Research on genes identification for antiseptic action in some basil genotypes

 Corresponding author: Ligia Ion Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Research on genes identification for antiseptic action in some basil genotypes Mădălin Vasile Chiuoaru, Adrian Chira, Daniel Nagy, Gabriela Butnariu and Ligia Ion * University of Agronomic Sciences and Veterinary Medicine of Bucharest, faculty of Horticulture, 59 Mărăști Blvd, District 1, Bucharest, Romania. World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 Publication history: Received on 06 July 2025; revised on 12 August 2025; accepted on 15 August 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.2.2959 Abstract The studies undertaken by the present work refer to 21 domestic and foreign genotypes, in which a screening was done with 8 molecular markers, for the investigation of the genome in terms of highlighting the gene that controls the antiseptic action and the resistance of plants to biotic factors. The collection of basil genotypes is not very large in Romania. There are only 150 autochthonous basil genotypes. The genomic DNA isolated from the basil genotypes, “Aromat de Buzau”, “BZ 1”, “Hof Igal 2”, “Gea”, “LBRS 2”, “LBVS 1”, etc. was used to implement of RADP molecular markers to highlight the gene that controls antiseptic action. In order to determine, the DNA samples extracted from young leaves by the methods of Lodhi et al. 1994, modified by Pop et al., 2003 and Doyle & Doyle, 1990, were quantified/measured, amplified, and the amplification products (RAPDs) were electrophoretic ally separated in agarose gel. The research aimed to highlight the ObGAPDH gene in basil genotypes taken in the study using 8 specifically designed molecular markers. Keywords: Ocimum Basilico; Genotypes; Pathogen; Molecular Markers; Samples 1. Introduction Ocimum is a genus of aromatic plants and perennial and annual shrubs belonging to the Liliaceae family, comprising nearly 200 genera and 3,200 species. The number of species in the Ocimum genus is uncertain due to several taxonomic difficulties. Therefore, it could have 30–160 species. Ocimum basilicum L., or sweet basil, is an important species known for its medicinal properties and essential oil [15], [16]. Its flower has bilateral symmetry, with five petals and five sepals; the stem is erect, branched, solid, and hairy; the seeds are oval-shaped and black; the leaf is simple and opposite, with epidermal glands containing aromatic oil [12]. In recent years, Ocimum basilicum has been extensively studied for its various activities [4] [3]. In silico molecular docking is a rapidly developing field for understanding and predicting possible modes of interaction between a ligand and a target biomolecule [8, 9, 5, 11, 1], while molecular dynamics simulations have presented a plausible way to estimate the dynamic and energetic stability of a protein-ligand complex [1, 7, 17, 6, 13, 16, 10]. Therefore, a docking analysis of the main constituents of Ocimum basilica, linalool and estragole, with alpha-amylase and lipase together with their inhibitors was planned. The lipase inhibitory activity of plant extracts was estimated according to a reported protocol [14]. The DPPH free radical scavenging potential of polar and nonpolar extracts from Ocimum basilica leaves and flowers was determined according to the reported test [ 2] PCR which is used to rapidly make millions or billions of copies of a specific DNA sample, amplifying it exponentially in a series of temperature change cycles, thus allowing scientists to take a very small sample of DNA and amplify it to a quantity large enough to be studied. PCR technology was pioneered in 1983 by World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1267 American biochemist Kary Mullis at Cetus Corporation. The PCR process is now an indispensable procedure used in medical laboratory research in a wide variety of applications, including biomedical and forensic research. There are several PCR methods, but all are based on thermal cycling. These expose the reactants to repeated temperature changes to allow different reactions to occur—in particular, the melting and enzymatic replication of DNA. 2. Material and methods 2.1. Plant material consists of 21 genotypes of basil The research was conducted over three growing seasons, from 2021 to 2024, and involved measurements of the phenotypic characteristics of different basil genotypes, namely growth rate at 15 days, plant height, average bush diameter, average shoot length, and average number of shoots per plant. Other characteristics studied include the green mass weight of basil for the 16 genotypes studied over the three years allocated to the research. Other research addressed the average values of the germination period for the 16 genotypes studied over the three years allocated to the research. Table 1 Plant material basil plant biometrics for the 16 genotypes studied Nr cart. Genotypes studied Average height of the plant (cm) 1. Aromatic Basil from Buzau 75.50 2 Purple basil Serafim 77.10 3 Sweet basil L1 62.20 4 Limonero basil L2 73.30 5 Greek basil 30.21 6 Basil with salad leaves L4 84.40 7 Grand Vert Basil L5 60.30 8 Persian basil 52.48 9 Spicy Globe Basil L7 37.51 10 African basil L8 118.00 11 Holy basil var. Krishna L9 78.10 12 Macedonian basil 60.85 13 Dwarf basil Emarald 29.40 14 Dark Opal red basil 56.56 15 Siam Queen Basil 51.48 16 Basil cinnamon 110.50 17 BZ 1 60.22 18 Hofigal 2 71.13 19 Gea 81.11 20 LBVS 1 60.32 21 LBVS 2 62.22 (sutras/source: original) After the DNA isolation, the amount and purity of the DNA was determined for the 21 varieties studied. These determinations were made using the Nano Drop ND 33 000 device, with the Eppendorf Bio Photometer. The amount of DNA was expressed in ng/all and the purity was highlighted by realizing the ratio between two wavelengths, respectively 260/280. World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1268 After the phenotypic determinations were made, genotypic determinations followed, starting with the isolation of DNA from the basil genotypes studied. The DNA was quantified using a nanodrop and then revealed in a 2% electrophoresis gel. An amplification kit from Applied Biosystems with primers specific to the Ocimum Basilicum species was used to perform PCR. Following the optimization of amplification conditions, a PCR reaction was performed in a temperature gradient. Thus, a DNA sample was amplified at eight different temperatures: 51°C; 51.8°C; 53.1°C; 54.9°C; 57.4°C; 59.3°C; 60.5°C; 61°C (table 2). The PCR amplification products were migrated in 2% agarose gel and visualized under UV transilluminator. Considering the amplicon profile, the optimal primer hybridization temperature was selected. The amplification reaction was performed as follows: 20 all of reaction mixture was pipetted into each 0.2 all tube and 5 all of DNA was added, respectively 5 all of deionized H2O in the case of the negative control. The samples were vortexed and gently centrifuged, then placed in the PCR machine. The PCR machine was set according to the program in table 1. Table 2 PCR reaction scheme to optimize amplification conditions Temperature / Timp Denaturise initial 95 ºC, 10 min. Cyclura Denaturise: 95 ºC, 30 sec. Alimera primer: 5161 ºC, 30 sec. Extender: 72 ºC, 1 min. Nr. Cyclura 35 Extensae final 72 ºC, 15 min. (sutras/source: original) 3. Results and discussion Validation of amplicon identity is necessary to verify that the amplified DNA corresponds to the chosen target sequence. Agarose gel electrophoresis is a simple method for verifying the size of PCR products. However, sequencing the amplicons and comparing the results with those in databases is the most effective method of verifying the results obtained by PCR. The reaction products obtained by conventional PCR were sequenced after purification with the Wizard PCR Preps DNA Purification System (Promega). The markers and primers used for PCR amplification were labeled with Big Dye (ABI PRISM® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit, Applied Biosystems). Results regarding the quantification of genomic DNA at nanodrop for the studied basil genotypes (2021-2023) are summarized in Table 3. Table 3 Results regarding the quantification of genomic DNA at nanodrop for the studied basil genotypes Nr. Cart. Genotype Genomic and concentration (ng/ all) Report abs. 260/280 1.98 Report abs. 260/230 2,01 Absorption D260 24.532 1. Aromatic Basil from Buzau 1226.6 1.98 2.01 24.532 2. Purple basil Serafim 2108.2 1.96 2.02 42.164 3. Sweet basil L1 1630.2 1.97 1.98 32.603 4. Lemon basil L2 4847.3 1.7 1.64 96.945 5. Greek basil 259.3 1.71 1.12 5.185 6. Basil with salad leaves L4 429.7 1.71 0.97 8.593 7. Grand Vert Basil L5 870.6 1.71 1.05 17.411 World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1269 8. Persian basil 1481.2 1.86 1.43 29.625 9. Spicy Globe Basil L7 164 1.71 0.98 3.279 10. African basil L8 442.8 1.72 1.21 8.856 11. Holy basil var. Krishna L9 479.5 1.67 1.01 9.59 12. Macedonian basil 1478.7 1.82 1.36 29.575 13. Dwarf basil Emarald 261.6 1.82 1.38 5.231 14. Dark Opal red basil 224.2 1.34 1.71 4.485 15 Siam Queen Basil 794.6 1.82 1.26 15.892 16 Basil cinnamon 222.4 1.25 1.23 12.852 17 BZ 1 462.2 1.52 1.12 4.241 18 Hofigal 2 481.1 1.61 1.22 3.123 19 Gea 391.5 1.45 1.14 4.472 20 LBVS 1 473.4 1.71 1.21 4.732 21 LBVS 2 444.2 1.62 1.11 3.751 (sutras/source: original) Table 3 shows the results of genomic DNA quantification by nanodrop for the basil genotypes studied. The amount of genomic DNA varied greatly from genotype to genotype, with the highest value recorded for the Limonero basil genotype, with a value of 4847. The lowest values were recorded for the Sweet Basil genotype with values of 1630.2 ng/μl and the Persian Basil genotype with values of 1481.2 ng/all. The lowest values were recorded for the Spicy Globe basil genotypes with values of 164 ng/all, the Dark Opal red basil genotype with values of 224.2 ng/all, followed by the Greek basil genotype with values of 259.3 ng/all and Basil cinnamon 222.4 ng/all The final DNA solution is prepared according to the specific preparation method, either by resuspending the DNA sediment after its precipitation with alcohols (ethanol or isopropanol) in the presence of salts. The genetic material, DNA, was stored (at -20ºC) in TE solution, but it can also be stored in sterile deionized water. In the present experiment, the sediment was resuspended in 100 all TE pH 8.0. Table 4 Results of the unifactorial analysis for the factor regarding the quantification of genomic DNA at nanodrop, for the genotypes studied SOURCE OF THE VARIANT VARIANT GRADE of Liberty CORRECTED DISPERSION FISHER FACTOR CRITICAL FACTOR (SPA) (L) (S2) (F calculate) 10 % 5 % 1 % 0.1 % BETWEEN GROUPS (SYSTEMATICS) 5232179.07 14 373727.0763 0.5877 1.6620 1.9200 2.5200 3.3600 WITHIN GROUPS (RESIDUAL) 28618013.96 45 635955.8657 TOTAL 33850193.02 59 DIFERENCE LIMIT (DL) 1894.688 3 2271.3704 3033.7568 3969.8231 (sutras/source: original) Thus, Table 4. presents the univariate analysis between the factors of variation regarding the quantification of genomic DNA at nanodrop for the studied basil genotypes, which shows us a limit difference of 5% at 2271.3704, 1% at 3033.7568, and at 0.1% of 3969.8231, meaning the influence of factors on the resulting characteristics. World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1270 (sutras/source: original) Figure 1 Results of the unifactorial analysis for the factor regarding the quantification of genomic DNA at nanodrop, for the genotypes studied After DNA extraction, the samples were quantified spectrophotometrically using the Eppendorf Bio Photometer in the first part of the experiment and the Nanodrop NA 33 000 in the second part of the experiment. The spectrophotometric method is based on the fact that most biological substances have a characteristic absorption rate in the ultraviolet radiation range. Thus, the absorption rate of 260 nm corresponds to nucleic acids, that of 280 nm to proteins, and 230 nm to various contaminants. The optical density was measured at the absorption rates A 260 nm and A 280 nm, calculating the ratio between the two absorptions. (Figure1.) The univariate ANOVA analysis of the amount of genomic DNA extracted from different Ocimum basilicum genotypes revealed large variations between genotypes. The highest value was recorded in the Basil Limonero genotype (4847.3 ng/all), followed by Basil violet Serafim (2108.2 ng/all), Sweet basil (1630.2 ng/all), and Basil person (1481.2 ng/all). In contrast, the lowest values were found in Spicy Globe basil (164 ng/all), Dark Opal red basil (224.2 ng/all), and Greek basil (259.3 ng/all). However, only the difference between the Limonero basil genotype and the other genotypes exceeded the Limit Difference (LD) at p < 0.05 of 2271.3704 ng/all, indicating a statistically significant difference only in this case. Therefore, Basil Limonero stands out as having a significantly superior genetic capacity in terms of genomic DNA accumulation or stability, which may indicate a higher potential for biotechnological or genetic applications. Regarding the quantification of genomic DNA, the results were quite uneven, with values ranging from 4847.3 ng/µl for the "Basil Limonero" variety to 164 ng/µl for the "Basil Spicy Globe" variety. Table 5 Primers tested at Og-USAMVB Hort invest Nr Cart Primers Sequence (5’-3’) 1. Og A07 GAG ACG GGT A 2. ObGA08 ATG ACG TAG G 3. Og A09 CGG TAA CGC C 4. ObGA10 CTG ATC GCA C 5. Og A11 GAA TGC CCG T World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1271 6. ObGA12 CTG CAC CCA C 7. Og A13 ATG TGA CCG T 8. ObGB04 CGA CAG GTG A (sutras/source: original) However, given that small amounts of DNA in the order of 10–20 ng/µl are sufficient for SSR marker analysis, it is important to know the initial values in order to determine the necessary dilutions. For molecular marker analysis, the reaction mixture was prepared under a laminar flow hood under sterile conditions. A control sample containing all components of the reaction mixture except DNA was also taken into account. In order to amplify the samples obtained in the first part of the experiment, eight Deca nucleotide primers were used, each primer having 10 nucleotides (Table 5.) They were chosen from among those that presented polymorphic bands. These primers arrive at the laboratories in lyophilized form, each with a specific recipe for dilution when used. 3.1. Results obtained in the case of amplification of DNA extracted using the Doyle and Doyle method Of the primers studied, five primers (ObGA07, ObGA08, ObGA11, ObGA13, ObGA18, and ObGB04) amplified bands in only one variety, and two of the primers, ObGA09 and ObGA10, did not amplify any fragments in any of the varieties (Figure 1.6), and the amplified bands were unclear, making it impossible to assess them accurately. (sutras/source: original) Figure 2 Results concerning the bands obtained from three varieties of the test primers Og There could be an explanation related to the DNA samples, which did not have the necessary quantity or purity to produce polymorphism with the primers tested, even though they are universal and should have produced results. The polymerase used was Aplite Polymerase (Stoffel) and not Promega, and the amplification power may have been lower. (Figure 2.) 3.2. Analysis and processing of results obtained by RAPD The results refer to a total of 8 samples, of which 2 revealed polymorphism between genotypes, while the other 6 presented monomorphic bands, with a total of 75 bands generated, of which 66 were polymorphic (Table 6). Table 6 RAPD data obtained from image analysis (2023) Nr. Cart. Primer Total number of lanes Lanes/primer No. of polymorphic bands Bands/primer No. of monomorphic bands Bands/primer % polymorphism 1. Og A07 12 11 1 99.6 2. Og A08 9 8 1 88.8 3. Og A09 7 6 1 85.7 World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1272 4. Og A10 7 5 2 71.4 5. Og A11 11 10 1 92.8 6. Og A12 10 9 1 93.3 7. Og A13 8 8 0 100 8. Og B04 11 10 1 90.9 (sutras/source: original) In the laboratory, band detection was performed using the Faliscan program, by comparing them with a standard DNA, with a size between 100 and 4000 bp. All fragments resulting from amplification with polymorphic primers were between 200 and 2000 bp in length, with most between 300 and 1200 bp (in the first amplification case). 3.3. Interpretation of results Interpretation of the results obtained by the RAPD method involves the genomic DNA isolation protocol, the PCR reaction, the amplification program, and image acquisition using the Alpha Imager 2200 program. The smaller the genetic distance values in the table (binary matrix), the more closely related the genotypes are. These primary data were entered into the RAP Distance 1.04 program, which calculated the genetic distances based on the Jaccard coefficient (Table 7.). The formula used to calculate the Jaccard coefficient (Jij) is as follows: Jiji = Cin/ (in + Nj – Cin) where: Cin - represents the number of identical bands presented in the two genotypes; nj - represents the total number of bands identified in genotype I and j, respectively. Table 7 Results regarding the genetic distance of basil genotypes Nr cart. Genotypes Gene distance (come) Marker Og A07 Gene distance (come) Marker Og B04 1. Aromat de Buzău basil 1/04 1/42 2. BZ 1 1/04 1/42 3. Hof Igal 2 1/04 1/42 4. Gea 1/04 1/42 5. LBRS 2 1/12 1/62 6. LBVS 1 1/12 1/62 7. Serafim basil 1/12 1/62 8. Sweet basil 1/04 1/42 9. Basil Limonero 1/12 1/62 10. Greek basil L3 1/12 1/62 11. Basil Frunze de salat L4 1/21 1/69 12. Basil Grand Vert L5 1/21 1/69 13. Persin basil 1/21 1/69 14. Basil Spicy Globe L7 1/21 1/69 15. African basil 1/21 1/69 16. Basil Krishna L9 1/21 1/69 17. Basil Macedon 1/21 1/69 18. Basil Piti Emarald 1/21 1/69 World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1273 19. Dark Opal basil 1/21 1/69 20. Basil Siam Queen 1/21 1/69 21. Basil Cinnamon 1/21 1/69 (sutras/source: original) Table 7. shows the results of the gene distance calculation for the two markers that showed the highest percentage of polymorphism, ObGA07 and Og B04. Both molecular markers establish the presence of the ObGAPDH gene, but the significance of its expression covers more distant characters for foreign genotypes. (sutras/source: original) Figure 3 Results concerning the bands obtained from three varieties of the test primers Og Among the studied primers, five amplified bands in only one variety Hofigal 2, (ObGA07, ObGA08, ObGA11, ObGA13, ObGA18 and ObGB04), and 2 of the primers: ObGA09 and ObGA10 did not amplify any fragment in any of the varieties of basil (figure 3.), and the amplified bands had poor clarity, not being able to make a fair assessment of them. There could be an explanation related to the DNA samples not having the quantity or purity required to give polymorphism with the primers tested, although they are universal and should have given results, the polymerase used was Aplite Polymerase (Stoffel) and not Promega, and the amplification power may have been lower. 4. Conclusion In the laboratory, the bands were detected using the Faliscan program, by comparing them with a standard DNA sample, with a size between 100 and 4000 bp. All the fragments resulting from the amplification with the polymorphic primers were between 200 and 2000 bp in length, most of them between 300 and 1200 bp (in the first case of amplification). The lower the values of the genetic distances in the table (binary matrix), the more closely related the genotypes are. These primary data were entered into the RAPD distance 1.04 program which calculated the genetic distances based on the Jaccard coefficient. Rresults regarding the calculation of the genetic distance for the two markers that showed the highest percentage of polymorphism ObGA07 and Og B04. Both molecular markers establish the presence of the ObGAPDH gene, but the significance of its expression covers more distant characters for foreign genotypes. The choice of primers proved quite difficult, out of 8 primers used in total, only 2 gave distinct polymorphic bands, among them: ObGA07 and ObGB04. It turned out that the RAPD technique is a simple and fast method for revealing genetic polymorphism in basil genotypes according to the Doyle Doyle protocol or the CTAB method. The RAPD technique is extremely sensitive to working conditions (apparatus, reagents, solution concentrations, their pH, etc.). Optimizing the RAPD protocol can be an extremely difficult problem, changing the reaction components or the program used can have unpredictable consequences. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. World Journal of Advanced Research and Reviews, 2025, 27(02), 1266-1274 1274 References [1] Ahmed B., Ali Ashfaq U., Usman Mirza M. Medicinal plant phytochemicals and their inhibitory activities against pancreatic lipase: Molecular docking combined with molecular dynamics simulation approach. Nat. Prod. Res. 2018;32:1123–1129. doi: 10.1080/14786419.2017.1320786. [2] Brand-Williams W., Cuvelier M.-E., Berset C. 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