Introduction

Triticale (X Triticosecale Wittmack), successfully combines the hardiness and nutrient-use efficiency of rye (Secale spp.) with the grain yield of wheat (Triticum spp.) in a widely adapted and productive cereal crop. Modern triticale cultivars are the result of secondary crosses between primary triticales (the original fertile amphidiploid offspring of wheat and rye hybrids) and wheat, rye or other triticales. These modern cultivars produce higher biomass and yield than both progenitor species under most growing conditions (Mergoum and Gomez Macpherson 2004).

The purpose and process of creating primary triticales is similar to the purpose and process of synthetic wheat development. Difficulties and losses are experienced at all production stages, such that not all pollinated florets produce seed, not all seed contain viable embryos, and not all hybrid plants may undergo chromosome doubling. Success rates vary depending on genotype of the parents, their interaction and the parental cytoplasm (Cooper and Driscoll 1985; Oettler 1985; Sirkka et al. 1993). The crossability of the wheat parent, the inter-species interaction between parents and plant development of the haploids are all controlled by independent loci (Oettler 1983; Tikhenko et al. 2008). Although usually of poor agronomic value, primary triticales are easily crossable to existing triticales and are an effective conduit for expanding its genetic diversity. Continued introduction of beneficial alleles from both wheat and rye has the potential to greatly improve this synthetic crop, particularly in traits for which triticale is lacking key genes, such as grain quality.

Flour and dough characteristics of triticale are generally inferior to those of bread wheat (T. aestivum L.) for specific products, such as pan bread, which require strong gluten with high extensibility. Hexaploid triticale, 2n = 6x = 42, genome constitution AABBRR, lacks the D genome of bread wheat, 2n = 6x = 42 (AABBDD) where the most significant genes conferring desirable dough properties are contained. Nevertheless, genetic variability for dough properties exists within current triticale germplasm and the range of dough quality characteristics in modern triticale cultivars overlaps with that of wheat (Dennett et al. under review).

Gluten is formed from a polymeric backbone of HMW (high molecular weight) glutenin subunits which interact with LMW (low molecular weight) glutenins and gliadins through the formation of inter-chain disulphide bonds (Shewry and Halford 2001). The storage proteins of triticale are comprised of polymeric HMW glutenins and LMW glutenins from the AABB genome, HMW secalins and 75k γ-secalins from the RR genome, plus predominantly monomeric ω-secalins and gliadins, 40K γ-secalins, and α-, β-, and γ-gliadins. (Amiour et al. 2002; Peña et al. 1991; Salmanowicz and Dylewicz 2007). The interaction between these components is poorly understood in triticale, both in terms of genetic expression and quaternary linkages and structure. Formation of extensible and viscoelastic gluten is an essential component of bread and pasta products, particularly in modern high-throughput manufacturing systems.

Several studies of SDS-PAGE banding patterns concluded that the storage proteins of primary triticale are the sum of its two parents (Orth et al. 1974; Virdi and Larter 1984). When polymorphism was observed it was usually attributed to heterogeneity in the rye parent e.g. Virdi and Larter (1984). However Rozynek et al. (1998) found band intensities differed in the gliadin and secalin fractions between triticale and its parents, and, in some cases, differences in mobility between bands. Altpeter et al. (2004) and Field and Shewry (1987) concluded that mixed polymers were formed between rye and wheat storage proteins in transgenic rye and primary triticales respectively. Furthermore, expression of certain Glu-B1 prolamins and D-genome ω-gliadins appeared to be different in some lines resulting from tetraploid and octoploid triticale crosses (Bernard et al. 1990).

This study investigates glutenin and secalin interactions in wheat–rye hybrids made from modern wheat and rye genotypes, and examines the influence of the hybrid protein structures on gluten quality. The term ‘secaloglutenin’ is used here to collectively refer to the polymeric storage proteins of triticale, namely HMW glutenin and secalin from the 1A, 1B and 1R chromosomes, LMW glutenin from the 1A and 1B chromosomes, and 75k γ-secalin from the 2R chromosome.

Materials and methods

Primary triticale

Five durums (Triticum durum, 2n = 4x = 28) and two ryes (Secale cereale, 2n = 2x = 14) were chosen for hybridisation based firstly on their glutenin expression patterns and secondly on a preliminary crossability study. The durums were donated by Mike Sissons (DPI, Tamworth, Table 1), and an inbred maintainer rye (R8) and an inbred white-seeded rye (R11) were sourced from the University of Sydney rye breeding program (Tables 1, 2). Inbred ryes were used to obtain virtual homogeneity at the secaloglutenin loci thereby allowing assessment of inheritance from parent to hybrid.

Table 1 Allele designation for HMW and LMW alleles of the durum parents
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Table 2 HMW subunit designations and 75k γ-secalin alleles of the rye parents
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Pollen from four R11 plants and five R8 plants were crossed with the durum wheat parents. The resulting amphihaploids were treated in 0.02 % colchicine solution at 22 °C for 6 h. Up to five selfed seeds from each surviving fertile primary triticale, plus selfed seeds from the individual parental plants, were sown in the glasshouse. Note primaries 302030, 302035 and 302043 did not produce selfed seed, thus grain from unbagged heads was collected.

The new amphidiploids were sown in seedling trays then transplanted to the field at Cobbitty, NSW, Australia for a preliminary assessment of agronomic vigour and disease resistance. Lines were bulk harvested and the homogeneity between lines descended from the same primary plant was confirmed using SDS-PAGE of HMW and LMW glutenins. This was assessed on 3 grains from each line, thus up to 15 grains were tested per primary plant. Homogeneity among three selfed grains from each progenitor plant was also assessed. A total of 23 primary triticale lines were produced from eight cross combinations between the durum and rye progenitors (Table 3).

Table 3 Crossability of durum and rye parents
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A subset of 11 lines of sufficient vigour representing four cross combinations was grown at Roseworthy, SA, Australia under rainfed conditions. Depending on seed availability, between one and four 3.5 m rows of each line were sown in a red/brown loam soil, fertilised with 90 kg/ha DAP at planting (17.5 % nitrogen 20 % phosphorus 1.5 % sulphur) and topdressed with 80 kg/ha urea. Grain samples were ground on a Newport Scientific 600 Hammer Mill with 0.5 mm screen (Perten, Warriwood, NSW, Australia). Wholemeal flour protein content and hardness were estimated by NIR on an Inframatic 8100 PerCon (Perten, Hägersten, Sweden) which had been calibrated using a hard-grained bread wheat. Three triticale standards (Goanna, Rufus and Yowie) sourced from KV Cooper and MG Elleway (Sherlock, SA, Australia), and one wheat standard (Sunco) grown at Narrabri, NSW, Australia, were used as a comparison for current gluten strength in grain grown under commercial conditions. SDS-sedimentation was based on the method of Dick and Quick (1983), however using 3 mL indicator solution (0.001 % Bromophenol Blue) instead of distilled water, 0.75 g flour and 9 mL working solution (3 % SDS and 0.23 % lactic acid) to resolve differences in triticale flour, which has poor gluten formation. The test was performed in capped plastic test tubes of 16 mm diameter (Livingstone, Rosebery, NSW, Australia).

SDS-PAGE of glutenin profiles

The non-germ end of three seeds of the Cobbitty-grown durum parents, plus glasshouse-grown selfed grain from the exact rye parents and primary triticales, were divided using a scalpel, crushed with a pair of pliers and placed in 1.5 mL Eppendorf tubes. Non-glutenin proteins were removed by adding 200 μL of 50 % (v/v) propan-1-ol, vortexing then incubating at 65 °C in a water bath for 30 min, with vortexing approximately every 7 min. Tubes were then centrifuged at 5,000 rpm for 10 min and supernatant was discarded. This process was repeated using only 5 min incubation time, then repeated using distilled water and 5 min incubation, then samples were oven dried at 37 °C for 40 min to remove remaining liquid.

Extraction of glutenins proceeded with vortexing and incubation of the pellet for 5.5 min at 95 °C in 150 μL of Glutenin sample buffer (12.5 % 0.5 M Tris–HCl at pH 6.8, 25 % glycerol, 2 % SDS (w/v), 0.001 % bromophenol blue (w/v) and 2 % mercaptoethanol). Samples were then centrifuged at 14,000 rpm for 2.5 min.

Extracted glutenins were fractionated in a vertical CBS-Scientific Dual Adjustable Meg-Gel kit (CB-Scientific, CA, USA). The resolving gel contained 8 % Acr/Bis, 24 % 1.5 M Tris–HCl at pH 8.8, 0.1 % SDS, 0.12 % TEMED and 0.06 % APS. The stacking gel contained 4 % Acr/Bis, 25 % 0.5 M Tris–HCl at pH 6.8, 0.1 % SDS, 0.3 % TEMED and 0.15 % APS. Samples of 8 μL were resolved at 40 V overnight followed by 70 V for the final 4 h in electrode running buffer (0.3 % tris (w/v); 1.44 % glycine (v/v) and 0.1 % SDS (w/v)).

Gels were rinsed in water then stained in Coomassie blue solution (0.05 % Coomassie blue G-250, 10 % (w/v) phosphoric acid; 5 % aluminium sulphate and 20 % methanol) for approximately 60 min with gentle agitation then destained in water for 33 h aided by paper towelling and permanently stored between single layers of clear cellophane.

Individual parental lines were run in channels with their specific primary offspring between them. Wheat cultivars Chinese Spring, Veery and Sunstate, the rye cultivar Westwood and triticale cultivars Sirius, Titan and Presto 1R.1D5+10 were also run on each gel. The results were then confirmed in a second assessment using grains from the same seed increase. A single grain was assessed and Federation, Bowie, Gabo and Sunco were included as additional wheat standards. Allele nomenclature of McIntosh et al. (2008) is applied for wheat glutenins and 75k γ-secalins. No attempt was made to name the HMW secalins observed here because they differed from the standards. Arbitrary designations were given for purposes of this paper.

Results and discussion

Crossability of parental genotypes

Of the 198 crosses performed, 1198 seeds were produced, 541 embryos were rescued from these developing seeds (45 % of seeds) and 65 embryos developed into haploid plants (5.4 % of seeds) (Table 3). This success rate is comparable to previous reports (Oettler 1984; Cooper and Driscoll 1985; Balatero and Darvey 1993; Sirkka et al. 1993). Seed set was significantly different between durum lines (P = 0.004) however no difference was detected between rye parents (P = 0.286). The wheat parent has previously been reported to have a greater influence on success rates in primary triticale production than the rye parent (Oettler 1984; Balatero and Darvey 1993). Parental lines and their interaction also influenced the number of viable embryos formed within seeds (P = 0.042).

Although relatively few primary triticales have been produced in the past two decades, it remains a useful method of transferring traits from either durum or rye into high-yielding triticale genotypes. An inability to economically produce primary triticale from certain (poorly crossable) durum and rye genotypes has limited the usefulness of this technique. It is therefore essential that the inheritance and expression of target traits in the amphidiploid be well understood prior to investing in the creation of new primary germplasm.

Homogeneity in parental lines

Both HMW and LMW alleles exhibited no variability within the durum parental lines. The allele designations for their polymeric glutenins are provided in Table 1.

The HMW secalins of rye were comprised of x-type and y-type subunits (De Bustos and Jouve 2003). Most HMW secalins exhibited an electrophoretic mobility in between the Glu-A1 subunits (1 and 2*) and Glu-B1 subunits (6 + 8, 7 + 8, 7 + 9, 7 + 16, 7 + 18, 13 + 16, 20) (Fig. 1). The use of modern molecular markers has reduced the reliance on SDS-sedimentation for definition of secalins. However, for purposes of this study it was convenient to run parents and offspring in adjacent channels to reveal differences in the expression of multiple subunits clearly and concisely.

Fig. 1
figure 1

Storage proteins of durum, rye and their amphidiploid offspring. Identification of the secaloglutelin subunits are indicated on the figure, location of remnant monomeric secalins and gliadins after washing in 50 % propan-1-ol are indicated on the left. Lanes a and f, rye parent (R8); lanes c and d (durum parents D9 and D11 respectively); lane b, primary triticale (D9 x R8); lane e, primary triticale (D11 x R8); lane g, wheat standard with 1BL.1RS translocation (Veery); lane h, triticale standard (Titan)

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Segregation within each inbred rye plant was low: 55 % of parental rye plants were homogenous for HMW glutenins. The remainder expressed a maximum of two alleles amongst the tested seeds from each rye plant, and in most cases both the alleles were expressed in other parental plants of either R8 or R11. The fact that either of these alleles could have been potentially passed to the primary triticale from the parental plant was noted in those cases. Variation within individual parental plants was lower than variation within and between parental lines, however only seven and two alleles were observed at the Glu-R1 and Gli-R2 loci, respectively (Table 2). A star designation was given when two bands from different alleles were almost identical and hence may represent the same subunit.

There was a concerted effort to produced homogeneous rye parental lines for this study which exhibit a consistent secalin profile between individuals and can thus be compared to the resulting primary triticale. Although they are generally of poor agronomic vigour, inbred rye lines have been utilised in research and breeding for decades e.g. Oettler (1983). Significant progress in hybrid rye breeding over the last few decades has produced rye lines which are less susceptible to inbreeding depression. The challenge in this study was to stabilise the genome of the rye parents as much as possible without weakening their capacity to produce sufficient quantities of pollen. Rye parent R8 was an inbred maintainer rye from the University of Sydney Rye Breeding Program and still produced viable pollen after several generations of selfing. The second rye parental line (R11) was much more susceptible to inbreeding depression and hence was selfed fewer times before crossing. This is reflected in the slightly higher (but still low enough to be quantified) polymorphism in secalin subunits. Virdi and Larter (1984) concluded that while polymorphism in primary triticale can be reduced by use of inbred parents, it cannot be totally eliminated.

A much greater proportion of the total prolamin was removed from rye compared to durum by multiple washing with 50 % propan-1-ol. Rye differs from wheat in that a larger proportion of the polymers stabilised by disulphide bonds are soluble in ethanol (Field and Shewry 1987; Gellrich et al. 2003). Such polymers consist of the 75k γ-secalins and HMW secalins.

The 40k γ-secalin and S-rich gliadins (α-, β-, and γ-gliadins) were not removed particularly well by washing with 50 % propan-1-ol. Complete removal of alcohol-soluble storage proteins is difficult from manually crushed grains, although the possibility remains that some alcohol-soluble gliadins and secalins were incorporated into insoluble secaloglutenin aggregates in the developing grain and hence were unable to be removed without sonication (Altpeter et al. 2004; Field and Shewry 1987).

Expression of secaloglutenins in primary triticale

All primaries were found to express identical secaloglutenin subunits to their respective parental lines (Fig. 1). Thus if mixed polymers are formed in primary triticale, as hypothesised by Virdi and Larter (1984) and Rozynek et al. (1998), they were not apparent amongst the denatured secaloglutenins of this set of 23 primary triticale lines. There were five instances where a different Glu-R1 allele was expressed to the Glu-R1 allele of the parent in the adjacent channel, however these were all within the limited polymorphism observed in the R11 parent. Bands present in the LMW region that were present in durum but not in triticale were most likely gliadins which had not been completely removed rather than LMW glutenins which were not expressed in the amphidiploid. This is evidenced by the variable density and inconsistent presence of these extra bands.

It has been suggested that there is genetic suppression of certain prolamin genes in triticale, and/or chromosomal instability which may result in the loss of prolamin subunits (Field and Shewry 1987; Virdi and Larter 1984; Bernard et al. 1990; Rozynek et al. 1998; Zhang et al. 2008). However, these authors did not report the level of homozygosity in the rye parent and provided no evidence that differences in band intensity were not a result of environmental influences during grain protein accumulation. Our study did not find any evidence that secaloglutenin subunits are abnormally expressed in a primary triticale compared to its parents, but cannot rule out the possibility in other parts of the genome, particularly amongst the gliadins, 40k-γsecalins and ω-secalins. Differences in gene expression, genetic aberrations and novel alleles have been observed during the creation of synthetic wheat and in other triticale genes (Tikhenko et al. 2005; Dreisigacker et al. 2008).

Furthermore, whilst it is unlikely that there are systematic differences in genetic expression of secaloglutenin subunits, it remains probable that mixed aggregates at the ultrastructure level are formed between secalins and glutenins in the developing amphidiploid. The presence of 40k γ-secalins in the reduced protein fraction suggested this. Scanning electron micrographs of durum, rye and their derived primary triticale clearly reveal the differences in the ultrastructure of gluten produced from durum, rye and triticale (Orth et al. 1974). In a study of transgenic rye, mixed polymers were formed between rye secalins (including normally monomeric types) and HMW glutenins (Altpeter et al. 2004). The authors also observed an increase in the proportion of normally monomeric proteins in the polymeric protein fraction, suggesting 40k γ-secalins were incorporated into insoluble secaloglutenin aggregates. Similarly, Field and Shewry (1987) concluded that triticale formed at least some mixed polymers and oligomers based on by differences in solubility between triticale and its parents. They also found the ω-gliadins were present in lower amounts in triticale compared to the parental wheats, as was observed here by simple visual examination of band densities. Triticale has a different ratio of secaloglutenin to gliadin and different gliadin solubility compared to wheat, and different gliadins are found in different quantities among cultivars (Chen and Bushuk 1970; Gellrich et al. 2003; Salmanowicz and Nowak 2009).

Combined with this different secaloglutenin behaviour in the amphiploid genome, secaloglutenin mobility is not conclusive evidence of the presence of a certain subunit. Many subunits of similar mobility on SDS-PAGE may have different physical properties e.g. Subunit 10 from Glu-B3 and subunit 12 from Glu-B2. Hence the gluten strength of the amphiploids was examined by SDS-sedimentation.

Secalogluten strength

Field-grown grain was produced in sufficient quantities to examine secalogluten strength in 11 lines representing four cross combinations (Table 4). SDS-sedimentation revealed clear differences between primaries produced from different durum genotypes. Primaries from D2 had significantly greater gluten strength than primaries created from D12 or D9 (P < 0.001, Table 4). D2 has the favourable HMW glutenin alleles Glu-A1b (subunit 2*) and Glu-B1f (13 + 16) (Branlard and Dardevet 1985) however further investigation is required to confirm the source of the high gluten strength in this cross compared to the other crosses. Protein content was not a significant covariate in prediction of SDS-sedimentation (P = 0.738).

Table 4 SDS-sedimentation, protein content and hardness of wholemeal flour from a subset of triticale lines with one bread wheat and three triticale standards
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Several authors have noted the poor gluten quality of triticale, which has short mixing time and poor strength (Varughese et al. 1996). However, studies which have attempted to link the expression of specific glutenin alleles with this poor quality have either been inconclusive (Peña et al. 1991; Tohver et al. 2005) or could only observe an effect when there was an absence of allele expression (Ciaffi et al. 1991). Regardless of which loci or alleles have the greatest influence on gluten strength in triticale, our results suggest that the dough properties of triticale can be significantly improved by selecting parents of high gluten strength.

Conclusion

There was no evidence of genetic differences in secaloglutenin expression between triticale and its parental genotypes, however, it remains possible that the ultrastructure of the polymeric proteins is altered in the amphidiploid. Further investigation into the gluten strength of primary triticale compared to its parents should be conducted on a larger set of lines which has representatives of both rye parents in all crosses, and performed on grain sourced from replicated environments. The high protein content and high SDS-sedimentation of these primary triticales suggests they have the capacity to produce high volume bread, particularly those resulting from durum D2. These primaries are currently being crossed to locally adapted lines to introduce gluten strength into high-yielding backgrounds.