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Article

Traceability of Sicilian Durum Wheat Landraces and Historical Varieties by High Molecular Weight Glutenins Footprint

by
Giovanna Visioli
1,*,
Gianluigi Giannelli
1,
Caterina Agrimonti
1,
Alfio Spina
2 and
Gabriella Pasini
3
1
Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy
2
Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria (CREA), Centro di Ricerca Cerealicoltura e Colture Industriali, Corso Savoia 190, 95024 Acireale (CT), Italy
3
Dipartimento di Agronomia, Animali, Alimenti, Risorse Naturali e Ambiente, Università di Padova, Agripolis, viale dell’Università 16, 35020 Legnaro (PD), Italy
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(1), 143; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11010143
Submission received: 22 December 2020 / Revised: 11 January 2021 / Accepted: 11 January 2021 / Published: 14 January 2021
(This article belongs to the Special Issue Traceability and Control in Production and Quality of Value-Added Agricultural and Food Products)
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Abstract

:
Over the last new decade, there has been a strong interest in landraces and historical wheat varieties from farmers, manufacturers and consumers. They are agronomically and nutritionally interesting but the supply chain (from seed to end-product) is not solid and traceable. High molecular weight glutenins (HMW-GS) can act as markers to trace the varietal correspondence and to verify the genetic purity of the grain and consequently of the flours, marketed and labeled as mono-varietal. In the present work, HMW-GS of different durum wheat Sicilian landraces (Timilia, Russello, Perciasacchi) and one historical variety Margherito were analyzed. At first, specific protein profiles were assigned to each Sicilian landrace by SDS-PAGE and MALDI-TOF/MS analyses, thanks to the availability of pure seeds. Analysis of the protein profiles were then carried out from random samples of seed batches of the same landraces grown on a farm in South-East Sicily. The results highlighted the presence of different protein bands within the individual seed batches, which are reflected in complex profiles in the corresponding commercial flours labelled as mono-varietal. The bread wheat landrace Maiorca cultivated in the same farm was also found as a contaminant at different percentages in the durum wheat batches. The results of this study offer opportunities to improve the supply chain of the different Sicilian landraces or historical varieties cultivated, underlining the need for accurate controls from the field to the transformation process to be labelled as mono-varietal products.

1. Introduction

Genetic improvement programs of durum wheat, launched in Italy in the early 1900s, have had a significant impact on productivity and technological grain quality as a result of the needs of the processing industry and consumers’ demands [1,2]. Nevertheless, the domestication process of wheat and polyploidy speciation has caused a reduction of genetic variability and confined the cultivation of the “old landraces” to niche areas, with their maintenance being entrusted to public research institutions and custodian farmers [3]. Over the last new decade, there has been a strong interest in landraces and historical wheat varieties from farmers, manufacturers and consumers [3,4,5]. Indeed, a higher tolerance of landraces against biotic and abiotic stresses, compared to the modern varieties, has been demonstrated [6,7]. Moreover, durum wheat landraces have a higher content of phenols, known for their healthy properties, than modern varieties [4,8], as well as a higher concentration of minerals [9]. From a technological point of view, some old wheat varieties are also well suitable to pasta production, such as Cappelli [10], and for bread-making, such as Andriolo [11].
Sicily, a wide region of Southern Italy, is particularly rich in landraces of many agricultural species, especially durum wheat. Because of their adaptability to low agronomic input and sustainability for organic farming, several durum wheat landraces were recently re-discovered and re-employed [3].
Thanks to the conservation work of the seeds and the agricultural cultural heritage of the CREA (Research Centre for Cereal and Industrial Crops of Acireale (CT)), it has been possible to repopulate the countryside with the traditional grains that are grown in organic or natural environments, thanks to their characteristics of rusticity and tolerance to biotic and abiotic stresses. Today, the flours of Sicilian landraces such as Timilia, Russello, Perciasacchi and Maiorca, or of historical varieties such as Margherito and Cappelli, are commercial available to increase the supply of native varieties.
Since 2009, the Ministry of Agriculture, Food, Forestry and Tourism Policies of Italy established the National Register of the varieties of agricultural plant species (Legislative Decree n. 149) [12] for the registration of landraces (syn. local varieties) naturally adapted to local conditions and threatened by genetic erosion. Unfortunately, only some landraces appear to be registered and the seeds are produced and maintained among farmers. This implies that it is difficult to classify different landraces that can be mixed during harvesting by mechanical means or during storage of the grain, or to a much lesser extent, to any cross-pollination between different genotypes during farming.
In order to preserve these landraces and derived-products, already present in the market, the discovery of markers for their identification is necessary. While in the past wheat genetic diversity has been mostly evaluated using morphological and phenological descriptors, with the development of PCR, DNA-based markers have been abundantly utilized because they are easy to use and can be also applied to flour and end products. In wheat, single nucleotide polymorphisms (SNPs) are the most employed molecular markers [13,14,15,16,17].
Other markers utilized for the classification of wheat are the gluten proteins. Gluten is a protein complex, typical of cereals, made up of hundreds of protein components structured in the form of monomers or polymers. In general, gluten proteins are distinguished in polymeric alcohol-insoluble glutenins and monomeric alcohol-soluble gliadins. They form complexes among themselves through the formation of intra-chain or inter-chain disulfide bridges, hydrogen bonds and hydrophobic interactions, giving rise to a protein network that plays an important role in determining the quality of bread-making and pasta-making processes [18,19]. Glutenin can be divided into two categories: high MW subunits (HMW-GS, 70–140 kDa) and low MW subunits (LMW-GS, 30–50 kDa). Durum wheat HMW-GS is coded by the loci Glu-A1 and Glu-B1, which are positioned respectively on the long arm of chromosomes 1A and 1B. On each locus there are two genes closely associated with each other that code for a subunit of type x (x-type) and one of type y (y-type), which have a different electrophoretic mobility; the x subunits have a lower mobility with respect to the subunits y. Each HMW-GS protein variant determines different quality characteristics of the wheat flour, so once the allelic structure has been identified, it is also possible to assign to each profile a different quality level; for example, the allelic variation in the Glu-B1 locus coding for the Bx7 + By8 subunits is associated with a good quality of durum wheat; while the Bx20 profile is associated with a lower quality [20,21,22]. In this regard, HMW-GS are quality markers that are used in breeding programs, to select wheat cultivars that have advantageous technological characteristics [23].
The most used technique for identifying the composition of HMW-GS is SDS-PAGE (sodium dodecyl sulfate-poly-acrylamide gel electrophoresis) [24], but mass spectrometry (MS) methods are also currently used [25,26,27]. MS differs to the more conventional methods in terms of resolution, sensitivity, accuracy, throughput and precision in quantification of proteins.
The aim of this work is the application of SDS-PAGE and MALDI/TOF-MS to assign the allelic composition of HMW-GS of three durum wheat Sicilian landraces (Timilia, Russello, Perciasacchi) and one historical variety (Margherito) in order to trace the varietal correspondence and the genetic pureness of grains and flours labeled as mono-varietal, produced by a Sicilian organic management farm. The Maiorca Sicilian landrace was also used to verify the presence of possible hexaploid wheat contamination.

2. Materials and Methods

2.1. Materials

In this work, seed batches and whole flours labelled as mono-varietal of three Sicilian durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn.) landraces, including Timilia, Russello, Perciasacchi, one historical variety, Margherito, and one bread wheat (Triticum aestivum L. subsp. aestivum) Sicilian landrace, Maiorca, were supplied by a farm in the province of Ragusa (Sicily) that grows and grinds wheat.
Pure seeds of the same landraces/historical variety, in particular Timilia (syn. Tumminia), Russello (syn. Priziusa), Ruscìa (syn. Russello ibleo), Perciasacchi (syn. Strazzavisazzi), Margherito (syn. Bidì) and Maiorca propagated in large carefully purified plots derived from the collection at the CREA (Research Centre for Cereal and Industrial Crops of Acireale, CT). The pure seeds of commercial durum wheat reference varieties Aureo (Bx6 + By8), Biensur (Bx7 + By8) and Cappelli (Bx20 + By20) supplied by CREA were used for the comparison of landraces and historical variety protein assets.

2.2. Extraction and Quantification of Gluten Proteins in Seeds and Commercial Flours

Gluten proteins, gliadins, HMW-GS subunits and LMW-GS subunits were determined in the commercial flours labelled as mono-varietal from the different genotypes using the sequential extraction procedure [28]. An amount of 30 mg of flour was subjected to extraction with 1.5 mL of 550 mL·L−1 propan-2-ol for 20 min with continuous mixing at 65 °C, followed by centrifugation at 10,000× g for 5 min. This step was repeated three times, and the supernatants were combined and dried in a vacuum centrifuge to obtain the protein gliadin fraction. The remaining pellet containing the GS fractions was suspended in a 400 μL solution of 550 mL·L−1 propan-2-ol, 0.08 mol·L−1 tris (hydroxymethyl) aminomethane hydrochloric acid (Tris–HCl, pH 8.3) and 10 g·L−1 1.4-dithiothreitol (DTT, as reducing agent) and incubated for 30 min at 60 °C with continuous mixing. After centrifugation at 14,000× g for 5 min, the supernatant was transferred to a new tube. To precipitate HMW-GS, acetone was added to obtain a final concentration of 400·mL L−1, which was then centrifuged at 14,000× g for 10 min. The LMW-GS fraction was precipitated in the remaining supernatant by adding acetone to obtain a final concentration of 800·mL L−1, and this was then centrifuged at 10,000× g for 10 min. The GS fractions and gliadins were dissolved in 500 mL·L−1 acetonitrile (ACN) with 1 mL·L−1 trifluoroacetic acid (TFA); relative quantification was determined by colorimetric Bradford assay (Bio-Rad, Hercules, CA, USA). Five technical replicates were performed for each sample.
Single seeds of the different pure wheat landraces and commercial varieties from CREA and each single seed deriving from batches of grains labelled as mono-varietal from the farm (Ragusa-Sicily) were crushed in a mortal with a pestle until a fine powder was obtained. The procedure for extracting HMW-GS from seeds (30 mg) was the same as reported above. Gliadins and LMW-GS were discharged during the procedure while the HMW-GS pellet was air dried and left at −20 °C for SDS-PAGE and MALDI-TOF/MS protein analyses.

2.3. Proximate Composition of Commercial Wheat Flour Samples

Moisture content, total protein, total fat, total fiber, starch and ash content were determined according to AOAC [29]. All analyses were performed in triplicate for each sample and are expressed as the means.

2.4. HMW-GS Protein Separation by SDS-PAGE

SDS-PAGE was performed in a Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, CA, USA) on 80 g·L−1 acrylamide gels. Aliquots of dried HMW-GS were suspended in 20·μL of loading buffer containing 20 g L−1 SDS, 0.2 g·L−1 bromophenol blue, 1 mL·L−1 β-mercaptoethanol, 0.05 mol·L−1 Tris–HCl (pH 6.8) and 100 mL·L−1 glycerol and boiled at 95 °C for 5 min before loading onto the gels. A ColorBurst™ Electrophoresis Marker High Range (MW 30,000–220,000 Da) was used to detect HMW-GS. After electrophoretic separation at 40 mA, the gels were stained with brilliant blue G-colloidal solution (Sigma-Aldrich, Milan, Italy) fixed in 70 mL·L−1 acetic acid and 400 mL·L−1 methanol, and de-stained in 250 mL·L−1 methanol. Image Lab 4.5.1 software (Bio-Rad) was used to assess protein molecular weights (MWs) of HMW-GS single protein subunits on each gel.

2.5. MALDI-TOF/MS HMW-GS Identification

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis in linear mode was carried out as previously described [30] by using a 4800 Plus MALDI-TOF/TOF™ (AB SCIEX, Framingham, MA, USA) equipment. A mass range of 10,000–100,000 Da was used, and about 400 laser shots were averaged to improve the signal-to-noise level. Calibration was performed by a ProteoMass Protein MALDI-MS Calibration kit (Sigma Aldrich, St. Louis, MO, USA). The matrix solution was prepared by dissolving sinapinic acid (SA) in 50% ACN at a concentration of 10 μg·μL−1 according to Liu et al. (2009) [24]. One microliter of 1:10 (v/v) sample/matrix solution mixture was spotted directly onto a stainless steel MALDI target plate, and the solution was allowed to dry at room temperature. Positively charged ions were analyzed in linear mode. Three biological replicates for each sample were performed.

2.6. Statistical Analyses

Statistical analysis of the data was performed with the Statgraphics Centurion XIV software (StatPoint Technologies, Inc., Warrenton, VA, USA) and the results compared by one-way ANOVA. Significant differences were determined by Tukey’s test.

3. Results and Discussion

3.1. Landraces History

Table 1 shows the origin, historical information and local use of the Sicilian wheat landraces and commercial varieties chosen for this study [31]. All genotypes were the most cultivated landraces in Sicily until the middle of the last century [32]. Even now, in some areas of Sicily, the landraces or historical varieties of wheat make it possible to guarantee the production of artisanal pasta, bread and other typical bakery products that are also Italian specialties in the world. For example, the bread named ‘Pane Nero di Castelvetrano’ (Castelvetrano Black Bread), made with at least 20% of cultivar Timilia, has been declared a Slow Food Presidium, a recognition awarded to safeguard high quality artisan products (Slow Food Foundation for Biodiversity, International Presidia) [33]. Other durum wheat breads have been acknowledged by the denomination Traditional Agricultural Food Product. Among them, ‘Pane di Monreale’ is made with Timilia and Russello flour [34].
Mono-varietal flours and pasta related to some of these local Sicilian varieties are already available on the market but the lack of a traceability system starting from seed propagation does not give the guarantee that the transformed product is 100% the variety signed on the label.

3.2. Chemical Composition of Commercial Sicilian Wheat Flours

Table 2 shows the chemical characteristics of Sicilian durum wheat landraces labelled as Timilia, Russello, and Perciasacchi, of one historical durum wheat variety Margherito and of the bread wheat Sicilian landrace Maiorca deriving from commercial batches from the Sicilian organic farm. In particular, there is a high fiber content in all samples, since they are wholemeal flours, while the total protein content is at least 13 g 100 g−1 flour, which is considered a good value for technological quality for all the semolina samples. Specifically, Perciasacchi showed a very high protein content (around 16 g 100 g−1 flour), indicating a good quality in regards to gluten constituent and protein content for the pasta making process [19].
As far as the total gluten proteins are concerned, similar amounts were found in Margherito, Perciasacchi and Russello, while significantly lower amounts were found in Timilia (p ≤ 0.05) (Table 2). Besides the total protein amounts, by sequential gluten protein extraction, the relative abundance of the different gluten protein classes (gliadins, HMW-GS and LMW-GS) were also calculated. Margherito and Perciasacchi showed higher relative abundance of HMW-GS with respect to the other durum wheat landraces and, in addition, Margherito has the highest LMW-GS relative abundance. The bread wheat Maiorca showed the highest amount of HMW-GS as expected for hexaploid wheat. Both HMW and LMW-GS are important parameters affecting gluten strength, which is the ability of the proteins to form a tenacious network able to promote better extrusion properties and thus pasta with superior cooking properties [19]. Timilia instead showed the higher relative abundance of gliadin fraction with respect to the other landraces (Table 2), which render this variety more suitable for the production of leavened bread, since gliadins are mainly responsible for giving bread the ability to rise properly during baking [19].

3.3. Identification of HMW-GS Profiles in the Pure Seeds of the Durum Wheat Sicilian Landraces/Historical Varieties

HMW-GS were extracted both from pure seeds of each local variety from the CREA collection and from seeds of the three durum wheat commercial samples (Cappelli, Aureo and Biensur) with different known HMW-GS assets and separated in SDS-PAGE at 8%. These latter commercial varieties with different HMW-GS protein assets were utilized in order to compare protein profiles and to assign specific protein profiles to our unknown samples [35]. In particular, HMW-GS of the following durum wheat varieties were used as a comparison: Biensur (Bx7 + By8), Aureo (Bx6 + By8), Cappelli (Bx20 + By20). Based on the electrophoretic distribution of the HMW-GS of type Bx and By, it was possible to identify and attribute the asset similar to HMW-GS Bx20 of Cappelli to Margherito and Perciasacchi, the variant Bx13 + By16 to Russello (syn. Priziusa) as previously indicated by other authors [36], and an asset similar to Bx6 + By8 to Ruscìa (syn. Russello ibleo) and Timilia (Figure 1 and Table 3). For Timilia, different authors report an asset of Bx20 at locus B1 [37,38], while Muccilli et al. [39], analyzing 13 Timilia accessions from the gene bank of Experimental Wheat Crop Consortium Station of Sicily, showed that all of them contained HMW-GS assets of 75.2 kDa and 86.4 kDa, similar to a possible Bx6 and By8 asset, found also in our reference seed sample deriving from the same gene bank. Moreover, the presence of many accessions of Timilia (Timilia reste bianche, Timilia reste nere, Timilia SG1, Timilia SG2, Tumminia SG3) in the South of Italy with different HMW-GS assets could not be excluded. The data on the different landraces analyzed in this work by SDS-PAGE were also validated by the exact m/z calculated by MALDI/TOF-MS (Figure 2 and Table 3).

3.4. Analysis of HMW-GS Profiles in Commercial Flours and in the Corresponding Seed Batch of Each Wheat Sicilian Landrace/Historical Variety

Following the identification of the HMW-GS of the pure seeds of the gene bank of CREA in durum wheat local varieties, a characterization of the HMW-GS was carried out in the samples of commercial flours labeled as mono-varietal and in the corresponding batches of wheat grains, both supplied by the Sicilian farm. This latter approach was necessary to verify that all the seeds of the same grain batch, from which flour labeled as mono-varietal is produced, are characterized by the same HMW-GS profile. For this purpose, 15 to 20 seeds were randomly selected from the seed batches from which the flours labelled as mono-varietal derived. Figure 3 shows, for each Sicilian local variety, an example of the HMW-GS profile of the commercial flour labelled as mono-varietal, the HMW-GS profile of the reference pure seed deriving from the CREA gene bank and a number of HMW-GS profiles of single seeds from the different commercial batches labelled as Margherito, Perciasacchi, Russello, Timilia. The bread wheat Maiorca flour and seed batch was also analyzed, as bread wheat was cultivated in the same farm.
As far the commercial flour samples labelled as mono-varietal are concerned, many protein bands were present in the samples and it was not possible to assign a specific HMW-GS profile corresponding to any of the pure seeds of the wheat landraces (Figure 3). In addition, a contamination of bread wheat was evident in many of the commercial flours of the durum wheat landraces, which is visible by the presence of high molecular weight HMW-GS subunits typical of D genome in the gels.
In particular, for Margherito, many different profiles of both durum and bread wheat are present and only a few seeds from the seed batch corresponded to the reference profile Bx20 present in Figure 1. As an example, in Figure 3, five seeds have the Bx6 + By8 profile, and in two seeds, five HMW-GS bands were found, some of those corresponding to proteins encoded by the D genome typical of bread wheat. These data support the hypothesis that the grain of Margherito certified as mono-varietal not only has contamination by other durum wheat varieties, but also contamination from Maiorca, bread wheat landrace cultivated in the same farm (Figure 3).
For Perciasacchi, most of the seeds are characterized by a profile different from the reference Bx20 (Figure 3). Two seeds have a Bx20 profile, while the remaining have the Bx6 + By8 profile (Figure 3). Bread wheat contamination was also present in the batch.
Single seeds of the Ruscìa batch are characterized by two protein profiles shown in Figure 3. Most of the seeds have the Bx13 + By16 profile, associated with the reference Russello (Figure 1), while some samples have a profile Bx6 + By8 corresponding to Ruscìa (Figure 1). However, although most of the seeds appear to belong to one of the two landraces, there are some seeds having a different profile than the reference ones. In fact, the presence of seeds showing a profile similar to Bx20 and others with 5 bands typical of bread wheat Maiorca was also highlighted.
As far as Timilia is concerned, the image of the gel in Figure 3 showed that most of the seeds are characterized by the HMW-GS Bx6 + By8 profile, consisting of 2 bands, which correspond to the reference profile in Figure 1. However, some samples have a different profile, corresponding to Bx7 + By8, as shown in the reference gel (Figure 1), which do not belong to any of the landraces analyzed in this study. The same analysis was performed on a seed batch from bread wheat Maiorca cultivated in the same farm. Most of the HMW-GS protein profiles of Maiorca seeds are made up of 5 bands. However, few seeds have the Bx6 + By8 profile; while other seeds have a Bx20 profile. Both of these profiles were ascribed to durum wheat landraces.
The MALDI-TOF/MS analyses performed on a further 40 seed samples from the different seed batches allowed us to define the percentages of contamination found in the different grain batches (Figure 4). The Margherito batch has 47% of Margherito grain, 40% of Timilia grain and 13% of bread wheat. Perciasacchi batch has only 13% of Perciasacchi seed, 74% of Timilia profile and 13% of bread wheat. Russello constitutes 70% of the grain called Russello, although only the “word” Priziusa appears on the label, 70% is made up of 50% of the Priziusa and 20% of the Ruscìa accession; a 5% contamination is from bread wheat. Timilia constitutes 70% of the grain called Timilia, while 20% was from durum wheat and 10% was bread wheat. The bread wheat Maiorca is also represented by 67%, but otherwise has contamination from durum wheat Timilia and Margherito.

4. Conclusions

The environmental and historical characteristics of Italy guarantee a high degree of agricultural biodiversity. The benefits of biodiversity are reflected in services relating to the food supply chain and cultural interest. Indigenous varieties and historic wheat varieties are agronomical and nutritionally interesting [40] and there is currently a strong interest from consumers, farmers and producers. However, the production and marketing of certified seeds, as well as the correspondence with derived products, is not guaranteed due to a lack of traceability along the supply chain. Therefore, the flours labeled as mono-varietal for the production of pasta/bread (reporting the specific genotype on the label), may not fully correspond to the declared variety.
The results of this study showed that each flour examined (labeled as mono-varietal) is cross-contaminated by the other local varieties of durum wheat and by the native Maiorca bread wheat grown on the same farm. Therefore, contaminations may be due either to the exchange of genetically non-pure seeds between custodian breeders or to the mixing of seeds during harvesting by mechanical means, or, to a lesser extent, to cross-pollination between different genotypes. All that has been highlighted therefore opens new opportunities to improve the supply chain of local wheat varieties through a traceability system to verify the varietal identity from the seed to the final product. Finally, a solid certification system for these products will be able to protect both the farmer and the consumer: the farmer will have an economic advantage as the consumer will be willing to pay a higher price if he has the guarantee on the product that he expects to have nutritional and health benefits.

Author Contributions

Conceptualization, G.V. and G.P.; Data curation, G.V. and G.P.; Formal analysis, G.G., C.A. and G.P.; Funding acquisition, G.V. and G.P.; Investigation, G.V., C.A., A.S. and G.P.; Supervision, G.V. and G.P.; Validation, G.V. and G.P.; Writing—original draft, G.V.; Writing—review and editing, G.V., A.S. and G.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the local funding of research from University of Parma (Giovanna Visioli) and by Project BIRD203914 of the University of Padova (Gabriella Pasini).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Gianluca Paredi, Interdepartmental Measure Centre “Giuseppe Casnati”, University of Parma for the MALDI/TOF- MS spectra acquisition.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Subira, J.; Peña, R.J.; Álvaro, F.; Ammar, K.; Ramdani, A.; and Royo, C. Breeding progress in the pasta-making quality of durum wheat cultivars released in Italy and Spain during the 20th Century. Crop Pasture Sci. 2014, 65, 16–26. [Google Scholar] [CrossRef] [Green Version]
  2. Maccaferri, M.; Harris, N.S.; Twardziok, S.O.; Pasam, R.K.; Gundlach, H.; Spannagl, M.; Ormanbekova, D.; Lux, T.; Prade, V.M.; Milner, S.G.; et al. Durum wheat genome highlights past domestication signatures and future improvement targets. Nat. Genet. 2019. [Google Scholar] [CrossRef] [Green Version]
  3. Fiore, M.C.; Mercati, F.; Spina, A.; Blangiforti, S.; Venora, G.; Dell’Acqua, M.; Lupini, A.; Preiti, G.; Monti, M.; Pè, M.E.; et al. High-Throughput Genotype, Morphology, and Quality Traits Evaluation for the Assessment of Genetic Diversity of Wheat Landraces from Sicily. Plants 2019, 8, 116. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Lo Bianco, M.; Siracusa, L.; Dattilo, S.; Venora, G.; Ruberto, G. Phenolic fingerprint of Sicilian modern varieties and old durum wheat landraces: A tool to assess with diversity. Cereal Chem. 2017, 94, 1045–1051. [Google Scholar]
  5. Di Loreto, A.; Bosi, S.; Montero, L.; Bregola, V.; Marotti, I.; Sferrazza, R.E.; Dinelli, G.; Herrero, M.; Cifuentes, A. Determination of phenolic compounds in ancient and modern durum wheat genotypes. Electrophoresis 2018, 39, 2001–2010. [Google Scholar] [CrossRef]
  6. Newton, A.C.; Begg, G.; Swanston, J.S. Deployment of diversity for enhanced crop function. Ann. Appl. Biol. 2009, 154, 309–322. [Google Scholar] [CrossRef]
  7. Lopes, M.S.; El-Basyoni, I.; Baenziger, P.S.; Singh, S.; Royo, C.; Ozbek, K.; Aktas, H.; Ozer, E.; Ozdemir, F.; Manickavelu, A.; et al. Exploiting genetic diversity from landraces in wheat breeding for adaptation to climate change. J. Exp. Bot. 2015, 66, 3477–3486. [Google Scholar] [CrossRef]
  8. Dinelli, G.; Segura-Carretero, A.; Di Silvestro, R.; Marotti, I.; Arráez-Román, D.; Benedettelli, S.; Ghiselli, L.; Fernadez-Gutierrez, A. Profiles of phenolic compounds in modern and old common wheat varieties determined by liquid chromatography coupled with time-of-flight mass spectrometry. J. Chromatogr. A 2011, 42, 7670–7681. [Google Scholar] [CrossRef]
  9. Ficco, D.B.M.; Riefolo, C.; Nicastro, G.; De Simone, V.; Di Gesù, A.M.; Beleggia, C.; Platani, C.; Cattivelli, L.; De Vita, P. Phytate and mineral elements concentration in a collection of Italian durum wheat cultiv. Field Crop. Res. 2009, 111, 235–242. [Google Scholar] [CrossRef]
  10. Fois, S.; Schlichting, L.; Marchylo, B.; Dexter, J.; Motzo, R.; Giunta, F. Environmental conditions affect semolina quality in durum wheat (Triticum turgidum ssp durum L.) cultivars with differerent gluten strength and gluten protein composition. J. Sci. Food Agric. 2011, 91, 2664–2673. [Google Scholar] [CrossRef]
  11. Migliorini, P.; Spagnolo, S.; Torri, L.; Arnoulet, M.; Lazzerini, G.; Ceccarelli, S. Agronomic and quality characteristics of old, modern and mixture wheat varieties and landraces for organic bread chain in diverse environments of northern Italy. Eur. J. Agron. 2016, 79, 131–141. [Google Scholar] [CrossRef]
  12. Legislative Decree n. 149, of the 29/10/2009 GU n. 254. 31 October 2009. Available online: http://www.fao.org/faolex/results/details/en/c/LEX-FAOC090873/ (accessed on 13 January 2021).
  13. Trebbi, D.; Maccaferri, M.; de Heer, P.; Sørensen, A.; Giuliani, S.; Salvi, S.; Sanguineti, M.C.; Massi, A.; van der Vossen, E.A.G.; Tuberosa, R. High-throughput SNP discovery and genotyping in durum wheat (Triticum durum Desf.). Theor. Appl. Genet. 2011, 123, 555–569. [Google Scholar] [CrossRef] [PubMed]
  14. Oliveira, H.R.; Hagenblad, J.; Leino, M.W.; Leigh, F.J.; Lister, D.L.; Penã-Chocarro, L.; Jones, M.K. Wheat in the Mediterranean revisited—Tetraploid wheat landraces assessed with elite bread wheat Single Nucleotide Polymorphism markers. BMC Genet. 2014, 15, 54. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Wang, S.; Wong, D.; Forrest, K.; Allen, A.; Chao, S.; Huang, B.E.; Maccaferri, M.; Salvi, S.; Milner, S.G.; Cattivelli, L.; et al. Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array. Plant Biotech. J. 2014, 12, 787–796. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Gao, L.F.; Jia, J.Z.; Kong, X.Y. A SNP-Based Molecular Barcode for characterization of common wheat. PLoS ONE 2016, 11, e0150947. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Burridge, A.J.; Winfield, M.O.; Allen, A.M.; Wilkinson, P.; Barker, G.L.A.; Coghill, J.; Waterfall, C.; Edwards, K.J. High-Density SNP Genotyping Array for Hexaploid Wheat and Its Relatives. Methods Mol. Biol. 2017, 1679, 293–306. [Google Scholar] [PubMed]
  18. Wieser, H. Chemistry of gluten proteins. Food Microbiol. 2007, 24, 115–119. [Google Scholar] [CrossRef]
  19. Sissons, M. Role of durum wheat composition on the quality of pasta and bread. Food 2008, 2, 75–90. [Google Scholar]
  20. Gianibelli, M.C.; Larroque, O.R.; MacRitchie, F.; Wringley, W. Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chem. 2001, 78, 635–646. [Google Scholar] [CrossRef]
  21. Palumbo, M.; Spina, A.; Boggini, G. Bread-making quality of Italian durum wheat (Triticum durum Desf.) cultivars. Ital. J. Food Sci. 2002, 2, 123–133. [Google Scholar]
  22. Spina, A.; Ammar, K.; Peña, R.J.; Bentivenga, G.; Sciacca, F.; Virzì, N.; Palumbo, M. Durum wheat breeding lines with new HMW glutenin subunits combinations selected for bread-making quality. In Proceedings of the International Symposium Genetics and Breeding of Durum Wheat, Rome, Italy, 27–30 May 2013; pp. 583–587. [Google Scholar]
  23. Gao, L.; Ma, W.; Chen, J.; Wang, K.; Li, J.; Wang, S.; Bekes, F.; Appels, R.; Yan, Y. Characterization and comparative analysis of wheat high molecular weight glutenin subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS. J. Agric. Food Chem. 2010, 58, 2777–2786. [Google Scholar] [CrossRef] [PubMed]
  24. Liu, L.; Wang, A.; Appels, R.; Ma, J.; Xia, X.; Lan, P.; He, Z.; Bekes, F.; Yan, Y.; Ma, W. A MALDI/TOF based analysis of high molecular weight glutenin subunits for wheat breeding. J. Cereal Sci. 2009, 50, 295–301. [Google Scholar] [CrossRef] [Green Version]
  25. Cunsolo, V.; Foti, S.; Saletti, R.; Gilbert, S.; Tatham, A.S.; Shewry, P.R. Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix assisted laser desorption/ionisation mass spectrometry and high performance liquid chromatog-raphy/electrospray ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 442–454. [Google Scholar] [CrossRef] [PubMed]
  26. Lagrain, B.; Brunnbauer, M.; Rombouts, I.; Koehler, P. Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry. PLoS ONE 2013, 8, e58682. [Google Scholar] [CrossRef]
  27. Liu, L.; Ikeda, T.M.; Branlard, G.; Peña, R.J.; Rogers, W.J.; Lerner, S.E.; Kolman, M.A.; Xia, X.; Wang, L.; Ma, W.; et al. Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat. BMC Plant Biol. 2010, 10, 124. [Google Scholar] [CrossRef] [Green Version]
  28. Visioli, G.; Bonas, U.; Dal Cortivo, C.; Pasini, G.; Marmiroli, N.; Mosca, G.; Vamerali, T. Variations in yield and gluten proteins in durum wheat varieties under late-season foliar vs. soil application of nitrogen fertilizer in a northern Mediterranean environment. J. Sci. Food Agric. 2018, 98, 2360–2369. [Google Scholar] [CrossRef] [Green Version]
  29. Association of Analytical Chemistry. A.O.A.C. Official Method of Analysis, 17th ed.; Association of Analytical Chemistry: Washington, DC, USA, 2005. [Google Scholar]
  30. Visioli, G.; Comastri, A.; Imperiale, D.; Paredi, G.; Faccini, A.; Marmiroli, N. Gel-based and gel-free analytical methods for the detection of HMW-GS and LMW-GS in Wheat Flour. Food Anal. Methods 2016, 9, 469–474. [Google Scholar] [CrossRef]
  31. Venora, G.; Blangiforti, S. I Grani Antichi Siciliani: Manuale Tecnico per il Riconoscimento delle Varietà Locali dei Frumenti Siciliani; Le Fate: Ragusa, Italy, 2017; ISBN 978-88-940976-6-5. [Google Scholar]
  32. De Cillis, U. I Frumenti Siciliani; Tipografia Zuccarello & Izzi: Catania, Italy, 1942; ISBN 88-7751-229-6. [Google Scholar]
  33. Giancaspro, A.; Colasuonno, P.; Zito, A.; Blanco, A.; Pasqualone, A.; Gadaleta, A. Varietal traceability of bread ‘Pane Nero di Castelvetrano’ by denaturing high pressure liquid chromatography analysis of single nucleotide polymorphisms. Food Control 2016, 59, 809–817. [Google Scholar] [CrossRef]
  34. Melini, V.; Melini, F.; Acquistucci, R. Nutritional characterization of an Italian traditional bread from ancient grains: The case study of the durum wheat bread “Pane di Monreale”. Eur. Food Res. Technol. 2020. [Google Scholar] [CrossRef]
  35. Janni, M.; Cadonici, S.; Bonas, U.; Grasso, A.; Dahab, A.A.D.; Visioli, G.; Pignone, D.; Ceriotti, A.; Marmiroli, N. Gene-ecology of durum wheat HMW glutenin reflects their diffusion from the center of origin. Sci. Rep. 2018, 8, 16929. [Google Scholar] [CrossRef] [Green Version]
  36. De Santis, M.A.; Cunsolo, V.; Giuliani, M.M.; Di Francesco, A.; Saletti, R.; Foti, S.; Flagella, Z. Gluten proteome comparison among durum wheat genotypes with different release date. J. Cereal Sci. 2020, 96, 103092. [Google Scholar] [CrossRef]
  37. De Santis, M.A.; Giuliani, M.M.; Giuzio, L.; De Vita, P.; Lovegrove, A.; Shewry, P.R.; Flagella, Z. Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy. Eur. J. Agron. 2017, 87, 19–29. [Google Scholar] [CrossRef] [PubMed]
  38. Mefleh, M.; Paola Conte, P.; Fadda, C.; Giunta, F.; Motzo, R. From seed to bread: Variation in quality in a set of old durum wheat cultivars. J. Sci. Food Agric. 2020, 100, 4066–4074. [Google Scholar] [CrossRef] [PubMed]
  39. Muccilli, V.; Lo Bianco, M.; Cunsolo, V.; Saletti, R.; Gallo, G.; Foti, S. High Molecular Weight Glutenin Subunits in Some Durum Wheat Cultivars Investigated by Means of Mass Spectrometric Techniques. J. Agric. Food Chem. 2011, 59, 12226–12237. [Google Scholar] [CrossRef]
  40. Boukid, F.; Folloni, S.; Sforza, S.; Vittadini, E.; Prandi, B. Current Trends in Ancient Grains-Based Foodstuffs: Insights into Nutritional Aspects and Technological Applications. Compr. Rev. Food Sci. Food Saf. 2018, 17, 123–136. [Google Scholar] [CrossRef] [Green Version]
Agronomy 11 00143 g001 550
Figure 1. HMW-GS extracted from three commercial varieties of durum wheat (Biensur, Aureo, Cappelli) and from the durum wheat reference seeds (Ruscìa, Russello, Margherito, Perciasacchi and Timilia) obtained from CREA. M: markers weight.
Figure 1. HMW-GS extracted from three commercial varieties of durum wheat (Biensur, Aureo, Cappelli) and from the durum wheat reference seeds (Ruscìa, Russello, Margherito, Perciasacchi and Timilia) obtained from CREA. M: markers weight.
Agronomy 11 00143 g001
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Figure 2. Examples of HMW-GS spectra of: (A) Russello (syn. Priziusa) and (B) Ruscìa (syn. Russello ibleo) durum wheat landraces. Mass spectra obtained by MALDI-TOF/MS analysis in linear mode and the corresponding bands separated by SDS-PAGE.
Figure 2. Examples of HMW-GS spectra of: (A) Russello (syn. Priziusa) and (B) Ruscìa (syn. Russello ibleo) durum wheat landraces. Mass spectra obtained by MALDI-TOF/MS analysis in linear mode and the corresponding bands separated by SDS-PAGE.
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Figure 3. SDS-PAGE of single seed HMW-GS extraction from batches of three durum wheat Sicilian landraces (Perciasacchi, Russello, Timilia), one historical durum wheat variety (Margherito) and one bread wheat Sicilian landrace (Maiorca). F = flour labelled as mono-varietal; Ref: pure seed HMW-GS reference; RefI = pure seed HMW-GS reference Ruscìa (syn. Russello ibleo); RefP = pure seed HMW-GS pure seed Russello (syn. Priziusa).
Figure 3. SDS-PAGE of single seed HMW-GS extraction from batches of three durum wheat Sicilian landraces (Perciasacchi, Russello, Timilia), one historical durum wheat variety (Margherito) and one bread wheat Sicilian landrace (Maiorca). F = flour labelled as mono-varietal; Ref: pure seed HMW-GS reference; RefI = pure seed HMW-GS reference Ruscìa (syn. Russello ibleo); RefP = pure seed HMW-GS pure seed Russello (syn. Priziusa).
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Figure 4. Percentages of contamination found in the different grain batches of three durum wheat Sicilian landraces (Perciasacchi, Russello, Timilia), one historical durum wheat variety (Margherito) and one bread wheat Sicilian landrace (Maiorca).
Figure 4. Percentages of contamination found in the different grain batches of three durum wheat Sicilian landraces (Perciasacchi, Russello, Timilia), one historical durum wheat variety (Margherito) and one bread wheat Sicilian landrace (Maiorca).
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Table
Table 1. Taxonomy, origin and main local uses of Sicilian wheat landraces/historical and commercial varieties analyzed in this study.
Table 1. Taxonomy, origin and main local uses of Sicilian wheat landraces/historical and commercial varieties analyzed in this study.
Taxonomic ClassificationGenotypesGeographic or Genetic Origin and Historical InformationYear of ReleaseLocal Use
T. turgidum ssp. durumCappelliHistorical variety selected in Apulia from Tunisian landrace Jenah Rhetifah by Strampelli1915Dry pasta, artisanal breads
T. turgidum ssp. durumMargherito (syn. Bidì)Historical variety selected in Sicily from Tunisian landraces ‘Mahmoudi and Bidì’ by Santagati (Eastern Sicily) with name Margherito and Tucci (Western Sicily) with name Bidì<1915Dry pasta, fresh pasta, artisanal breads
T. turgidum ssp. turanicumPerciasacchi (syn. Strazzavisazzi)Indigenous landrace from Sicily<1900Dry pasta, fresh pasta, artisanal breads, sweet and savory baked goods
T. turgidum ssp. durumRussello (syn. Priziusa)Indigenous landrace from Sicily1927Dry pasta, fresh pasta, artisanal breads, sweet and savory baked goods
T. turgidum ssp. durumRuscìa (syn. Russello ibleo)Indigenous landrace from Sicily<1900Hard dough natural leaved bread, fresh pasta
T. turgidum ssp. durumTimilia (syn. Tumminia)Indigenous landrace from Sicily<1900‘Castelvetrano Black bread’, hard dough natural leaved bread, fresh pasta, craft beer
T. aestivum. ssp. aestivumMaiorcaIndigenous landrace from Sicily<1900Homemade cakes, typical Sicilian sweets
T. turgidum ssp. durumAureoCommercial variety (Italy) genealogy: Kofa/Svevo2009Mono-varietal dry pasta (Voiello®)
T. turgidum ssp. durumBiensurCommercial variety (France) genealogy: not available2001Dry and fresh pasta
Table
Table 2. Proximate composition and gluten proteins quantification (Gliadins, high molecular weight glutenins—HMW-GS and low molecular weight glutenins -LMW-GS) of Sicilian commercial wheat flours.
Table 2. Proximate composition and gluten proteins quantification (Gliadins, high molecular weight glutenins—HMW-GS and low molecular weight glutenins -LMW-GS) of Sicilian commercial wheat flours.
Landrace/Historical VarietyTotal Protein Content 1Total Gluten Proteins 2Gli 3HMW-GS 3LMW-GS 3HMW/LMW-GSTotal GS/GliAsh 1Lipids 1 Fibre 1 Starch 1
Margherito12.9 b31.3 a 81.4 d3.1 b15.4 a0.2 b0.2 b2.0 a2.3 a13.1 a66.3 b
Perciasacchi15.9 a31.2 a 83.7 b3.7 b12.6 c0.3 b0.2 b1.8 b2.0 a 9.8 c70.3 a
Russello13.0 b31.2 a82.6 c1.6 c15.8 a0.1 c0.2 b1.6 b2.2 a12.1 b64.2 b
Timilia13.6 b29.4 b85.7 a1.3 c13.1 b0.1 c0.2 b1.8 b2.2 a 12.8 b65.1 b
Maiorca13.2 b23.1 c74.8 d9.3 a15.2 a0.5 a0.3 a1.9 b2.1 a10.6 c68.6 b
Within each parameter, different letters indicate significant differences (Tukey test, p < 0.05; n = 5). 1 Expressed as g 100 g−1 of dry matter. 2 Total gluten proteins correspond to the sum of the three different protein fractions (gliadins, HMW-GS and LMW-GS) expressed in mg g−1. 3 Percentage of the different gluten classes with respect to the total amount extracted2.
Table
Table 3. HMW-GS profiles assigned to the reference seeds of landraces and historical cultivars both with SDS-PAGE profiles in comparison with commercial durum wheat varieties and by MALDI-TOF/MS analyses.
Table 3. HMW-GS profiles assigned to the reference seeds of landraces and historical cultivars both with SDS-PAGE profiles in comparison with commercial durum wheat varieties and by MALDI-TOF/MS analyses.
Modern and Historical Cultivars and LandracesHMW-GS 1m/z2
BiensurBx7 + By882.927; 74.862
AureoBx6 + By886.510; 74.899
CappelliBx20 + By2082.467; 75.304
MargheritoBx20 + By2082.475; 75.488
PerciasacchiBx20 + By2082.423; 75.060
RuscìaBx6 + By886.026; 75.060
RusselloBx13 + By1682.612; 76.944
TimiliaBx6 + By886.618; 74.374
1 High molecular weight glutenin subunit assets (HMW-GS) assigned by SDS-PAGE. 2 m/z obtained by spectra from MALDI/TOF-MS analysis in linear mode.
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Visioli, G.; Giannelli, G.; Agrimonti, C.; Spina, A.; Pasini, G. Traceability of Sicilian Durum Wheat Landraces and Historical Varieties by High Molecular Weight Glutenins Footprint. Agronomy 2021, 11, 143. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11010143

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Visioli G, Giannelli G, Agrimonti C, Spina A, Pasini G. Traceability of Sicilian Durum Wheat Landraces and Historical Varieties by High Molecular Weight Glutenins Footprint. Agronomy. 2021; 11(1):143. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11010143

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Visioli, Giovanna, Gianluigi Giannelli, Caterina Agrimonti, Alfio Spina, and Gabriella Pasini. 2021. "Traceability of Sicilian Durum Wheat Landraces and Historical Varieties by High Molecular Weight Glutenins Footprint" Agronomy 11, no. 1: 143. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11010143

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