Assessment of genetic diversity in bread and durum wheat landraces based on biochemical and in silico analysis of glutenin and gliadin proteins

Leena M. SAIT; Areej S. Jalal; Nora M. AL ABOUD; Eman FAYAD; Mohammed ALQURASHI; Esmael M. ALYAMI; Fatmah Ahmed SAFHI

doi:10.15835/nbha53414855

Authors

Leena M. SAIT King Abdulaziz University, College of Science & Arts, Biological Sciences Department, Rabigh, 21911 (SA) https://orcid.org/0000-0002-5817-3903
Areej S. Jalal Princess Nourah bint Abdulrahman University, College of Science, Department of Biology, Riyadh, 11671 (SA)
Nora M. AL ABOUD Umm Al-Qura University, Faculty of Science, Department of Biology, Makkah, 21955 (SA)
Eman FAYAD Taif University, College of Sciences, Department of Biotechnology, Taif, 21944 (SA)
Mohammed ALQURASHI Taif University, College of Sciences, Department of Biotechnology, Taif, 21944 (SA)
Esmael M. ALYAMI 1) King Khalid University, College of Science, Department of Biology, PO Box 960, Asir, Abha, 61421; 2) King Khalid University, Research Centre for Advanced Materials Science (RCAMS), Abha, 61413 (SA)
Fatmah Ahmed SAFHI Princess Nourah bint Abdulrahman University, College of Science, Department of Biology, Riyadh, 11671 (SA)

DOI:

https://doi.org/10.15835/nbha53414855

Keywords:

bioinformatics and biochemical analysis, cluster analysis, durum wheat, gluten proteins, processing quality, SDD-PAGE

Abstract

This study assessed the genetic diversity between durum wheat landraces (L1-L12) and bread wheat landraces (L1-L12) using a comparative biochemical (SDS-PAGE) and bioinformatics analysis of their major gluten proteins: LMW-GSs, HMW-GSs, and gliadin fractions. Biochemical analysis revealed significant polymorphism in both species, with approximately 24-26% of the assessed loci showing variability. Cluster analysis based on the overall protein profiles successfully grouped all genotypes into three distinct clusters. Specific focus on the HMW-GSs and omega-gliadins revealed molecular markers related to end-use quality. Significant allelic polymorphism was detected at the Glu-1 loci: the Glu-A1 subunit 1 was fixed in durum wheat but rare in bread wheat, while Glu-A1 subunit 2 showed the inverse distribution. Diverse alleles (7, 8, 17, 18) were found at Glu-B1. Furthermore, bread wheat generally had five omega-gliadin subunits, compared to only four in durum wheat. Crucially, hierarchical clustering using the combined HMW-GS and omega-gliadin data provided a robust molecular signature, cleanly separating all genotypes into two clusters (Durum and Bread). This demonstrates the effectiveness of these two protein groups in distinguishing the wheat types, reflecting quality-related genetic differences. The subsequent bioinformatics analysis of 3D structures showed that most fractions (LMW-GSs, gamma-gliadin, and alpha/beta-gliadin) are highly conserved, with only minor variations in the N-terminal regions. In contrast, HMW-GSs and omega-gliadin exhibited significant structural divergence in their central repetitive domains. HMW-GS variations in beta-turn and beta-spiral distribution directly correlated with dough viscoelastic properties. Notably, durum omega-gliadin was found to be longer and contained more rigid polyproline type II helices, highlighting key structural differences influencing wheat quality.

References

Adzhubei AA, Sternberg MJ, Makarov AA (2013). Polyproline-II helix in proteins: structure and function. Journal of Molecular Biology 425:2100-2132. https://doi.org/10.1016/j.jmb.2013.03.018

Al-Khayri JM, Alshegaihi RM, Mahgoub EI, Mansour E, Atallah OO, Sattar MN, … Hassanin AA (2023a). Association of high and low molecular weight glutenin subunits with gluten strength in tetraploid durum wheat (Triticum turgidum spp. durum L.). Plants 12:1416. https://doi.org/10.3390/plants12061416

Al-Khayri JM, Alwutayd KM, Safhi FA, Alqahtani MM, Alshegaihi RM, Abd El-Moneim D, … Hassanin AA (2023b). Assessment of intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on omega, gamma and alpha-gliadin profiles. PeerJ 11:e16330. https://doi.org/10.7717/peerj.16330

Alboukadel K, Fabian M (2016). Factoextra: extract and visualize the results of multivariate data analyses. CRAN: Contributed Packages. https://doi.org/10.32614/cran.package.factoextra

Altenbach SB, Chang H-C, Yu XB, Seabourn BW, Green PH, Alaedini A (2019). Elimination of omega-1,2 gliadins from bread wheat (Triticum aestivum) flour: effects on immunogenic potential and end-use quality. Frontiers in Plant Science 10:580. https://doi.org/10.3389/fpls.2019.00580

Bhat GR, Sethi I, Rah B, Kumar R, Afroze D (2022). Innovative in silico approaches for characterization of genes and proteins. Frontiers in Genetics 13:865182. https://doi.org/10.3389/fgene.2022.865182

Bouabdellah N, Chacón E, Benavente E, Ruiz M, Giraldo P, Pascual L (2024). Image-assisted quantification of high and low molecular weight glutenin fractions in wheat by SDS-PAGE. Journal of Cereal Science 118:103977. https://doi.org/10.1016/j.jcs.2024.103977

Broccanello C, Bellin D, DalCorso G, Furini A, Taranto F (2023). Genetic approaches to exploit landraces for improvement of Triticum turgidum ssp. durum in the age of climate change. Frontiers in Plant Science 14:1271. https://doi.org/10.3389/fpls.2023.1101271

Bushuk W, Zillman RR (1978). Wheat cultivar identification by gliadin electrophoregrams. I. apparatus, method and nomenclature. Canadian Journal of Plant Science 58:505-515. https://doi.org/10.4141/cjps78-076

Desai SA, Patel VP, Bhosle K, Sapkal SP, More MJ (2025). Chapter 10 - Computational tools in genomics and proteomics. In: Shahiwala A, Surti N (Eds.). Challenges in delivery of therapeutic genomics and proteomics (2nd ed). Academic Press pp 489-518. https://doi.org/10.1016/B978-0-443-27416-9.00002-2

Dice LR (1945). Measures of the amount of ecologic association between species. Ecology 26:297-302. https://doi.org/10.2307/1932409

Franaszek S, Salmanowicz B (2021). Composition of low-molecular-weight glutenin subunits in common wheat (Triticum aestivum L.) and their effects on the rheological properties of dough. Open Life Sciences 16:641-652. https://doi.org/10.1515/biol-2021-0059

Guzmán C, Crossa J, Mondal S, Govindan V, Huerta J, Crespo-Herrera L, … Ibba MI (2022). Effects of glutenins (Glu-1 and Glu-3) allelic variation on dough properties and bread-making quality of CIMMYT bread wheat breeding lines. Field Crops Research 284:108585. https://doi.org/10.1016/j.fcr.2022.108585

Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. https://doi.org/10.1038/227680a0

Lafiandra D, Kasarda DD (1985). One-and two-dimensional (two-pH) polyacrylamide gel electrophoresis in a single gel: separation of wheat proteins. Cereal Chem 62:314-319.

Nazarzadeh Z, Onsori H, Akrami S (2020). Genetic diversity of bread wheat (Triticum aestivum L.) genotypes using RAPD and ISSR molecular markers. Journal of Genetic Resources 6:69-76. https://doi.org/10.22080/jgr.2020.18262.1172

Ozuna CV, Iehisa JCM, Giménez MJ, Alvarez JB, Sousa C, Barro F (2015). Diversification of the celiac disease α-gliadin complex in wheat: A 33-mer peptide with six overlapping epitopes, evolved following polyploidization. The Plant Journal 82:794-805. https://doi.org/10.1111/tpj.12851

Palombieri S, Bonarrigo M, Potestio S, Sestili F, Messina B, Russo G, …Masci S (2024). Characterization among and within sicilian tetraploid wheat landraces by gluten protein analysis for traceability purposes. Plants 13:741. https://doi.org/10.3390/plants13050741

Pandey V, Kapoor S, Patwa N, Gupta OP, Gopalareddy K, Ram S, Singh GP (2022). Molecular, biotechnological and omics-based interventions for improving wheat grain quality: advances and way forward. In: Kashyap PL, Gupta V, Prakash Gupta O, Sendhil R, Gopalareddy K, Jasrotia P, Singh GP (Eds.). New horizons in wheat and barley research. Springer Singapore, Singapore pp 759-787. https://doi.org/10.1007/978-981-16-4449-8_29

Patel MJ, Chakrabarti-Bell S (2013). Flour quality and dough elasticity: Dough sheetability. Journal of Food Engineering 115:371-383. https://doi.org/10.1016/j.jfoodeng.2012.10.038

Patil VR, Talati JG, Singh C, Parekh VB, Jadeja GC (2015). Genetic variation in glutenin protein composition of Aestivum and Durum wheat cultivars and Its relationship with dough quality. International Journal of Food Properties 18:2393-2408. https://doi.org/10.1080/10942912.2014.980948

Payne PI, Holt LM, Law CN (1981). Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin. Theoretical and Applied Genetics 60:229-236. https://doi.org/10.1007/BF02342544

Payne PI, Lawrence GJ (1983). Catalogue of alleles for the complex gene loci, Glu-A1, Glu-B1, and Glu-D1 which code for high-molecular-weight subunits of glutenin in hexaploid wheat. Cereal Research Communications 11:29-35.

Ribeiro M, Carvalho C, Carnide V, Guedes-Pinto H, Igrejas G (2011). Towards allelic diversity in the storage proteins of old and currently growing tetraploid and hexaploid wheats in Portugal. Genetic Resources and Crop Evolution 58:1051-1073. https://doi.org/10.1007/s10722-010-9642-9

Robin X, Waterhouse AM, Bienert S, Studer G, Alexander LT, Tauriello G, … Pereira J (2024). The SWISS-MODEL repository of 3D protein structures and models. In: Daina A, Przewosny M, Zoete V (Eds). Open access databases and datasets for drug discovery. Wiley‐VCH GmbH pp 175-199. https://doi.org/10.1002/9783527830497.ch6

Ruiz M, Giraldo P (2021). The influence of allelic variability of prolamins on gluten quality in durum wheat: An overview. Journal of Cereal Science 101:103304. https://doi.org/10.1016/j.jcs.2021.103304

Subedi M, Ghimire B, Bagwell JW, Buck JW, Mergoum M (2023). Wheat end-use quality: State of art, genetics, genomics-assisted improvement, future challenges, and opportunities. Frontiers in Genetics 13:1032601. https://doi.org/10.3389/fgene.2022.1032601

Tosi P, Masci S, Giovangrossi A, D’Ovidio R, Bekes F, Larroque O, … Shewry P (2005). Modification of the low molecular weight (LMW) glutenin composition of transgenic durum wheat: effects on glutenin polymer size and gluten functionality. Molecular Breeding 16:113-126. https://doi.org/10.1007/s11032-005-5912-1

Zhang S, Shan X, Wang Y, Lu T, Xu D, Gong H, … Gu YQ (2025). Recent duplications and rare structural variations revealed by comparative sequence analysis of low molecular weight glutenin subunits (LMW-GS) genes re-identified using LMWgsFinder in 26 genomes of the grass family. Theoretical and Applied Genetics 138:128. https://doi.org/10.1007/s00122-025-04919-7

Zhou Z, Geng S, Guan H, Liu C, Qin M, Li W, … Hou J (2022). Dissection of the genetic architecture for quantities of gliadins fractions in wheat (Triticum aestivum L.). Frontiers in Plant Science 13:826909. https://doi.org/10.3389/fpls.2022.826909