as in durums (CARRILLO, 1990b; Ruiz and
CARILLO, 1993). Only one genotype DDK-1001 showed the presence of gamma gliadin 45, which is associated with good pasta quality in durums. The genotypes analyzed showed five different patterns of LMW glutenin B subunits and LMW-5 type was most common. Although three different gamma gliadins were observed at Gli-B1 loci, at Glu-3 locus five different patterns were observed. This may be due to the fact that, the LMW B glutenin subunit pattern is a mixture of products coded by Glu-A3, Glu-B3 and Glu-B2 loci. All the three released varieties included in the present analysis are derived from a cross between dicoccum and durum cultivars. These released varieties show some distinct alleles derived from durum parents which are absent in other dicoccums. For example, MACS-2574 showed 7-51 (Fig. 2, lane F) at Gli-A1 locus, DDK1001 showed g - 4 5 and also linked Glu-B3 alleles. It also showed 830 bp band after PCR amplification using Glu-B3 specific primers (D'OVIDIO and PORCEDDU, 1996) (data not shown). Dicoccums have high seed weight and protein content (Anonymous, 1999) and they also have the potential to grow in adverse conditions. The presence of novel patterns of LMW glutenins and g-gliadins is interesting and their relationship with technological quality of dicoccum wheats needs to be investigated further.
ACKNOWLEDGEMENTS The authors thank Dr. G. Branlard, Station d'Amelioration des Plantes, Clermont-Ferrand, France and Directorate of Wheat Research, Karnal, India for providing standards for present study. This work was supported by Department of Biotechnology, Government of India, New Delhi. Research Fellowship to MD Oak under this project is acknowledged.
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