Evaluation of molecular diversity of central European maize cultivars

Evaluation of molecular diversity of central European maize cultivars Želmíra Balážová*, Martin Vivodík, Zdenka Gálová Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Biochemistry and Biotechnology, Tr. A. Hlinku 2, 949 76, Nitra, Slovak Republic *Corresponding author: Martin Vivodík , Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Biochemistry and Biotechnology, Tr. A. Hlinku 2, 949 76, Nitra, Slovak Republic. Phone: +421 37 641 4269, E-mail: vivodikmartin@gmail.com Received: 05 May 2015; Revised: 18 October 2015; Accepted: 18 October 2015; Published Online: 20 October 2015 Emirates Journal of Food and Agriculture. 2016. 28(2): 93-98 doi: 10.9755/ejfa.2015.05.204 http://www.ejfa.me/ Balážová, et al.: RAPD analysis of maize 94 Emir. J. Food Agric ● Vol 28 ● Issue 2 ● 2016 relationships among them. In the case of maize, there are a lot of papers (Osipova et al., 2003; Carvalho et al., 2004, Asif et al., 2006; Bruel et al., 2007, Abuali et al., 2011, AlBadeiry et al., 2013, Molin et al., 2013) that have reported the application of the RAPD marker technique for maize molecular identification, and the technique was proved to be effective for or verification of purity and would improve the efficiency of breeding programmes (Asif et al., 2006). The aim of this study was to detect genetic variability among the set of 40 maize genotypes using 13 RAPD markers and to testify an usefulness of chosen RAPD markers for genetic diversity study. MATERIAL AND METHODS Plant material and extraction of genomic DNA Maize genotypes (40) were obtained from the Gene Bank VURV Praha-Ruzine (Czech Republic) and from the Gene Bank in Piesťany, the Slovak Republic (Table 1). Genomic DNA was isolated from the 14 days leaves with GeneJET Plant Genomic DNA Purification Mini Kit according to the manufacturer’s instructions. RAPD amplification and gel electrophoresis Amplification of RAPD fragments was performed according to Gajeraa et al. (2010) (Table 2) using decamer arbitrary primers (Operon technologies Inc, USA; SIGMA-D, USA). Polymerase chain reactions (PCR) were carried out in 25 μl of following mixture: 10.25 μl deionized water, 12.5 μl Master Mix (Promega, USA), 1.25 μl of genomic DNA, 1 μl of 10 pmol of primer. Amplification was performed in a thermocycler (Biometra, Germany) with initial denaturation at 94 °C for 5 min, 42 cycles of denaturation at 94 °C for 1 min, primer annealing at 38 °C for 1 min, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min. Amplified products were separated in 1.5% agarose in 1× TBE buffer. The gels were stained with ethidium bromide and documented using gel documentation system Grab-It 1D pre Windows. Data analysis The RAPD bands were scored as present (1) or absent (0), each of which was treated as an independent character regardless of its intensity. A dendrogram based on hierarchical cluster analysis using the unweighted pair group method with arithmetic average (UPGMA) with the SPSS professional statistics version 17 software package was constructed. For the assessment of the polymorphism between genotypes maize and usability RAPD markers in their differentiation we used diversity index (DI) (Weir, 1990), the probability of identity (PI) (Paetkau et al., 1995) and polymorphic information content (PIC) (Weber, 1990). Table 1: List of 40 analyzed genotypes of maize Genotypes Country of origin Year of registration 1. Feheres Sarga Filleres Hungary 1965 2. Mindszentpusztai Feher Hungary 1964 3. Zakarpatskaja Union of Soviet Socialist Republics 1964 4. Przebedowska Burskynowa Poland 1964 5. Krasnodarskaja Union of Soviet Socialist Republics 1964 6. Mesterhazy Sarga Simaszemu Hungary 1964 7. Slovenska biela perlova Czechoslovakia 1964 8. Zuta Brzica Yugoslavia 1975 9. Zloty Zar Poland 1964 10. Slovenska Florentinka Czechoslovakia 1964 11. C.44 Juhoslavska Yugoslavia 1964 12. Kostycevskaja Union of Soviet Socialist Republics 1964 13. Mindszentpusztai Sarga Lofogu Hungary 1964 14. Stodnova Czechoslovakia 1964 15. Slovenska žltá Slovak Republic 1964 16. Slovenska krajová velkozrná Slovak Republic 1964 17. Partizanka Union of Soviet Socialist Republics 1964 18. Voroneskaja Union of Soviet Socialist Republics 1964 19. Kocovska Skora Slovak Republic 1964 20. Milada Czechoslovakia 1964 21. Moldavskaja Union of Soviet Socialist Republics 1964 22. Bučiansky Konský Zub Slovak Republic 1964 23. Hodoninský konský zub žltý Czechoslovakia 1964 24. M Silokukurica Hungary 1964 25. Valticka Czechoslovakia 1964 26. Przebedowska Biala Poland 1964 27. Toschevska Slovak Republic 1964 28. Šamorinsky konský zub Hungary 1964 29. Wielkopolanka Poland 1964 30. Czechnicka Poland 1964 31. Manalta Czechoslovakia 1964 32. Zlota gorecka Poland 1964 33. Celchovicka ADQ Czechoslovakia 1964 34. Belaja mestnaja Union of Soviet Socialist Republics 1964 35. Bučanská žltá Slovak Republic 1964 36. Iregszemeseil 2 hetes Hungary 1964 37. Dnepropetrovskaja Union of Soviet Socialist Republics 1964 38. Bezuncukskaja Union of Soviet Socialist Republics 1964 39. Mikulická Czechoslovakia 1964 40. Aranyozon sarga lofogu Hungary 1964 RESULTS AND DISCUSSION Our study dealt with detection of genetic polymorphism in maize cultivars using RAPD markers. For the differentiation of forty maize genotypes thirteen RAPD markers (Table 1) Balážová, et al.: RAPD analysis of maize Emir. J. Food Agric ● Vol 28 ● Issue 2 ● 2016 95 were chosen according to Gajeraa et al. (2010). PCR amplifications using 13 RAPD primers produced 92 DNA fragments that could be scored in all genotypes (Figure 1). Chosen primers amplified DNA fragments across 40 maize genotypes studied, with the number of amplified fragments ranged from 5 (OPA-02, OPB-08, OPD-07) to 10 (OPA-13), and the amplicon size varying from 100 to 2500 bp. Of the 92 amplified bands, all 92 were polymorphic, with an average of 7.08 polymorphic bands per primer. The polymorphic information content (PIC) values varied from 0.709 (OPB-08) to 0.872 (OPA-13), with an average of 0.801 and index diversity (DI) value varied from 0.718 (OPB-08) to 0.874 (OPA-13) with an average of 0.808 (Table 3). Similar values of DI and the PIC were detected by other authors (Osipova et al., 2003; De Vasconcelos et al., 2008; Mukharib et al., 2010; Al-Badeiry et al., 2013; Molin et al., 2013; Mrutu et al., 2014) and these values presented a high level of polymorphism of maize genotypes detected by RAPD markers. Our results based on the values of DI and PIC showed that RAPD markers are suitable marker system to distinguish maize genotypes. Osipova et al. (2003) used RAPD markers to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. De Vasconcelos et al. (2008) used the RAPD technique to evaluate somaclonal variation in maize plants derived from tissue culture from the maize inbred line L48 (derived from Suwan). Forty seven different decamer oligonucleotide primers generated 221 amplification products, 130 of them being polymorphic. Al-Badeiry et al. (2013) used RAPD markers to fingerprint 20 varieties of maize. Twenty operon primers generated informative RAPD patterns and selected for further RAPD analysis. The largest number of polymorphic bands (20 bands) was produced by primer OPX-04 while, the lowest number of polymorphic bands (1 band) was produced by primer OPA-03. The primers of the most interest of this purpose were those that produced more variety specific DNA profiles, such as OPD-03, OPE-18, OPF-05, OPL-11 and OPX-04. In our study we have detected 7 polymorphic alleles by primer OPA-03. Much higher number of alleles (7) compared to Al-Badeiry et al. (2013), who detected only one allele, can be caused by diverse set of maize varieties used for analysis. Mrutu et al. (2014) assessed the genetic diversity of maize hybrids grown in Southern highlands of Tanzania by using RAPD markers. Twelve maize samples (six inbreds and six hybrids) were collected and used in this study. A total of 123 bands were produced of which 98 (80%) were polymorphic. The aim of Molin et al. (2013) was to estimate the genetic diversity across 48 varieties of maize landraces cultivated at different locations in the States of Rio Grande do Sul (RS) and Paraná (PR) by means of different marker system including random amplified polymorphic DNA (RAPD). Maize landrace accessions were genotyped using the 30 RAPD primers. RAPD analysis resulted in amplification of 335 fragments polymorphic fragments and a polymorphic index of 81.9%. Similar level of polymorphism (84.44%) obtained also Bruel et al. (2007). A dendrogram prepared based on hierarchical cluster analysis using UPGMA algorithm separated 40 maize genotypes into two clusters. First cluster contained two maize genotypes Mikulická and Aranyozon sarga lofogu. from Czechoslovakia and Hungary, respectively. Second cluster was subdivided in two subclusters (2a and 2b). Table 2: List of RAPD primers Primers Primer sequence (5 ́‐3 ́) Molecular weight range (bp) OPA-02 TGCCGAGCTG 300-2000 OPA-03 AGTCAGCCAC 250-900 OPA-13 CAGCACCCAC 400-2000 OPB-08 GTCCACACGG 400-1700 OPD-02 GGACCCAACC 500-2000 OPD-07 TTGGCACGGG 200-1000 OPD-08 GTGTGCCCCA 300-1700 OPD-13 GGGGTGACGA 100-1500 OPE-07 AGATGCAGCC 200-1300 OPF-14 TGCTGCAGGT 150-2500 SIGMA-D-01 AAACGCCGCC 300-2000 SIGMA-D-14 TCTCGCTCCA 400-800 SIGMA-D-P TGGACCGGTG 200-2500 Table 3: The statistical characteristics of the RAPD markers used in maize Primers Number of alleles DI PIC PI OPA-02 5 0.768 0.755 0.041 OPA-03 7 0.826 0.820 0.007 OPA-13 10 0.874 0.872 0.006 OPB-08 5 0.718 0.709 0.032 OPD-02 6 0.765 0.751 0.049 OPD-07 5 0.725 0.723 0.026 OPD-08 8 0.834 0.829 0.006 OPD-13 9 0.856 0.849 0.005 OPE-07 7 0.835 0.829 0.006 OPF-14 8 0.865 0.862 0.003 SIGMA-D-P 7 0.839 0.833 0.005 SIG


INTRODUCTION
Maize (Zea mays) is one of the world's most important crop plants after wheat and rice, which provides staple food to large number of human population in the world (Ahmad et al., 2011).It is belonging to the family of Poaceae.In developing countries maize is a major source of income to many farmers (Tagne et al., 2008).The genetic diversity observed across landraces is the most important part of maize biodiversity, and local races represent an important fraction of the genetic variability exhibited by this genus.However, few agronomic and genetic data exist for such collections, and this scarcity has limited the use, management, and conservation of this germplasm.In addition, a few improved genotypes with narrower genetic variability are quickly replacing maize landraces (Pollack, 2003).
Since 1990, random amplified polymorphic DNA (RAPD) markers have been successfully applied for identification of DNA polymorphism in various plant species (Williams et al., 1990).They are often used for screening of a wide range of genetic stocks in order to find linkage with traits of agronomic significance (Masojć et al., 2001).
RAPD technique requires only small amounts of DNA sample without involving radioactive labels and are simpler as well as faster.RAPD has proven to be quite efficient in detecting genetic variations and used for diversity assessment and for identifying germplasm in a number of plant species (Gajeraa et al., 2010;El Kichaoui et al., 2013;Srivashtav et al., 2013;Omalsaad et al., 2014;Vivodík et al., 2014;Žiarovská et al., 2014).Suitability of RAPD markers for the construction of genetic maps, fingerprinting and phylogenetic studies has been proved by many authors.In cereal crops, such as wheat (Saleh, 2012;Bibi et al., 2012;Cifci et al., 2012), barley (Bakht et al., 2011), rye (Persson et al., 2002;Petrovičová et al., 2014), the technique has been applied to identify cultivars and revealing phylogenetic relationships among them.In the case of maize, there are a lot of papers (Osipova et al., 2003;Carvalho et al., 2004, Asif et al., 2006;Bruel et al., 2007, Abuali et al., 2011, Al-Badeiry et al., 2013, Molin et al., 2013) that have reported the application of the RAPD marker technique for maize molecular identification, and the technique was proved to be effective for or verification of purity and would improve the efficiency of breeding programmes (Asif et al., 2006).
The aim of this study was to detect genetic variability among the set of 40 maize genotypes using 13 RAPD markers and to testify an usefulness of chosen RAPD markers for genetic diversity study.

Plant material and extraction of genomic DNA
Maize genotypes (40) were obtained from the Gene Bank VURV Praha-Ruzine (Czech Republic) and from the Gene Bank in Piesťany, the Slovak Republic (Table 1).Genomic DNA was isolated from the 14 days leaves with GeneJET Plant Genomic DNA Purification Mini Kit according to the manufacturer's instructions.

RAPD amplification and gel electrophoresis
Amplification of RAPD fragments was performed according to Gajeraa et al. (2010) (Table 2) using decamer arbitrary primers (Operon technologies Inc, USA; SIGMA-D, USA).Polymerase chain reactions (PCR) were carried out in 25 μl of following mixture: 10.25 μl deionized water, 12.5 μl Master Mix (Promega, USA), 1.25 μl of genomic DNA, 1 μl of 10 pmol of primer.Amplification was performed in a thermocycler (Biometra, Germany) with initial denaturation at 94 °C for 5 min, 42 cycles of denaturation at 94 °C for 1 min, primer annealing at 38 °C for 1 min, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min.Amplified products were separated in 1.5% agarose in 1× TBE buffer.The gels were stained with ethidium bromide and documented using gel documentation system Grab-It 1D pre Windows.

Data analysis
The RAPD bands were scored as present (1) or absent (0), each of which was treated as an independent character regardless of its intensity.A dendrogram based on hierarchical cluster analysis using the unweighted pair group method with arithmetic average (UPGMA) with the SPSS professional statistics version 17 software package was constructed.For the assessment of the polymorphism between genotypes maize and usability RAPD markers in their differentiation we used diversity index (DI) (Weir, 1990), the probability of identity (PI) (Paetkau et al., 1995) and polymorphic information content (PIC) (Weber, 1990).

RESULTS AND DISCUSSION
Our study dealt with detection of genetic polymorphism in maize cultivars using RAPD markers.For the differentiation of forty maize genotypes thirteen RAPD markers (Table 1) were chosen according to Gajeraa et al. (2010).PCR amplifications using 13 RAPD primers produced 92 DNA fragments that could be scored in all genotypes (Figure 1).Chosen primers amplified DNA fragments across 40 maize genotypes studied, with the number of amplified fragments ranged from 5 (OPA-02, OPB-08, OPD-07) to 10 (OPA-13), and the amplicon size varying from 100 to 2500 bp.Of the 92 amplified bands, all 92 were polymorphic, with an average of 7.08 polymorphic bands per primer.The polymorphic information content (PIC) values varied from 0.709 (OPB-08) to 0.872 (OPA-13), with an average of 0.801 and index diversity (DI) value varied from 0.718 (OPB-08) to 0.874 (OPA-13) with an average of 0.808 (Table 3).
Similar values of DI and the PIC were detected by other authors (Osipova et al., 2003;De Vasconcelos et al., 2008;Mukharib et al., 2010;Al-Badeiry et al., 2013;Molin et al., 2013;Mrutu et al., 2014) and these values presented a high level of polymorphism of maize genotypes detected by RAPD markers.Our results based on the values of DI and PIC showed that RAPD markers are suitable marker system to distinguish maize genotypes.Maize landrace accessions were genotyped using the 30 RAPD primers.RAPD analysis resulted in amplification of 335 fragments polymorphic fragments and a polymorphic index of 81.9%.Similar level of polymorphism (84.44%) obtained also Bruel et al. (2007).
A dendrogram prepared based on hierarchical cluster analysis using UPGMA algorithm separated 40 maize genotypes into two clusters.First cluster contained two maize genotypes Mikulická and Aranyozon sarga lofogu.from Czechoslovakia and Hungary, respectively.Second cluster was subdivided in two subclusters (2a and 2b).RAPD molecular markers have been used in population genetic studies (Žiarovská et al., 2013;Pawar et al., 2013;Petrovičová et al., 2014;Kallamadi et al., 2015).Some researchers have considered RAPD markers to represent segments of DNA with noncoding regions and to be selectively neutral (Vivodík et al., 2014), and some studies have shown that RAPD markers are distributed throughout the genome and may be associated with functionally important loci (Penner, 1996).

CONCLUSION
The analysis showed that the RAPD markers are very effective molecular markers for the assessment of the genetic diversity in maize and for differentiation of a set of maize genotypes.
A dendrogram based on UPGMA analysis separated 40 maize genotypes into two subclusters.The primers used in our analysis recorded 100 per cent polymorphism.RAPD markers can be used to identify diverse sources in crop germplasm collections or to select groups of genotypes with desirable characters and contrasting phenotypes, if large number are employed.RAPD markers are useful in the assessment of maize diversity, the detection of duplicate sample in genotypes collection, and the selection of a core collection to enhance the efficiency of genotypes management for use in maize breeding and conservation.

Table 3 : The statistical characteristics of the RAPD markers used in maize
Bruel et al. (2007)ined two genotypes of different origin from former Czechoslovakia and Yugoslavia, and subcluster 2b was further subdivided into two subclusters, subcluster 2ba with two genotypes from Slovakia and former Union of Soviet Socialist Republics, and subcluster 2bb with 34 maize genotypes.Two genotypes of 2bb subcluster (Kostycevskaja and Mindszentpusztai Sarga Lofogu) from former Union of Soviet Socialist Republics and Hungary, were genetically the closest (Fig 2).Dendrogram partially reflects the country of origin of studied maize genotypes.PCR amplification products of 18 genotypes of mayze produced with RAPD primer OPA-02.Lane M is 1-kb DNA ladder and lanes 1-18 are maize genotypes (Table 2).Dendrogram of 40 maize genotypes prepared based on 13 RAPD markers.CZE -Czechoslovakia, HU -Hungary, POL -Poland, SUN -Union of Soviet Socialist Republics, SK -Slovakia, YUG -Yugoslavia De Vasconcelos et al. (2008) based on cluster analyses divided the maize samples into three distinct groups, considering an upper limit of 0.38 genetic distances.Molin et al. (2013) using 30 RAPD primers studied the genetic diversity across 48 varieties of maize landraces cultivated at different locations in the States of Rio Grande do Sul (RS) and Paraná (PR) by means of different marker system including random amplified polymorphic DNA (RAPD).Regarding the RAPD dendrogram, groups comprising accessions from RS prevailed, whereas SSR comprised varieties from both collection sites.Mrutu et al. (2014)based on RAPD analysis assessed the genetic diversity of maize hybrids grown in Southern highlands of Tanzania.The range of genetic similarity of the studied samples calculating based on Jaccard's similarity coefficient was from 0.32 to 0.95.The UPGMA analysis indicated higher similarity between the hybrids than the inbreds.Bruel et al. (2007)constructed dendrogram using UPGMA clustering method.They found out that 16 lines separated into five distinct groups, which were in agreement with the heterotic patterns described based on the genealogy of the lines.