Identification of vetch species of the genus Vicia L. by the composition of the 11S globulin basic polypeptides of seeds using V. narbonensis complex and V. pannonica Crantz as an example

Background. The maintenance of purity, control of identity, and reproduction of seeds of different taxonomic groups of plants entering the collection requires simple and rapid screening methods that allow preliminary identification and registration of the material. The most suitable method for these purposes is SDS-electrophoresis of seed storage proteins, but its variants known from the literature mainly include analysis of the total protein pattern, which is usually very heterogeneous and thus complicates the interpretation of the obtained results. The objective of the work was to find a clearly identifiable and species-specific group of polypeptides of a particular protein in this multicomponent pattern. Materials and methods. The objects of the study were seed proteins of 47 accessions of five species of Vicia L. subgenus Vicia, analyzed by SDS-electrophoresis in 12.5% PAAG. Results and discussion. A comparison of patterns of the total extracted seed proteins separated by electrophoresis in the presence and absence of 2-mercaptoethanol identified a group of the 11S globulin (legumin) basic polypeptides, which allows visual unambiguous determination of a species (both self- and cross-pollinated) to which vetch seeds belong. It was found that the accessions differed only in the intensity of individual bands within the species, but not in the band composition, indicating the species-specific pattern of the 11S globulin basic polypeptides. Clear differences in the basic polypeptides composition were observed between the closely related species of the V. narbonensis complex, which are difficult to distinguish in the field. The species specificity of the patterns in this group of polypeptides correlates with the reproductive isolation of the species. In turn, cultivated and weedy accessions of Pannonian vetch, V. pannonica Crantz, of different geographical origins from the VIR collection with a likely predominance of cross-pollination showed homogeneity in the patterns of the legumin basic polypeptides. The patterns of the 11S globulin basic polypeptides of V. hyaeniscyamus Mout., V. narbonensis L., V. serratifolia Jacq., V. johannis Tamamschjan in Karyagin, and V. pannonica were recorded using the “soybean scale” in the form of a “polypeptide formula”. Conclusion. Therefore, the proposed method, including the identification of the 11S globulin basic polypeptides in the multicomponent pattern of seed proteins, can be recommended for rapid screening of seed collections of both self-pollinated and cross-pollinated species of the genus Vicia. This simple method is also promising for identifying species of other dicotyledonous crops.

1. Aleksandrova T. Vetches cultivated in the Russian Federation. In: International congress on oil and protein crops. Abstract book; 2018 May 20-24; Chisinau, Republic of Moldova. European Association for Research on Plant Breeding; Scientific Association of Geneticists and Breeders of the Republic of Moldova; 2018. p.59.

2. Ali H.B., Osman S.A. Genetic relationship study of some Vicia species by FISH and total seed storage protein patterns. Journal of Genetic Engineering and Biotechnology. 2020;18(1):37. DOI: 10.1186/s43141-020-00054-6

3. Bechkri S., Medoukali I., Khelifi D. Ecogeographic variability and genetic diversity associated with seed albumins, globulins and prolamins patterns in Vicia taxa from Algeria. Botanical Studies. 2017;58:1-5. DOI: 10.1186/s40529-017-0177-7

4. Bennett S.J., Maxted N. An ecogeographic analysis of the Vicia narbonensis complex. Genetic Resources and Crop Evolution. 1997;44:411-428. DOI: 10.1023/A:1008688919569

5. Bosmali I, Lagiotis G, Haider N, Osathanunkul M, Biliaderis C, Madesis P. DNA-based identification of Eurasian Vicia species using chloroplast and nuclear DNA barcodes. Plants. 2022;11(7):947. DOI: 10.3390/plants11070947

6. Bouabid S., Jemai L., Zoghlami Khélil A. Evaluation of the breeding system of two Vicia narbonensis L. accessions from East Mediterranean region. Euro-Mediterranean Journal for Environmental Integration. 2019;4:14. DOI: 10.1007/s41207-018-0095-4

7. Breeding systems table. Available from: https://cropgenebank.sgrp.cgiar.org/images/ file/forage_legumes/breeding%20systems%20table.pdf. [accessed Nov. 15, 2024].

8. Cooke R.J. (ed.). Handbook of variety testing. Electrophoresis handbook: variety identification., Cambridge: Chemistry and QA Department, National Institute of Agricultural Botany; 1992. Available from: https://www.seedtest.org/en/ publications/handbooks.html [accessed Nov. 15, 2024]

9. Cooke R.J. Gel electrophoresis for the identification of plant varieties. Journal of Chromatography A. 1995;698(1-2):281-299. DOI: 10.1016/0021-9673(94)00649-T

10. Dhull S.B., Kidwai M.K., Noor R., Chawla P., Rose P.K. A review of nutritional profile and processing of faba bean (Vicia faba L.). Legume Science. 2022;4(3):e129. DOI: 10.1002/leg3.129

11. Eggi E.E. Identification of cultivars of narrow-leaved lupine (Lupinus angustifolius L.) using the electrophoretic profile of polypeptides in seed proteins: guidelines (Identifikatsiya sortov lyupina uzkolistnogo (Lupinus angustifolius L.) s ispol'zovaniem elektroforeticheskogo spektra polipeptidov belkov semyan: metodicheskiye ukazaniya). I.P. Gavrilyuk (ed). St. Petersburg: VIR; 2013. [in Russian]

12. Eggi E.E. Electrophoresis of seed proteins for the cultivar identification of high polimorphing crops on the example of eastern galega (Galega orientalis Lam.). Agrarnaya Rossiya = Agrarian Russia. 2015;(11):14-20. [in Russian]. DOI: 10.30906/1999-5636-2015-11-14-20

13. Eggi E.E., Aleksandrova T.G. Determination of an unknown accession of common vetch for compliance with the variety Yubileinaya 110 according to morphology and electrophoresis of seed protein. Legumes and Groat Crops. 2019;4(32):71-81. [in Russian]. DOI: 10.24411/2309-348X-2019-11135

14. Eggi E.E., Aleksandrova T.G., Semenova E.V. The SDS electrophoresis method for monitoring and maintaining the purity of legume collections. In: 125 Years of Applied Botany in Russia: Book of abstracts of the Conference; 2019 November 25-28; St. Petersburg, Russia. St. Petersburg: VIR; 2019. p.80. [in Russian]. URL: http://www.vir.nw.ru/wp-content/uploads/2019/11/Sbornik-tezisov_125-let-prikladnoj-botaniki-v-Rossii-2.pdf [дата обращения: 19.11.2024].

15. El-Badan G.E., Amin A.W., Ashour F.M., El-Sadek L.M. Allele frequency and genetic diversity of some species of genus Vicia L. using SDS-PAGE technique. Egyptian Journal of Botany. 2021;61(2):491-511. DOI: 10.21608/ejbo.2021.47720.1579

16. El-Badan G.E., Amin A.W., Ashour F.M., El Sadek L.M. Characterization of the genetic diversity of some species of genus Vicia using ISSR and ITS molecular techniques. Egyptian Journal of Botany. 2022;62(2):475-492. DOI: 10.21608/ ejbo.2022.84745.1732

17. Enneking D., Maxted N. Narbon bean: Vicia narbonensis L. (Leguminosae). Evolution of Crop Plants. 1995:316-21.

18. Firincioğlu H.K., Ünal S., Doğruyol L. Phenotypic variation of Vicia pannonica Crantz (var. pannonica and var. purpurascens) in central Turkey. Journal of Central European Agriculture. 2011;12(1):82-91. DOI: 10.5513/JCEA01/12.1.882

19. Gianfranco V., Ravalli C., Cremonini R. The karyotype as a tool to identify plant species: Vicia species belonging to Vicia subgenus. Caryologia. 2008;61(3):300-319. DOI: 10.1080/00087114.2008.10589642

20. Han S., Sebastin R., Wang X., Lee K.J., Cho G.T., Hyun D.Y., Chung J.W. Identification of Vicia species native to South Korea using molecular and morphological characteristics. Frontiers in Plant Science. 2021;12:608559. DOI: 10.3389/fpls.2021.608559

21. Hasan A.A., Haddad D.A., Ali L.M. The phylogenetic relationships between species of Vicia L. based on morphological characteristics and proteins present in seeds. Asian Journal of Research in Botany. 2023;6(2):225-232. Available from: http://science.sdpublishers.org/id/eprint/1656/1/Hasan622023AJRIB106921.pdf [accessed Oct. 31, 2024].

22. Heim U., Schubert R., Bäumlein H., Wobus U. The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes. Plant Molecular Biology. 1989;13:653-663. DOI: 10.1007/BF00016020

23. Horstmann C., Schlesier B., Otto A., Kostka S., Müntz K. Polymorphism of legumin subunits from field bean (Vicia faba L. var. minor) and its relation to the corresponding multigene family. Theoretical and Applied Genetics. 1993;86;867-874. DOI: 10.1007/BF00212614

24. Ibañez S., Medina M.I., Agostini E. Vicia: a green bridge to clean up polluted environments. Applied Microbiology and Biotechnology. 2020;104;13-21. DOI: 10.1007/s00253-019-10222-5

25. Ilieva A., Naidenova G. Phenotypic evaluation of variability in quality traits of Hungarian vetch (Vicia pannonica ssp. pannonica Crantz) accessions. Bulgarian Journal of Crop Science. 2016;53(4):63-67.

26. Interactive agricultural ecological atlas of Russia and neighboring countries. Available from: https://agroatlas.ru/en/content/related/Vicia_pannonica/index.html [accessed Nov. 12, 2024].

27. Khalid U., Waheed M.Q., Parveen N., Arif M.A.R., Arif A. Estimation of genetic diversity using seed storage protein (SSP) profiling in wild and cultivated species of Cicer L. Molecular Biology Reports. 2023;50(5):4175-4185. DOI: 10.1007/s11033-023-08358-9

28. Khalik K.N.A., Al-Gohary I.H. Taxonomic relationships in some Vicia species from Egypt, based on seed morphology and SDS-PAGE of seed proteins. Acta Scientiarum-Biological Sciences. 2013;35(4):603-611. DOI: 10.4025/actascibiolsci.v35i4.19345

29. Konarev A.V., Tomooka N., Vaughan D.A. Proteinase inhibitor polymorphism in the genus Vigna subgenus Ceratotropis and its biosystematic implications. Euphytica. 2002;123:165-177. DOI: 10.1023/A:1014920309710

30. Konarev Al.V. Insect and fungal enzyme inhibitors in study of variability, evolution and resistance of wheat and other Triticeae Dum. cereals. In: P.R. Shewry, A.S. Tatham (eds). Wheat Gluten. Cambridge: Royal Society of Chemistry; 2000. p.526-530. DOI: 10.1039/9781847552372-00526

31. Konarev Al.V., Eggi E.E., Aleksandrova T.G. Proteolytic degradation of seed proteins of vetch species (Vicia L. subgenus Vicia) of section Peregrinae Kupicha during SDS-electrophoresis and its prevention. Plant Biotechnology and Breeding. 2024;7(3):5-18. [in Russian]. DOI: 10.30901/2658-6266-2024-3-o2

32. Konarev V.G. (ed.). Identification of varieties and registration of the gene pool of cultivated plants based on seed proteins (Identifikatsiya sortov i registratsiya genofonda kul’turnykh rasteniy po belkam semyan). St. Petersburg: VIR; 2000. [in Russian]

33. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685. DOI: 10.1038/227680a0

34. Maxted N. A phenetic investigation of Vicia L. subgenus Vicia (Leguminosae, Vicieae). Botanical Journal of the Linnean Society. 1993;111(2):155-182. DOI: 10.1006/bojl.1993.1013

35. Mazin A.M., Eggi E.E. Comparative evaluation of red clover genotypes with the "Pskovskiy mestnyy dvuukosnyy" variety. Fodder production. 2021;(6):16-21. [in Russian]. DOI: 10.25685/KRM.2021.35.75.001

36. McGregor S.E. Clover and some relatives. In: McGregor S.E. Insect pollination for cultivated crop plants. Chapter 3. Agricultural Research Service, U.S. Department of Agriculture; 1976. Available from: https://www.ars.usda.gov/ ARSUserFiles/20220500/OnlinePollinationHandbook.pdf [accessed Nov. 12, 2024].

37. Mirali N. The use of SDS-PAGE seed protein variability within and among accessions to predict the mating system of some Vicia species. Journal of Genetics and Breeding. 2005;59(3/4):253.

38. Potokina E.K.; Aleksandrova T.G. The pollination features in the annual species of the genus Vicia. (Osobennosti opyleniya u odnoletnikh vidov roda Vicia (Fabaceae)). Botanical journal = Botanicheskii zhurnal. 1996;81(1):74-79. [in Russian]

39. Potokina E., Endo Y., Eggi E., Ohashi H. Electrophoretic patterns of seed proteins in the East Asian Vicia species (Leguminosae) and their systematic utility. Journal of Japanese Botany. 2003;78(1):29-37.

40. Salehi B., Abu‐Reidah I.M., Sharopov F., Karazhan N., Sharifi‐Rad J., Akram M., Daniyal M., Khan F.S., Abbaass W., Zainab R., Carbone K., Fahmy N.M., Al-Sayed E., El-Shazly M., Lucarini M., Durazzo A., Santini A., Martorell M., Pezzani R. Vicia plants – A comprehensive review on chemical composition and phytopharmacology. Phytotherapy Research. 2021;35(2):790-809. DOI: 10.1002/ptr.6863

41. Shevkani K., Singh N., Chen Y., Kaur A., Yu L. Pulse proteins: Secondary structure, functionality and applications. Journal of Food Science and Technology. 2019;56:2787-2798. DOI: 10.1007/s13197-019-03723-8

42. Shewry P.R., Napier J.A., Tatham A.S. Seed storage proteins: structures and biosynthesis. The Plant Cell. 1995;7(7):945-956. DOI: 10.1105/tpc.7.7.945

43. Shutov A.D., Kakhovskaya I.A., Braun H., Bäumlein H., Müntz K. Legumin-like and vicilin-like seed storage proteins: evidence for a common single-domain ancestral gene. Journal of Molecular Evolution. 1995;41:1057-1069. DOI: 10.1007/BF00173187

44. Tarlakovskaya A.M., Eggi E.E., Gavriluyk I.P. Belyaeva Zh.I. (comp.) Identification of pea varieties by electrophoresis of seed proteins: guidelines (Identifikatsiya sortov gorokha metodom elektroforeza belkov semyan: metodicheskiye ukazaniya). V.G. Konarev (ed.). Leningrad: VIR; 1990. [in Russian]

45. Tripathy S.K. Legume seed storage proteins – A review. Advances in Bioresearch. 2018;9(6):152-162. DOI: 10.15515/abr.0976-4585.9.6.152162

46. Tucci M., Capparelli R., Costa A., Rao R. Molecular heterogeneity and genetics of Vicia faba seed storage proteins. Theoretical and Applied Genetics. 1991;81:50-58. DOI: 10.1007/BF00226111

47. Vishnyakova M.A., Seferova I.V., Buravtseva T.V., Burlyaeva M.O., Semenova E.V., Filipenko G.I., Aleksandrova T.G., Egorova G.P., Yankov I.I., Bulyntsev S.V., Gerasimova T.V., Drugova E.V. VIR global collection of grain legume crop genetic resources: replenishment, conservation and studying: methodological guidelines. M.A. Vishnyakova (ed.). 2nd ed. St. Petersburg: VIR, 2018. [in Russian]

48. Warsame A.O., Michael N., O’Sullivan D.M., Tosi P. Seed development and protein accumulation patterns in faba bean (Vicia faba, L.). Journal of Agricultural and Food Chemistry. 2022;70(30):9295-9304. DOI: 10.1021/acs.jafc.2c02061

49. Zha F., Rao J., Chen B. Modification of pulse proteins for improved functionality and flavor profile: A comprehensive review. Comprehensive Reviews in Food Science and Food Safety. 2021;20(3):3036-3060. DOI: 10.1111/1541-4337.12736