J. Plant Biochemistry & Biotechnology Vol. 3, 47-51, January 1994

Seed Storage Protein Profiles of Seven Indian Wheat (Triticum aestivum L.) Varieties

G Sreeramulu, 0 Vishnuvardhan and Nagendra K Singh· Molecular Biology Unit, Central Food Technological Research Institute, Mysore 570 013, India

Seed storage protein profiles, Including trltlcln, gliadin and glutenin, of seven Indian wheat varieties were Investigated using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (50S-PAGE). A strategy was developed for half-seed analysis of the three seed storage protein classes. The results are presented In the form of a key for Identification of these varieties based on their seed protein composition. A minimum of 10 seeds of each variety were analysed to check for possible intravarietal heterogeneity, and all the varieties except HUW-12 were homogeneous. The HUW-12 was a mixture of two 'biotypes' with respect to the low molecular weight (LMW) glutenin subunits coded by the Glu-A3 locus. The varieties were also analysed for the presence of rye chromatin using a rye-specific repetitive DNA probe (pAW-161), and two of the recently released varieties HUW-206 and HUW-318 were found to possess rye chromatin. Based on the DNA dot-blot results, presence of rye secalins and the absence of chromosome 1 B-speclflc gliadlns It was concluded that these two varieties possess 1BL-1RS wheat-rye translocation. Both of these varieties also have high molecular weight (HMW) glutenin subunits 5 + 10 which may be necessary for compensating the loss of dough strength associated with the wheat-rye translocations.

Key words: wheat, Triticum aestivum, tritiein, gliadin, glutenin, seealin, DNA dot blotting, wheat-rye translocation.

The suitability of a wheat variety for different end­uses viz. bread, chapati, biscuit, or noodle is deter­mined to a large extent by its seed protein compo­sition. The major components of wheat seed proteins are alcohol-soluble prolamins (gliadin and glutenin) which are responsible for the visco-elastic properties of the wheat flour doughs (1, 2). A right balance of these proteins produces the desired baking quality (3). However, due to limiting amounts of essential amino acids lysine and threonine these proteins have rela­tively poor nutritional quality for monogastric animals and humans (4). In contrast to the prolamins, triticin is a minor wheat storage globulin with superior nu­tritional quality (5-7). While triticin is relatively poorly studied, extensive intervarietal polymorph isms has been reported for the gliadin and glutenin, and is being used for the varietal identification and selection of supe­rior dough properties in developed countries (8).

Very limited information is available on the allelic variation for these proteins among the Indian wheat varieties. In this paper, we present complete seed storage protein profiles of seven Indian wheat vari­eties released from the Banaras Hindu University. In addition, genomic DNA dot-blots of these varieties were probed for the presence of rye chromatin. The 1 BL-1 RS wheat-rye translocations have shown consistent yield advantage and higher disease resistance as

'Corresponding author

compared to the normal wheat lines in extensive field trials (9), but produce weak doughs not suitable for commercial bread-making (10, 11). Although this trans­location is now being used by the Indian wheat breed­ers, the extent of its prevalence in the commercially released varieties is not known.

Materials and Methods Wheat seedS'--Samples of seven wheat varieties, HUW-12, HUW-37, HUW-55, HUW-206, HUW-213, HUW-234 and HUW-318 were obtained from Dr Ramdhari, Senior Wheat Breeder, Department of Ge­netics and Plant Breeding, Banaras Hindu University, Varanasi, India. Control wheat varieties, Chinese Spring, Hira, Gabo, Gabo 1DL-1 RS, Lerma Rojo, Orca and Halberd were obtained from Dr KW Shepherd of the University of Adelaide, Australia. Protein extraction and SDS-PAGE-The endosperm half of a single wheat kernel was crushed into a fine powder. First, triticin was extracted with 1 ml of 1 M NaCI at 55°C overnight. After a 10 min centrifuga­tion (12,000 x g), triticin was precipitated from the supernatant by adding 6 ~I of glacial acetic acid, washed with 70% ethanol and dissolved in SDS-PAGE sample buffer [4% (w/v) SDS, 15% (v/v) glycerol, 0.001 % (w/v) bromophenol blue, 60 mM Tris-HCI, pH 6.8] for electrophoresis. The gliadins and gluten ins were extracted from the residue according to the method described elsewhere (12).

48 J Plant Biochem Biotech

The discontinuous system of SDS-PAGE used to fractionate the proteins was based on Lawrence and Shepherd (13), as modified before (5, 12). The stack­ing gel had 3% acrylamide, 0.08% bisacrylamide, 0.1 % (w/v) SDS, 125 mM Tris-HCI (pH 6.8). The separat­ing gel (150 x 150 x 1.5 mm) contained 0.1 % (w/v) SDS and 375 mM Tris-HCI (pH 8.9). Separating gels of three different concentrations were used for the maximum resolution of triticin (10% acrylamide, 0.08% bisacrylamide), gliadin (10% acrylamide, 0.15% bisacrylamide) and glutenin (7.5-13% acrylamide and 0.1-0.2% bisacrylamide gradient) bands. IsolatIon of genomic DNA and dot-blot analysis­The genomic DNA for dot blotting was prepared by a simple "miniprep" method. Briefly, a small portion of leaf (- 0.5 g) was homogenized in 0.6 ml DNA extraction buffer (4% sarkosyl; 100 mM NaCI; 10 mM EDTA, pH 8.0; 100 mM Tris-HCI, pH 8.5) in an Eppendorf tube and extracted twice with phenol : chloroform : isoamyl alcohol (25:24:1). The aqueous phase was transferred to a new tube and DNA was precipitated by adding 0.1 volume of 3M sodium ac­etate (pH 4.8) and an equal volume of propan-2-01. The DNA pellet was recovered by centrifugation, washed with 70% ethanol, vacuum dried and dissolved in 10 mM Tris-HCI, 1 mM EDTA, pH 8.0 (TE). The filter for DNA dot-blot was prepared according to (7). The insert (- 400 bp) of clone pAW-161 was excised by restriction digestion with Bam HI, electroeluted from the cut out agarose gel piece, and then used as a probe for rye DNA. The hybridization and detection was done using Digoxygenin DNA labelling and de­tection kit (Boehringer & Manheim, Germany). Prehybridization (6 h) and hybridization (16 h) were carried out at 68°C according to the manufacturers'

protocol. After hybridization, the filter was washed twice (5 min each) with 2 x SSC, 0.1 % SDS and then twice (15 min each) with 0.1 x SSC, 0.1% SDS at 68°C. The detection was done according to manufac­turers' instructions.

Results and Discussion The seven wheat varieties were analysed for their seed storage protein profiles, including triticin, gliadin and glutenin patterns. It has been shown earlier that a number of modern Australian wheat varieties are heterogeneous for their seed protein composition; the variant forms of a variety are called 'biotypes' (14). Hence, we analysed ten random seeds of each va­riety to check for the homogeneity of their ,seed protein composition. A complete set of data on triticin, glia­din/secalin and glutenin subunits was obtained from the endosperm half of the individual seeds. The overall results are summarised in Table 1, and details on each class of proteins is described below: Tritlcln (TrI-1~ There was no heterogeneity for triticin banding pattern (triplet bands) within any of the seven wheat varieties analysed (Results not shown). Out of the two functional triticin genes described earlier (5, 7, 15), the Tri-A 1 is identical in all the seven variet­ies (Table 1), whereas, two different banding patterns (alleles) were observed for the Tri-D1. Thus, HUW-206 and HUW-318 have the Chinese Spring pattern and the remaining five varieties possess' the India-115 pattern. The allelic designations of triticin are according to (15). ro-glladins (GII-1) and ro-secalins (Sec-1~Unreduced prolamins were extracted from the residue after the triticin extraction using 50% propan-1-01 (12). The SDS­PAGE patterns of the unreduced prolamins from seven

Table 1. Allelic composition of seven Indian wheat varieties for twelve different seed storage protein lOCI"

Glutenin

Triticin Cll-gliadins/secalins HMW subunits LMW subunits Variety

Tri-A1 Tri-D1 GIi-B1 GIi-D1 Sec-1 Glu-A1 Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3 Glu-4

HUW-12A cs 9 9 2" 7+8 2+12 e b b w HUW-12B cs 9 9 2" 7+8 2+12 c b b w HUW-37 cs 9 9 2" 7+8 2+12 c b b w HUW-55 cs h55 h55 17+18 5+10 d h55 h55 n HUW-206 cs cs 9 + 7+9 5+10 c b w HUW-213 cs h213 9 2" 7+8 2+12 c h213 b w HUW-234 cs h213 9 2" 7+8 2+12 c h213 b w HUW-318 cs cs 9 + 7+9 5+10 c b n

"The gene and allele symbols are taken from the references as follows: Genes Tri-A " Tri-01 and alleles cs. i for triticin, alleles 9 for gliadins, and gens Glu-A3, Glu-B3, Glu-03 for LMW glutenin subunits (15); genes Gli-B1, GIi-01 for gliadins (21); Sec-1 for ro-secalins (22); genes Glu-Af. Glu-Bt, Glu-Dt and alleles 2", 7 + 8, 2 + 12 etc for HMW subunits (18); alleles a. b. c. d for LMW subunits (19). Symbols Glu-4, h55, h213, w (wide doublet) and n (narrow doublet are new for this study, - = absent, + = present.

varieties were compared with those of the control varieties of known patterns (Fig. 1, lanes e-h). As with the triticin, the prolamin pattern of each variety was also homogeneous. In the co-gliadin region, the slowest moving bands are coded by genes at the GIi-Bf lo­cus on chromosome 1 S (labelled GIi-S1 in Fig. 1), while another set of bands moving slightly faster than these are coded by the GIi-Df locus on chromosome 1 D (5, 15). Products of the G/I-A f genes were diffi­cult to study in the present SOS-PAGE system, and hence these have not been considered here.

GU-81 [

GIUlI

• b e d • II ~ I ~ I

GII - glled h.

h, -.fl- .r ­gllodlno

Fig. 1. SDS-PAGE patterns of unreduced prolamins (50% propan-l-ol extracts). Lanes: a-HUW-12 biOtype A; b-HUW-12 biotype 8; c-HUW-37; d-HUW-55; e-Chinese Spring; f-Gabo; g-Gabo 1 DL-1 RS; h-Orca; i-HUW-206; j-HUW-213; k-HUW-234; I-HUW-318. The GIi-81 , GIi-D1 (oo-gliadins) and the Sec-1 (00-

secalins) zones are indicated.

For the GIi-Bf locus, varieties HUW-12 and HUW-37 (Fig. 1, lanes a-c) showed Gabo type 'g' allele (lane f, allele deSignations according to 15). Variet­ies HUW-55, HUW-213 and HUW-234 (Fig. 1, lanes d, j and k) possessed novel alleles at this locus, and these have been deSignated 'h55' (HUW-55) and 'h213' (HUW-213 and HUW-234). The gliadins of the latter two varieties were similar and they lacked a major Gli-B 1 band (Fig. 1, lanes j. k). It has been shown earlier that 1 BL-1 RS Wheat-rye translocation lines lack the Gli-B1 bands and have the Sec-1 (secalins) pro­teins instead (16). Two of the seven varieties viz. HUW-206 and HUW-318 lacked the Gli-S1 co-gliadins and showed rye co-secalin bands (Fig. 1, lanes i and I, compare with a known 1 OL-1 RS variety in lane g), suggesting that these have 1 BL-1 RS wheat-rye trans­location. This is co,'firmed below by DNA dot-blotting for the presence of -ye chromatin (see Fig. 3). The Gli-D1 allele of all th.l varieties except HUW-55 was identified as Gabo ('g') type (15). Variety HUW-55 seems to possess a novel allele "'hich we have ten­tatively designated as h5f=

Seed Storage Proteins of Indian Wheats 49

HMW (Glu-1) and LMW (Glu-3) subunits of gluteni~Both the HMW and LMW subunits of glutenin were analysed on a single gel using a sim­plified one-step SOS-PAGE procedure (12). These gels also gave information on a new group of LMW sub­units (Glu-4) which have not been fully characterized as yet (12, 17). Here we have used the numbering system of Payne and Lawrence (18) for the HMW subunits and an alphabetic nomenclature system for the alleles of LMW subunits (19). Since G/u-3 genes for the LMW subunits are very tightly linked with the G/I-1 genes for gliadins on each of the chromosomes 1 A, 1 Band 10 (15), the Gli-1 bands were used as markers to identify some of the G/u-83 and G/u-D3 alleles. The G/u-A3 alleles were easily identified without the inference from gliadin bands.

The allelic constitution of the HMW subunits (G/u-f loci) of seven wheat varieties are shown in Fig. 2 and Table 1. We detected two alleles for the Glu-A 1 (subunits 1 and 2*), three for the Glu-81 (sub­units 7 + 8, 7 + 9 and 17 + 18) and two for the Glu-D1 (subunits 2 + 12 and 5 + 10). To use the key in Table 1, it is better to first determine the al­lelic constitution of the Glu-D1 locus (2 + 12 vs 5 + 10), followed by the Glu-A1 locus and finally the G/u­B1 locus. It is important to make the determinations in this sequence as it helps to ascertain the identity of the Glu-A 1 al\ele, because subunits 2 and 2* have

Glu-l [- : 1

abed.lglIl, kim

Fig. 2. SDS-PAGE patterns of reduced and alkylated glutenin subunits. Lanes: a-HUW-12 biotype 'A'; b-HUW-12 biotype 8; c-HUW-37; d-HUW-55; e-Orca; f-Halberd; g-Chinese Spring; h­Halberd; i-Arcana; j-HUW-206; k-HUW-213; I-HUW-234; m-HUW-318. The HMW subunits (Glu-1) are numbered according to (18). The alleles of LMW subunits (Glu-3) are indicated as follows: ---t = Glu-A3 (lane a- null allele Glu-A3e; lane b- Glu­A3c; lane d- Glu-A3d). > = Glu-83 (lane c- Glu-B3b; lane d­Glu-83h55; lane k- Glu-83h213). ~ = Glu-D3 (lane d- Glu­D3h55; lane j- Glu-D3b). w = wide doublet and n = narrow doublet of the Glu-4 protein bands.

50 J Plant Biochem Biotech

very similar electrophoretic mobility. Thus, in the pres­ence of subunits 2 + 12, a uniform 10% acrylamide/ 0.15% bisacrylamide gel' and non-alkylated samples were used to determine the presence/absence of 2*. It was not necessary to run a second gel with sub­units 5 + 10. The HMW subunit composition of vari­eties HUW-12 (biotypes A and B), HUW-37, HUW-213 and HUW-234 are identical (2 + 12, 2* and 7 + 8, Fig. 2 lanes a-c, k, I and Table 1). The two vari­eties with 1 BL-1 AS wheat-rye translocation (HUW-206 and HUW-318) have the same HMW subunits 5 -+- 10, 1 and 7 + 9 (Fig. 2, lanes j and m), and HUW-55 has a unique HMW subunit composition of 5 + 10, 2* and 17 + 18 (Fig. 2, lane d). The presence of HMW subunits 5 + 10 in both the varieties carrying wheat-rye translocation, and that of subunits 2 + 12 in all the normal wheat varieties except HUW-55 is significant. Subunits 5 + 10 with their capacity to produce stronger doughs (20)· may be essential for compensating the quality defect associated with these translocations (10, 11).

Variety HUW-12 was a mixture of two biotypes with respect to the Glu-A1 LMW subunits. Thus, biotype 'A' has the null allele Glu-A3e (Fig. 2, lane a) and biotype 'B' has the Glu-A3c allele (Fig. 2, lane b, band shown with arrow). More than 20 seeds of this vari­ety were analysed and the frequency of biotype was apprClximately 50% each. All the remaining varieties except HUW-55 also possessed the Glu-A3c allele. HUW-55 has the slowest moving subunit Glu-A3d for this locus (6). For the Glu-B3 subunits, HUW-12 and HUW-37 have the Glu-83b allele (Fig. 2 lanes a-c, bands marked with arrowheads in lane c). HUW-55 and t.lUW-213/HUW-234 possessed novel alleles for this locus, which have been tentatively designated as Glu-83h55 and Glu-83h213, respectively. Varieties HUW-206 and HUW-318 were negative for the Glu­B3 proteins as expected due to the replacement of wheat chromosome arm 1 BS by the rye chromosome arm 1 RS in these lines (Fig. 2, lanes j, m). The chromosome 1 D coded LMW subunits (Glu-D3) of all the varieties except HUW-55 were identical (the Glu­D3b allele). The novel allele in HUW-55 is tentatively designated as Glu-D3h55 (Fig. 2, lane d).

The alkylated preparations of glutenin were also useful for analysing a new group of subunits which have been tentatively designated as Glu-4. The ge­netics and identity of these proteins is less well un­derstood at present, and at this stage we distinguish the two forms of these bands by letters 'w' (for wide doublet) and 'n' (for narrow doublet). Extraction of triticin with 1 M NaCI significantly improved the glutenin resolution by preventing contamination with some of the albumin/globulins, in addition to providing informa­tion on the triticin variation. However, the extraction of triticin has to be done at 55°C as higher temperature created difficulties with the subsequent resuspension

of residue in the 50% propan-1-01 solutions due to the gelatinization of starch. Presence of rye chromatin-The genome of cereal rye possesses specific repetitive DNA sequences. Two types of rye-specific repetitive DNAs have been iso­lated and cloned (P Langridge, personal communica­tion). The first category of repeats are interspersed throughout the genome and the second category rep­resents telomeric repeats which are present at the telomeric regions of all the seven rye chromosomes. The function of these repetitive DNAs is not well understood as yet but they can be used very effec­tively as markers for the presence of rye chromatin in wheat background. One of such clones (pAW-161) was used as probe to confirm the presence of rye chromosome arm 1 RS in the varieties HUW-213 and HUW-318 which were thought to have 18L-1 RS wheat­rye trans locations from the protein data above (see Fig. 1). The pAW-161 insert hybridized very strongly to the genomic DNAs isolated from these two vari­eties but not at all to the DNAs of other five variet­ies. This confirmed the presence of rye translocations in these two varieties (Fig. 3).

In conclusion, the seed storage protein profiles can be used for the identification of wheat varieties. Al­though the seven varieties analysed in this study had related pedigrees, it was still possible to distinguish them from each other except for HUW-213 and HUW-234 which showed identical patterns. In addition, the varieties carrying 1 BL-1 RS Wheat-rye translocation were also easily identified on the basis of seed storage protein data. Presently we are analysing a large num-

HUW· 12

HUW-37

HUW-55

HUW-206 •

HUW·213

HUW-234

HUW-3l8 •

pAW· 161 _

Fig. 3. DNA dot-blot analysis for the presence of rye chromatin in seven Indian wheat varieties. Denatured genomic DNAs (5

I-Ig) were spotted on a nylon membrane in grids and probed with digoxygenin-Iabelled insert of the rye repetitive DNA clone

pAW-161. Sixty nanograms of the pAW-161 plasmid was spotted

as a positive control.

bar of Indian wheat varieties for a complete cataloguing of their seed protein alleles.

Acknowledgements We are grateful to Dr Ramdhari, Banaras Hindu University for supplying seeds of Indian wheats and Dr KW Shepherd, the University of Adelaide for the control wheat samples. We also

thank Dr P Langridge for the kind gift of rye repetitive DNA probe pAW-161.

Received 15 September, 1993; revised 22 November, 1993

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Seed Storage Proteins of Indian Wheats 51

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