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© 2025 by the authors; licensee Asian Online Journal Publishing Group 
 

Agriculture and Food Sciences Research 
Vol. 12, No. 1, 49-53, 2025 

ISSN(E) 2411-6653/ ISSN(P) 2518-0193 
DOI: 10.20448/aesr.v12i1.6818 

© 2025 by the authors; licensee Asian Online Journal Publishing Group 

 
 

 
 
 
The potential of specific profilin based fingerprinting differs in legume species 

 
Adam Kovacik1   

Lenka Kucerova2     

Alzbeta Jauschova3   

Jana Ziarovska4   

 

 
( Corresponding Author) 

 
1,2,3,4Institute of Plant and Environmental Sciences, Faculty of Agrobiology and Food Resources, Slovak University 
of Agriculture in Nitra, Slovakia. 
1Email: adam.kovacik@uniag.sk  
2Email: xkucerova@uniag.sk  
3Email: xjauschova@uniag.sk  
4Email: jana.ziarovska@uniag.sk  

 
Abstract 

One requirement for using germplasm in agricultural development initiatives is its 
characterization, which necessitates knowledge of the genetic polymorphism and relationships 
among the individual varieties. Up to now, different DNA markers were utilized for this purposed, 
one of the newest are those for coding regions. Here, we aimed to investigate polymorphism and 
genetic relationships among 24 varieties of Cicer arietinum L. and 23 varieties of Pisum sativum L. 
using profilin based fingerprinting. PCR approach was used to generate homologue amplicons of 
plant profilins and UPGMA grouping for visualization of obtained fingerprint similarity. 
Amplification results showed different results for analysed legume species, where the higher 
polymorphism at the level of 96% was obtained within the accessions of pea varieties, as for 
chickpea fingerprints a very similar profiles were generated with only a limited amplicons of a 
total of three different length of 150 bp, 182 bp and 348 bp.  This information could be useful in 
breeding strategies for the improvement of chickpea and pea accessions. 

 
Keywords: Cicer arietinum L., Length polymorphism, Pisum sativum L., Profilin, DNA markers, Fingerprinting. 

 
Citation | Kovacik, A., Kucerova, L., Jauschova, A., & Ziarovska, J. 
(2025). The potential of specific profilin based fingerprinting differs 
in legume species. Agriculture and Food Sciences Research, 12(1), 49–
53. 10.20448/aesr.v12i1.6818 
History:  
Received: 15 November 2024 
Revised: 28 May 2025 
Accepted: 13 June 2025 
Published: 25 June 2025 
Licensed: This work is licensed under a Creative Commons 

Attribution 4.0 License  
Publisher:  Asian Online Journal Publishing Group 
 

Funding: This research is supported by the Grant Agency of Faculty of 
Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, 
Slovakia (Grant number: GA FAPZ 12/2024). 
Institutional Review Board Statement: Not applicable. 
Transparency: The authors confirm that the manuscript is an honest, 
accurate, and transparent account of the study; that no vital features of the 
study have been omitted; and that any discrepancies from the study as planned 
have been explained. This study followed all ethical practices during writing. 
Competing Interests: The authors declare that they have no competing 
interests. 
Authors’ Contributions: All authors contributed equally to the conception 
and design of the study. All authors have read and agreed to the published 
version of the manuscript. 

 

Contents 
1. Introduction ...................................................................................................................................................................................... 50 
2. Materials and Methods ................................................................................................................................................................... 50 
3. Results and Discussion ................................................................................................................................................................... 51 
References .............................................................................................................................................................................................. 52 
 

 

 

 

 

 

 

mailto:adam.kovacik@uniag.sk
mailto:xkucerova@uniag.sk
mailto:xjauschova@uniag.sk
mailto:jana.ziarovska@uniag.sk
https://creativecommons.org/licenses/by/4.0/
https://creativecommons.org/licenses/by/4.0/
https://www.doi.org/10.20448/aesr.v12i1.6818
https://orcid.org/0009-0004-7474-6056
https://orcid.org/0009-0001-9997-8214
https://orcid.org/0009-0008-2929-6403
https://orcid.org/0000-0002-0005-9729


Agriculture and Food Sciences Research, 2025, 12(1): 49-53 

50 
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Contribution of this paper to the literature 
Allergen based DNA marker techniques provide a valuable insight to polymorphism of plant coding 
regions. Here, profilin based polymorphism was firstly applied to analyse the variability among pea and 
chickpea germplasm. 

 
1. Introduction 

Genetic diversity of plants and its description is still of great importance in the research. Plants are inevitable 
parts of our daily lives, but many of their characteristics are still unknown. The group of legumes provide a basic 
part of human nutrition in many areas worldwide [1] that is why the interest in their germplasm genetic 
variability is actual and different studies were published previously [2-5]. Especially molecular and DNA marker-
based characterization of legume germplasm has the great potential in determining of genetic diversity and 
identification of unique variants that can be used in breeding programmes. In this study, selected varieties of Cicer 
arietinum L. and Pisum sativum L. were analysed. Both of this species are well characterized in their germplasm 
variability using different DNA based markers. Investigation of chickpea gerplasm by RAPD (Randomly Amplified 
Polymorphic DNA) provided a low degree of polymorphism with high coefficients of genetic similarity [6]. Inter-
simple sequence repeat (ISSR) markers were found to be dependent on the characteristics of their 3´anchores 
sequences in the ability to generate polymorphism in chickpea accessions and a combination of the principal 
component anaysis (PCA) together with cluster analysis in data analysis is important for informativeness of 
generated data and extracting the valuable informations [7, 8]. In this technique, more primers need to be used to 
analyse germplasm thoroughly [9]. Retrotransposon based DNA marker techniques such as iPBS (inter Primer 
Binding Sites polymorphism) were reported to be able to generate up to the 100% polymorphism in chickpea 
variability analysis [10]. Start codon targeted (SCoT) polymorphism is efficient in genetic diversity analysis of 
chickpea depending on the primer combinations and the genetic similarity can range from 84 to 98%, but many of 
primer combination can be monomorphic in its results [11]. Investigation of pea germplasm by RAPD were 
performed previously with good results in obtained polymorphism where clear pattern of clustering according to 
the source of germplasm were showed in different studies [12-14]. Microsatellite based markers were reported to 
differentiate successfully among parent and hybrid genotypes of pea [15] and provided a high level of 
polymorphism among pea genotypes [16]. SCoT are more informative marker compared to ISSR and RAPD 
markers for discrimination and identification of P. sativum subspecies [17]. Retrotransposon based marker 
techniques were proved to assess distinctness of pea germplasm, especially in cases where the time frame plays an 
important role. RBIP technique (Retrotransposon based information polymorphism) was reported to be robust and 
easy to score method, while multilocus IRAP (Inter retrotransposon amplified polymorphism) produced 
informative fingerprint already in a single analysis [18].  

DNA markers are actually developed based on in silico prediction and the of potential of these markers was 
reported to be efficient [19]. In silico approach in allergen coding-based DNA markers is based on the identification 
of conserved parts in the amino acid and genomic sequences of individual homologs of plant allergens.  Finding the 
similarity in alignments, specific as well as degenerate primers are designed [20]. Plant allergens and their protein 
and nucleotide sequences share a high degree of homology [21, 22] what allow to predict DNA markers for them, 
too. Previously, Bet v 1 based amplified polymorphism was conformed to be informative in various of plant species, 
as the genomic sequences of this pollen allergen of birch has the high level of conservation in its epitops [23, 24]. 
Here, an abundant plant allergen profilin wase used based on the designation of primer pair to its specific 
conservative part. When using a nonspecific profilin based primer pairs, the profilin based amplified polymorphism 
method (PBAP) was effective generating of specific fingerprints in all of the analysed genotypes of soybean and 
groundnut [25, 26] here, specific one was used. 

The aims of the present study were (1) analyzing of the potential of specific legume profilin based marker 
technique to reveal the polymorphism among legume species and genotypes, (2) characterizing and comparing the 
polymorphism based on the specific profilin based fingerprints generated for Cicer arietinum L. and Pisum sativum, 
L. 

 

2. Materials and Methods 
2.1. Biological Material 

Seeds of legume species Cicer arietinum L. and Pisum sativum L. were obtained from GeneBank of Slovak 

Republic, Piešťany. Different numbers of individual varieties were randomly selected to be able analyse the natural 
intraspecie variability as old land, ancient as well as modern varieties were represent in a final number of 24 for 
chickpea and 23 for pea. Young plants were obtained in vitro from sterilized seeds in basal Murashige and Skoog 
medium [27] with day-length 15h and 20°C.  

 

2.2. DNA Extraction 
Total genomic DNA was extracted by GeneJET™Plant Genomic DNA Purification Mini Kit (Thermo 

Scientific, Waltham, MA, USA) following the manufacturer instruction. Quantity and quality of extracted DNA 
was analysed spectrophotometrically by NanoPhotometer™ (Implen) and functionality in PCR was checked by 
ITS primers [28].  
 

2.3. Specific Profilin Based Polymorphism Analysis 
Primers for specific profilin based polymorphism fingerprints were designed using the in silico analysis of their 

conserved sequences [29]. In PCRs, DreamTaq™ DNA polymerase together with 600 nmol of each primer was 

utilized. PCR conditions of time and thermal profile was: 95 ◦C for 5 minutes (95 ◦C for 45 seconds; 55 ◦C for 45 

seconds; 72 ◦C for 35 seconds) 40x plus final 72 ◦C for 10 minutes. Obtained amplicons were separated in 2% 
agarose gel stained by GelRed®. Amplified fingerprints were transformed into binary matrices and based on this, 



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distance matrices and dendrograms of genetic dissimilarity were prepared using UPGMA analysed based on  
Jaccard index [30]. Dendrograms of profilins were created by DendroUPGMA software [31].  

 

3. Results and Discussion 
In the case of obtained fingerprints for Cicer arietinum L., a very similar profiles were generated with only 

a limited amplicons of a total of three different length of 150 bp, 182 bp and 348 bp. A total of six groups were 
obtained in the constructed dendrogram, that are part of three branches with the sparation of varieties Calia, 
Sefiros and Farihame with the same fingerprint profile (Figure 1). The first group of genotypes were characterized 
with only one amplified fargment with the length of 150 bp from which genotypes Yialousa and Kalika were 
separeted having one more amplicon with the length of 348 bp. Second branch of the dendrogram comprises from 
groups with amplicon of the 182 bp length and those that have this one plus amplicon with the lenght of 348 bp.  

 

 
Figure 1. Dendrogram of  chickpea accession using the profilin specific fingerprinting. 

 
For Pisum sativum L., very different specific profilin fingerprints were obtained. For individual varieties, from 7 

to 12 amplicons were generated with the length within the range from 95 bp up to the 1350 bp. The polymorphism 
in the analysed set of pea varieties was of 96%. Different groups were obtained in the constructed dendrogram, 
with a separation of varieties Lancet, Libochovicky urodny, Frostar and Parade (Figure 2). The most of analysed 
accessions were grouped with the average Jaccard coefficient of genetic similarity of 0.47 and two of analysed pea 
varieties (Cicero and Skagid) have the same profile of generated amplicons. An amplicon of the length 95 bp was 
amplified in all of the analysed pea varieties.  

Profilins belong to actin binding molecules and are defined as plant panallergens, what provide them as 
markers universal in their use [32]. For legume species - soybean and groundnut genotypes, profilins were 
previously utilized by using degenerated primer pair in PBAP (profilin based amplified polymorphism) [25, 26]. 
For both of this species, polymorphic profiles were generated. In the set of groundnut accessions, all were 
distinguishable but profiling based marker technique, but in soybean, a couple of varieties (Sciaming and Krajina) 
produced the same fingerprint profiles. PBAP fingerprints of Arachis hypogaea L. were distributed in the length 
ranging from 78 bp up to the 1642 bp and for soybean from 118 bp up to 1000 bp. This correspond to the finding of 
this study, that profilin based fingerprints are specie specific for legumes with different levels of polymorphism 
generated. Such differences in the ability of generating polymorphism and distinguishing of analyzed genotypes are 
in concordance with natural variability of profiling homologs in plants. Multiple proteins belonging to profilins 
were found to have their sequences in higher plants. They are divided into two classes that differ in their distinct 
expression patterns, mainly in vegetative and reproductive plant tissues [33]. Actuallly, more than four hundred 
plant profilin proteins sequences are stored at NCBI (National Center for Biotechnology Information) gene 
database [34]. It was reported previously that even small changes in the sequence of amino acid can alter the 



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biochemical properties of profilin substantially and a results in sequence diversity among profiling genes with their 
evolutionary origin in the polyphyletic mode [35]. 

 

 
Figure 2. Dendrogram of  pea accession using the profilin specific fingerprinting. 

 
The results obtained in this study provide data that profilin based generated polymorphism characteristics 

provide a good source of germplasm variability in the analysis of legumes. 
 

References 
[1] F. Stagnari, A. Maggio, A. Galieni, and M. Pisante, "Multiple benefits of legumes for agriculture sustainability: An overview," 

Chemical and Biological Technologies in Agriculture, vol. 4, pp. 1-13, 2017.   

[2] A. Savić, B. Pipan, M. Vasić, and V. Meglič, "Genetic diversity of common bean (Phaseolus vulgaris L.) germplasm from Serbia, as 
revealed by single sequence repeats (SSR)," Scientia Horticulturae, vol. 288, p. 110405, 2021.   

[3] K. Haliloğlu, A. Türkoğlu, H. I. Öztürk, G. Özkan, E. Elkoca, and P. Poczai, "iPBS-retrotransposon markers in the analysis of 
genetic diversity among common Bean (Phaseolus vulgaris L.) germplasm from Türkiye," Genes, vol. 13, no. 7, p. 1147, 2022.   

[4] Z. Hromadová, L. Mikolasova, Z. Balazova, M. Vivodík, M. Chnapek, and Z. Gálová, "Genetic diversity analysis of common bean 
(Phaseolus vulgaris L.) genotypes using scot polymorphism," Journal of Microbiology, Biotechnology and Food Sciences, vol. 12, no. 1, 
pp. e5919-e5919, 2022.   

[5] M. Vivodík, Ž. Balážová, M. Chňapek, Z. Hromadová, L. Mikolášová, and Z. Gálová, "Molecular characterization and genetic 
diversity study of soybean (Glycine max L.) cultivars using RAPD markers," Journal of Microbiology, Biotechnology and Food Sciences, 
vol. 12, no. Special issue, p. Article e9219, 2022.   

[6] R. Singh, C. Durga Prasad, V. Singhal, and G. J. Randhawa, "Analysis of genetic diversity in Cicer arietinum L using Random 
Amplified polymorphic DNA markers," Journal of Plant Biochemistry and Biotechnology, vol. 11, pp. 109-112, 2002.  

[7] S. Bhagyawant and N. Srivastava, "Genetic fingerprinting of chickpea (Cicer arietinum L.) germplasm using ISSR markers and 
their relationships," African Journal of Biotechnology, vol. 7, no. 24, pp. 4428-4431, 2008.  

[8] P. Yadav, K. K. Yadav, and S. S. Bhagyawant, "Molecular assessment of wild and cultivated Cicer species using ISSR markers," 
Ecological Genetics and Genomics, vol. 25, p. 100137, 2022.   

[9] A. R. Bagheri, V. Ghasemi Omran, and M. Haghpanah, "Chickpea (Cicer arietinum L.) genetic diversity detected by Inter Simple 
Sequence Repeat markers," Journal of Plant Molecular Breeding, vol. 9, no. 1, pp. 35-41, 2021.   

[10] E. E. Andeden, F. S. Baloch, M. Derya, B. Kilian, and H. Özkan, "iPBS-Retrotransposons-based genetic diversity and relationship 
among wild annual Cicer species," Journal of Plant Biochemistry and Biotechnology, vol. 22, pp. 453-466, 2013.   

[11] A. M. Serag, "Molecular characterization of ten cicer arietinum L. genotypes using SCOT marker," Journal of Agricultural Chemistry 
and Biotechnology, vol. 12, no. 1, pp. 1-4, 2021.  

[12] O. Koveza, Z. Kokaeva, F. Konovalov, and S. Gostimsky, "Identification and mapping of polymorphic RAPD markers of pea (Pisum 
sativum L.) genome," Russian Journal of Genetics, vol. 41, pp. 262-268, 2005.  

[13] B. Thakur, S. Sharma, I. Sharma, P. Sharma, and S. M. Zargar, "Diversity analysis of pea genotypes using RAPD markers," Legume 
Research-An International Journal, vol. 41, no. 2, pp. 196-201, 2018.  



Agriculture and Food Sciences Research, 2025, 12(1): 49-53 

53 
© 2025 by the authors; licensee Asian Online Journal Publishing Group 

 

 

[14] G. A. Wani, B. A. Mir, and M. A. Shah, "Evaluation of diversity in pea (Pisum sativum L.) genotypes using agro-morphological 
characters and RAPD analysis," International Journal of Current Research and Review, vol. 5, no. 10, p. 17, 2013.  

[15] A. Badr, H. Ahmed, M. Hamouda, and S. F. Badr, "Genetic diversity among varieties and hybrid lines of pea (Pisum sativum L.) as 
revealed by morphological traits and SSR markers," Egyptian Journal of Botany, vol. 55, pp. 17-29, 2015.   

[16] A. Mohamed et al., "Evaluation of the genetic relationships of some endangered Tunisian peas adapted to arid regions and Turkish 
accessions revealed by inter simple sequence repeat (ISSR) markers," Polish Journal of Environmental Studies, vol. 32, no. 3, pp. 
2251–2260, 2023.   

[17] S. A. Osman and H. Ali, "Genetic relationship of some Pisum sativum subspecies using different molecular markers," Jordan 
Journal of Biological Sciences, vol. 14, no. 1, pp. 65-74, 2021.  

[18] P. Smýkal, J. Horáček, R. Dostálová, and M. Hýbl, "Variety discrimination in pea (Pisum sativum L.) by molecular, biochemical and 
morphological markers," Journal of Applied Genetics, vol. 49, no. 2, pp. 155-166, 2008.  

[19] J. Žiarovská and L. Zeleňáková, "Application of genomic data for PCR screening of Bet v 1 conserved sequence in clinically 
relevant plant species. In D. Vlachakis (Ed.), Systems Biology," IntechOpen. 2019, pp. 65–82. 

[20] L. Hovaňáková, L. Klongová, and J. Žiarovská, "In silico prediction of sequential similarities of selected lipid transfer proteins," 
Agrobiodiversity for Improving Nutrition, Health and Life Quality, vol. 8, no. 1, pp. 1-8, 2024.  

[21] J. A. Jenkins, S. Griffiths-Jones, P. R. Shewry, H. Breiteneder, and E. C. Mills, "Structural relatedness of plant food allergens with 
specific reference to cross-reactive allergens: An in silico analysis," Journal of Allergy and Clinical Immunology, vol. 115, no. 1, pp. 
163-170, 2005.  

[22] M. Nedyalkova, M. Vasighi, A. Azmoon, L. Naneva, and V. Simeonov, "Sequence-based prediction of plant allergenic proteins: 
Machine learning classification approach," ACS Omega, vol. 8, no. 4, pp. 3698-3704, 2023.   

[23] J. Žiarovská and L. Urbanová, "Utilization of Bet v 1 homologs based amplified profile (BBAP) variability in allergenic plants 
fingerprinting," Biologia, vol. 77, no. 2, pp. 517-523, 2022.   

[24] L. Urbanová and J. Žiarovská, "Variability of DNA based amplicon profiles generated by Bet v 1 homologous among different 
vegetable species," Acta Fytotechnica et Zootechnica, vol. 24, no. 1, pp. 1-6, 2021.  

[25] A. Kováčik, J. Žiarovská, and L. Urbanová, "Variability of allergen-based length polymorphism of Glycine max L. varieties," 
Biology and Life Sciences Forum, vol. 30, no. 1, p. 20, 2024.  

[26] J. Žiarovská et al., "Polymorphism of Bolivian accessions of Arachis hypogaea L. revealed by allergen coding DNA markers," Plant, 
Soil and Environment, vol. 69, no. 12, pp. 615-627, 2023.   

[27] T. Murashige and F. Skoog, "A revised medium for rapid growth and bio assays with tobacco tissue cultures," Physiologia 
Plantarum, vol. 15, no. 3, pp. 473-497, 1962.   

[28] T. J. White, T. Bruns, S. Lee, and J. Taylor, "Amplification and direct sequencing of fungal ribosomal RNA genes for 
phylogenetics," PCR protocols: a guide to methods and applications, vol. 18, no. 1, pp. 315-322, 1990.  

[29] L. Klongová, A. Kováčik, L. Urbanová, M. Kyseľ, E. Ivanišová, and J. Žiarovská, "Utilization of specific primers in legume 
allergens based polymorphism screening," Science, Technology and Innovation, vol. 13, no. 2, pp. 12-21, 2021.   

[30] P. Jaccard, "New research on floral distribution," Bulletin de la Société Vaudoise des Sciences Naturelles, vol. 44, pp. 223–270, 1908.   
[31] S. Garcia-Vallvé, J. Palau, and A. Romeu, "Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in 

Escherichia coli and Bacillus subtilis," Molecular Biology and Evolution, vol. 16, no. 9, pp. 1125-1134, 1999.   

[32] S. Čerteková, A. Kováčik, L. Klongová, and J. Žiarovská, "Utilization of plant profilins as DNA markers," Acta Fytotechnica et 
Zootechnica, vol. 26, no. 3, pp. 324–331, 2023.  

[33] M. K. Kandasamy, E. C. McKinney, and R. B. Meagher, "Plant profilin isovariants are distinctly regulated in vegetative and 
reproductive tissues," Cell Motility and the Cytoskeleton, vol. 52, no. 1, pp. 22-32, 2002.  

[34] J. C. Jimenez-Lopez, S. Morales, A. J. Castro, D. Volkmann, M. I. Rodríguez-García, and J. d. D. Alché, "Characterization of 
profilin polymorphism in pollen with a focus on multifunctionality," PloS One, vol. 7, no. 2, p. e30878, 2012.   

[35] D. K. Pandey and B. Chaudhary, "Evolution of functional diversity among actin-binding profilin genes in land plants," Frontiers in 
Cell and Developmental Biology, vol. 8, p. 588689, 2020.   

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

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