Acta agriculturae Slovenica, 119/1, 1–8, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2425 Original research article / izvirni znanstveni članek Comparative assessment of ISSR, DAMD and RAPD markers for evalu- ation of genetic diversity of gerbera (Gerbera jamesonii Bolus ex Hooker f.) cultivars Abbas SAIDI 1, 2, Melika HAJKAZEMIAN 1 Received November 15, 2021; accepted December 06, 2022. Delo je prispelo 15. november 2021, sprejeto 6. decembra 2022 1 Faculty of Life Sciences and Biotechnology, Department of Plant Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran 2 Corresponding author, e-mail: abbas.saidi@gmail.com Comparative assessment of ISSR, DAMD and RAPD markers for evaluation of genetic diversity of gerbera (Gerbera jameso- nii Bolus ex Hooker f.) cultivars Abstract: Genetic diversity is the best way to improve available genetic resources for breeding programs in gerbera. In present study, genetic diversity of 22 gerbera cultivars was investigated using inter-simple sequence repeat (ISSR), directly amplified minisatellite DNA (DAMD), and Random Amplified Polymorphic DNA (RAPD) markers. Average polymorphism information content (PIC) for ISSR, DAMD and RAPD mark- ers was 0.40, 0.41 and 0.40, respectively. Cluster analysis for ISSR, DAMD and RAPD divided the cultivars into three dis- tinct clusters. The comparative analysis of the three markers (ISSR, DAMD and RAPD) showed that DAMD had superior- ity over RAPD and ISSR in characterization of genetic diver- sity in Gerbera. To our knowledge, this is the first report of a comparison of performance among DAMD, ISSR and RAPD techniques on a set of gerbera genotypes. Overall, our results showed that DAMD markers well represented different geno- types of gerbera diversity. Key words: Gerbera; DAMD; genetic diversity; ISSR; DAMD Primerjalno preverjanje ISSR, DAMD in RAPD markerjev za vrednotenje genetske raznolikosti sort gerbere (Gerbera ja- mesonii Bolus ex Hooker f.) Izvleček: Genetska raznolikost je izhodišče razpoložljivih genetskih virov v žlahtniteljskih programih gerbere. V študiji smo genetsko raznolikost 22 sort gerbere preučevali z označe- valci ISSR (inter-simple sequence repeat, regije med mikrosa- teliti), DAMD (direct amplified minisatellite DNA, pomnoženi minisateliti) in RAPD (Random Amplified Polymorphic DNA, naključno pomnožena polimorfna DNA). Popreprečna infor- macijska vrednost polimorfizma (PIC) je bila za ISSR, DAMD in RAPD markerje 0,40; 0,41 in 0,40. Klasterska analiza ISSR, DAMD in RAPD markerjev je sorte razdelila v tri različne skupine. Primerjalna analiza treh markerjev (ISSR, DAMD in RAPD) je pokazala, da so imajo DAMD markerji prednost pred RAPD in ISSR za določitev genetske raznolikosti pri ger- beri. Pričujoča raziskava je ena prvih, ki primerja učinkovitost DAMD, ISSR in RAPD markerjev na izbranem naboru geno- tipov gerber. Naši rezultati so pokazali, da so markerji DAMD najbolje prikazali raznolikost genotipov gerbere. Ključne besede: Gerbera; DAMD; genetska raznolikost; ISSR; DAMD Acta agriculturae Slovenica, 119/1 – 20232 A. SAIDI and M. HAJKAZEMIAN 1 INTRODUCTION Gerbera (Gerbera jamesonii Bolus  ex  Hooker f.)) belongs to the Compositae family and is one of the top five cut flowers in the world in terms of production and consumption, which has a great economic value in the international flowering industry (Aghdam et al., 2019). Gerbera (Compositae), is native to tropical regions of South America, Africa and Asia (Danaee et al., 2011). It is a diploid species with somatic chromosome number of 2n = 50. Gerbera is commercially important and it is considered a tender perennial plant. Gerbera was intro- duced to China in the 1980s and has been used as a mod- el organism in surveying flower formation (Yang, 2012). Evaluation of genetic diversity and genetic relationships among cultivars are necessary tools for future breeding and improvement programs. Selection is essential for germplasm breeding and is effective only when the visible variation in the population is highly heritable (Kumari et al., 2011). Detection of genetic diversity and classifica- tion of genetic resources (germplasm) are important and essential strategies in breeding and preservation of plant genetic resources. Breeding of gerbera is based on cross- ing among cultivars and thereby phenotypic selection of new and improving progeny, followed by producing new cultivars. Molecular markers are an appropriate tool for measuring the diversity of plant species. Important fac- tors such as low assay cost, affordable hardware, through- put, convenience and ease of assay development, make this technique an important aid to breeding and culti- var development (Rafalski, 2002). ISSRs can be targeted towards sequences, which are reported to be abundant in the genome and can overcome the technical difficul- ties of RFLP. In recent years, many new alternative and promising marker techniques have been developed in line with the rapid growth of genomic research. Minis- atellite DNAs are tandemly repeated regions of genomes and are used in a procedure denoted directed amplifica- tion of minisatellite-region DNA (DAMD). This tech- nique utilized help effectively in genetic diversity analy- sis in gerbera. Genetic diversity in the gerbera has been reported using diverse molecular markers such as RAPD (Prajapati et al., 2014), and EST-SSR (Gong et al., 2010), ISSR (Li et al., 2004). In the present study, RAPD, ISSR and DAMD markers were used as useful tools for genetic variability analysis to provide more comprehensive in- sight regarding the genetic relationships and germplasm management of the cultivars utilized in this study (Ku- mar et al., 2011). Here, we report for the first time the use of the DAMD marker-based technique for surveying genetic diversity in gerbera cultivars. The objectives of the present research were to evaluate genetic diversity of gerbera using three markers namely ISSR, DAMD, and RAPD. The efficiencies of these markers were also evalu- ated and compared for diagnostic fingerprinting of the gerbera cultivars. 2 MATERIALS AND METHODS The plant material used in this study included twen- ty-two cultivars of G. jamesonii were collected from the Research Centre for Plants and Flowers (RCPF), Mahal- lat, Iran. Names of the cultivars are given in Table 1. Genomic DNA was extracted from young leaves of plants according to the modified CTAB method as de- scribed by Lassner et al. (1989). The purified total DNA was quantified by agarose-gel electrophoresis using a known concentration of uncut λ DNA as a standard. A set of 30 RAPD primers were procured of which 17 prim- ers gave clear and polymorphic patterns. The polymor- phic primers were then used for further analysis of 22 cultivars (Table 2). PCR amplifications were performed in 25 μl reactions containing 30 ng sample DNA, 2.5 μM primer, 200 μM of each dNTP, 1.5–2.5 mM MgCl2 (Mag- nesium chloride) and 1.5 unit of Taq DNA polymerase (Cinnagene, Iran). Thermal cycling included 3 min at 94 °C followed by 35 cycles of denaturation at 93 °C for 45 s, annealing at optimum temperature for 45 s, extension at 72 °C for 90 s, and a final extension cycle at 72 °C for 10 min. PCR products were separated on 1.5 % agarose gels, stained with ethidium bromide and scored for the presence or absence of bands. Six ISSR and eight DAMD primers were selected for final amplification (Table 2). The amplification was per- formed in a thermal cycler (Eppendorf, Germany) with the following conditions: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, an- Sample NO. Genotype Sample NO. Genotype 1 Rosalin 12 Cacharlle 2 Sorbet 13 Hooper 3 Souvenir 14 Nuance 4 Dune 15 Quote 5 Intense 16 Esmara 6 Aquamelone 17 Sazo 7 Edelweiss 18 Pink Elegance 8 Carambole 19 Essendre 9 Balance 20 Cabana 10 Stanza 21 Klimanjaro 11 Double Dutch 22 Red-417 Table 1: Names of the studied cultivars in this research Acta agriculturae Slovenica, 119/1 – 2023 3 Comparative assessment of ISSR, DAMD and RAPD markers for evaluation of genetic diversity of gerbera ... cultivars No. Name primer Seq (5’-3’) No. Name primer Seq (5’-3’) ISSR RAPD 1 UBC828 TGTGTGTGTGTGTGTGA 17 OPE-10 CACCAGGTGC 2 UBC822 TCTCTCTCTCTCTCTCA 18 OPF-02 GAGGATCCCT 3 UBC801 ATATATATATATATATT 19 OPG-19 GTCAGGGCAA 4 UBC874 CCCTCCCTCCCTCCCT 20 OPK-17 CCCAGCTGTG 5 UBC816 CACACACACACACACAT 21 OPL-08 AGCAGGAGGA DAMD 22 OPM-03 GGGGGATGAG 7 URP2F GTGTGCGATCAGTTGCTGGG 23 OPN-04 GACCGACCCA 8 URP9R AGGACTCGATAACAGGCTCC 24 OPQ-11 TCTCCGCAAC 9 URP25F GGCAAGCTGGTGGGAGGTAC 25 OPR-10 CCATTCCCCA 10 URP30F TACATCGCAAGTGACACAGG 26 OPR-17 CCGTACGTAG 11 URP13R AATGTGGGCAAGCTGGTGGT 27 OPAA-04 AGGACTGCTC 12 URP17R GATGTGTTCTTGGAGCCTGT 28 OPAF-10 GGTTGGAGAC 13 URP 6R GGACAAGAAGAGGATGTGGA 29 OPAD-06 AAGTGCACGG 14 URP2F AAGAGGCATTCTACCACCAC 30 OPAE-05 CCTGTCAGTG RAPD 15 OPB-07 GGTGACGCAG 16 OPD-06 ACCTGAACGG Table 2: RAPD, ISSR, and DAMD primers used for amplification Fig. 1: The RAPD pattern obtained with OPZ-17 primer (a), the ISSR pattern obtained with UBC822 primer (b), and the DAMD pattern obtained with URP17R (c) Acta agriculturae Slovenica, 119/1 – 20234 A. SAIDI and M. HAJKAZEMIAN nealing at 48 °C (for ISSR analysis) and 50 °C (for DAMD analysis) for 1min each, extension at 72 °C for 2 min and final extension at 72 °C for 7 min. The PCR products ob- tained were separated on 2 % agarose gel using 1 × TBE buffer at a constant voltage of 100 V for one hour. The gel stained with ethidium bromide and visualized using a gel documentation. The amplified RAPD, ISSR and DAMD fragments obtained were scored for presence (1) or absence (0) of bands. Tree construction following an NJ tree using a similarity matrix was performed through Splits Tree. The dissimilarity matrix thus obtained was subjected to cluster analysis using the un-weighted neighbor-joining analyses (UNJ), followed by bootstrap analysis with 1,000 permutations to obtain a dendrograme for all 22 geno- types. Mantel statistic was used to compare the similarity primer No. of poly- morphic bands No. of mono- morphic bands No. of amplified bands PIC value Polymorphism (%) MI ISSR UBC828 4 0 4 0.33 100 1.34 UBC822 9 0 9 0.44 100 3.96 UBC801 5 2 7 0.38 100 1.93 UBC874 10 0 10 0.44 100 4.94 UBC816 7 0 7 0.42 71 2.97 DAMD URP2F 7 0 7 0.39 100 2.76 URP9R 10 0 10 0.43 100 4.39 URP25F 5 0 5 0.38 100 1.93 URP30F 7 0 7 0.41 100 2.93 URP13R 10 1 11 0.43 90 4.32 URP17R 9 0 9 0.43 100 3.92 URP 6R 6 0 6 0.40 100 2.41 RAPD OPB-07 9 0 9 0.42 100 3.86 OPD-06 8 0 8 0.35 100 2.87 OPE-10 9 1 10 0.39 90 3.56 OPF-02 11 1 12 0.43 91 4.8 OPG-19 10 0 10 0.44 100 4.47 OPL-08 7 1 8 0.44 87 3.01 OPZ-17 13 0 17 0.48 100 6.71 OPM-03 7 1 8 0.39 87 2.75 OPN-04 7 0 7 0.41 100 2.87 OPQ-11 6 1 7 0.39 85 2.38 OPR-10 5 0 5 0.37 100 1.86 OPR-17 7 1 8 0.44 87 3.01 OPAA-04 7 0 7 0.41 100 2.87 OPAF-10 8 0 8 0.42 100 3.42 OPAD-06 6 0 6 0.36 100 2.18 OPAE-05 2 0 2 0.24 100 0.48 Table 3: DAMD, ISSR, and RAPD markers used for genetic diversity analysis Acta agriculturae Slovenica, 119/1 – 2023 5 Comparative assessment of ISSR, DAMD and RAPD markers for evaluation of genetic diversity of gerbera ... cultivars matrices as well as the dendrogrames produced by the ISSR, DAMD, and RAPD techniques. All these proce- dures were performed by appropriate routines in NTSYS- pc version 2.0. Polymorphic information content (PIC) values were calculated for each ISSR, DAMD, and RAPD primers according to the formula: PIC = 1 – ∑(Pij)2; where Pij is the frequency of the ith (frequency of the ith pattern ) pattern revealed by the jth (primer summed across all patterns) primer summed across all patterns revealed by the primers. The Mantel test of significance (Mantel 1967) was also used to compare each pair of similarity matrices produced. 3 RESULT AND DISCUSSION The 17 primers produced a total of 143 reliable fragments of which 138 were polymorphic. The num- ber of fragments generated by these RAPD primers was found to range from 2 to 15 bands. OPZ-17 primer produced the maximum number of polymorphic bands and OPAE-05 primer generated the minimum num- ber of polymorphic bands. The RAPD pattern obtained with OPZ-17 primer is shown in Fig1a. The percentage of polymorphism ranged from 85 % for OPQ-11 to 100 % for OPN-04, OPR-10, OPAF-10, OPZ-17, OPAE-05, OPA-02, OPK-07, OPL-08, OPG-19, OPB-07, OPAD-06, and OPD-06 with an average of 96 % polymorphism per primer. The polymorphism information content ranged from a 0.44 (OPG-19, OPL-08, OPZ-17, and OPR-17) to 0.24 (OPAE-05) with an average of 0.40, indicating hy- pervariability among the individuals studied. The mark- er index (MI) for RAPD was the highest for the primer OPZ-17 (6.71) and the lowest for the primer OPZ-17 (0.48). An average MI of 2.52 per primer was observed. Cluster analysis showed similar grouping pattern. A total 10 ISSR primers were screened of which five primers showed polymorphic bands. A total of 57 polymorphic bands were detected amongst 22 gerbera cultivars using 5 ISSR markers. Average polymorphism percentage was 94 % across all cultivars. The number of products generated per primer was found to range from 4 (UBC 828) to 10 (UBC 874) bands. The ISSR pattern obtained with UBC822primer is shown in Fig1b. Poly- morphism information content (PIC) values ranged from 0.33 to 0.44, with an average value of 0.40 per prim- Fig. 2: Dendrogram of the 22 gerbera genotypes based on the dissimilarity matrix developed using ISSR markers Acta agriculturae Slovenica, 119/1 – 20236 A. SAIDI and M. HAJKAZEMIAN er (Table 3). Cluster analysis grouped Gerbera cultivars into three distinct clusters (Fig. 2). Cluster I and II each contained seven cultivars. The marker index was found to be the highest for primer UBC874 (4.94) and the low- est for the primer UBC828 (1.34) with an average MI of 3.03 per primer. Cluster III showed similar grouping pat- terns with those obtained by RAPD data. Cluster I and II included 8 and 7 cultivars, respec- tively. Cluster III contained 3 cultivars (Fig 3). Seven primers generated a total of 55 bands of which 54 bands were polymorphic. The number of amplified products generated by DAMD primers were in the range of 5–10 bands. Primers URP13R and URP9R generated the maximum (10 bands) and primer URP25F generated the minimum (5 bands) number of amplicons. Average polymorphism percentage was 99 % across all cultivars. PIC values ranged from 0.38 to 0.43, with an average value of 0.41 per primer (Table 4). The DAMD pattern obtained with URP17R primer is shown in Fig. 1c. Based on un-weighted neighbour-joining method, all 22 ger- bera varieties were grouped into three distinct clusters (Fig. 3). The estimates of MI were found to be the highest for primer URP9R (4.39) and the lowest with the primer Fig. 3: Dendrogram of the 22 gerbera genotypes based on the dissimilarity matrix developed using RAPD markers URP25F (1.93) with an average MI of 3.24 per primer. Cluster I contained seven cultivars, cluster II included 11 cultivars, and cluster III included four cultivars showing relatively a similar grouping patterns with clusters III and IV of ISSR and cluster I of DAMD markers (Fig 4). Our study using three marker systems suggested the presence of a significant polymorphism and revealed high level of variability in surveyed gerbera genotypes which agrees with those reported by Prajapati (2014). This is the first research to utilize RAPD, ISSR and DAMD molecu- lar markers to examine the extent of genetic variability among germplasm of gerbera. In the current study, three different molecular markers, ISSR, DAMD, and RAPD were employed for the analysis of the genetic variability of a set of 22 gerbera cultivars. The results of our study suggests that these markers can be used in breeding pro- grammes to reliably distinguish studied gerbera varieties. A comparison of level of polymorphism and effective performance of ISSR, DAMD, and RAPD showed that each of the three markers can be detected genetic rela- tionships and discriminating efficacy within cultivars. All of the three markers were used to show high level of pol- ymorphism. Previous studies also revealed that DAMD Acta agriculturae Slovenica, 119/1 – 2023 7 Comparative assessment of ISSR, DAMD and RAPD markers for evaluation of genetic diversity of gerbera ... cultivars techniques were able to provide more reliable diversity information compared to RAPD or ISSR techniques (Pakseresht et al., 2013) and are useful as tools for study- ing the genetic diversity of different plant germplasm. In comparison with those studies, we observed that the av- erage polymorphism rate for RAPD, ISSR, and DAMD markers in gerbera cultivars were relatively high. ISSR and DAMD showed higher polymorphism percentage than RAPD but PIC in three markers equal. These results suggested the presence of a considerable polymorphism in two markers at studied and revealed a high potential of genetic diversity in the existing gerbera germplasm. Mantel coefficient correlation test showed higher positive correlation between ISSR and DAMD metrices, indicat- ing a consistent relationship between genetic distances from both marker systems. The correlation coefficient (r) was 0.73 between ISSR and RAPD, 0.80 between DAMD and ISSR, and 0.77 (significant p > 0.01) between RAPD and DAMD. All three molecular marker types showed positive but significant correlation with each other. Based on Mantel test results, the rate of genetic diversity for genotypes was in ISSR and DAMD approximately equal but RAPD was less than two markers. We predict that the source of detected diversity is various, as each tech- nique targets different regions of the genome. Results of mantel coefficient correlation test showed higher positive correlation between ISSR and DMAD metrices, indicat- ing a consistent relationship between genetic distances from both marker systems. This higher correlation may have been attributable to similarity in DNA sequence variation at primer binding sites between the ISSR and DAMD techniques. It is interesting to note that all three (ISSR, DAMD, and RAPD) datasets showed high levels of correlation. This is not surprising as these markers are known to target different genomic fractions involving repeat and/or unique sequences, which may have been differentially evolved or preserved in due course of natu- ral or human selection. These techniques were more in- formative and effective than the previously used molecu- lar methods to study variation in gerbera, such as SSR (Hajibarat et al., 2014), and RAPD markers (Feghhi et al., 2014, Prajapati et al., 2014, Saidi et al., 2018). Discord- ance between dendograms obtained by ISSR and DAMD with RAPD could be explained by the different nature of each technique, region coverage of genome by each marker, level of polymorphism and the number of loci. Fig. 4: Dendrogram of the 22 gerbera genotypes based on the dissimilarity matrix developed using DAMD markers Acta agriculturae Slovenica, 119/1 – 20238 A. SAIDI and M. HAJKAZEMIAN Our results substantiate the previous reports by cluster- ing genotypes using different marker systems in chickpea (Pakseresht et al., 2013), and Lenti (Seyedimoradi and Talebi 2014). Although the level of diversity for the three marker techniques was approximately equal, we offered that there are several possible explanations for such re- sults: some of them linked with stricter of different mo- lecular markers that designed from different regions of genome. Comparison with MI between ISSR, DAMD, and RAPD shows that DAMD has higher marker rate than RAPD and ISSR. The MI, which can be a resolving power to distinguish among different accessions (Khoda- dadi et al., 2011), was different in three marker markers (Table 3). Our study revealed that the resolving power of DAMD markers is higher than ISSR primers. Infor- mation about current genetic diversity permits the clas- sification of our available germplasm into various/het- erotic groups, which is particularly important to hybrid/ cross-breeding programs in gerbera. DAMD markers are usually reproducible, while primer length and annealing temperature are not the sole factors determining repro- ducibility (Saidi et al., 2017). In conclusion, DAMD marker analysis was suc- cessfully developed to evaluate the genetic relationships among the gerbera cultivars. Polymorphism percentage revealed by DAMD was so abundant and could be used for molecular genetics study of the gerbera cultivars, pro- viding high-valued information for the management of germplasm, improvement of the current breeding strate- gies, and conservation of the genetic resources of Gerbera species. 4 REFERENCES Aghdam, M., Asil, M. H., Ghasemnezhad, M., & Mirkalaei, S. A. A. M. (2019). Effects of pre-harvest applications of dif- ferent source of calcium on the cell wall fractions and stem bending disorder of Gerbera (Gerbera jamesonii L.) cultivar flowers. Advances in Horticultural Science, 33(1), 57-65. Danaee, E., Mostofi, Y., & Moradi, P. (2011). Effect of GA3 and BA on postharvest quality and vase life of gerbera (Gerbera jamesonii. cv. Good Timing) cut flowers. Horticulture, En- vironment, and Biotechnology, 52(2), 140-144. https://doi. org/10.1007/s13580-011-1581-7 Feghhi, S. M. A., Norouzi, P., Saidi, A., Zamani, K., & Amiri, R. (2012). Identification of SCAR and RAPD markers linked to Rz1 gene in sugar beet using BSA and two genetic dis- tance estimation methods. Electronic Journal of Plant Breed- ing, 3(1), 598-605. Gong, L., & Deng, Z. (2010). EST-SSR markers for gerbera (Gerbera hybrida). Molecular Breeding, 26(1), 125-132. htt- ps://doi.org/10.1007/s11032-009-9380-x Hajibarat, Z., Saidi, A., Hajibarat, Z., & Talebi, R. (2014). Genet- ic diversity and population structure analysis of landrace and improved chickpea (Cicer arietinum) genotypes using morphological and microsatellite markers. Environmental and Experimental Biology, 12(4), 161-166. Khodadadi, M., Fotokian, M. H., & Miransari, M. (2011). Ge- netic diversity of wheat (Triticum aestivum L.) genotypes based on cluster and principal component analyses for breeding strategies. Australian Journal of Crop Science, 5(1), 17-24. Kumar, S., Kumaria, S., Sharma, S. K., Rao, S. R., & Tandon, P. (2011). Genetic diversity assessment of Jatropha curcas L. germplasm from Northeast India. Biomass and Bioen- ergy, 35(7), 3063-3070. https://doi.org/10.1016/j.biombi- oe.2011.04.025 Lassner MW, Peterson P, Yoder JI (1989) Simultaneous ampli- fication of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their prog- eny. Plant Molecular Biology Reporter, 7, 116–128. https:// doi.org/10.1007/BF02669627 Li, Z. J., Xiong, L., & Gui, M. (2004). Optimization of the genomic DNA extraction and concentration of DNA tem- plate for ISSR-PCR amplification of Gerbera jamesonii cul- tivars. Acta Botanica Yunnanica, 26(4), 439-444. Mantel, N. (1967). The detection of disease clustering and a generalized regression approach. Cancer Research, 27(2_ Part_1), 209-220. Pakseresht, F., Talebi, R., & Karami, E. (2013). Comparative as- sessment of ISSR, DAMD and SCoT markers for evaluation of genetic diversity and conservation of landrace chickpea (Cicer arietinum L.) genotypes collected from north-west of Iran. Physiology and Molecular Biology of Plants, 19(4), 563- 574. https://doi.org/10.1007/s12298-013-0181-7 Prajapati, P., Singh, A., Patel, N.L., Singh, D., & Srivastav, V. (2014). Evaluation of genetic diversity in different geno- types of Gerbera jamesonii Bolus using random ampli- fied polymorphic DNA (RAPD) markers. African Journal of Biotechnology, 13, 1117-1122. https://doi.org/10.5897/ AJB2013.13347 Rafalski, J.A. (2002). Novel genetic mapping tools in plants: SNPs and LD-based approaches. Plant Science, 162, 329– 333. https://doi.org/10.1016/S0168-9452(01)00587-8 Saidi, A., Eghbalnegad, Y. & Hajibarat, Z. (2017). Study of ge- netic diversity in local rose varieties (Rosa spp.) using mo- lecular markers. Banat′s Journal of Biotechnology, 16, 148- 157. https://doi.org/10.7904/2068-4738-VIII(16)-148 Saidi, A, Daneshvar, Z. & Hajibarat, Z. (2018). Comparison of genetic variation of Anthurium (Anthurium andraeanum) cultivars using SCoT, CDDP and RAPD markers. Plant Tissue Culture and Biotechnology, 28, 171-82. https://doi. org/10.3329/ptcb.v28i2.39676 Seyedimoradi, H. & Talebi, R. (2014). Detecting DNA polymor- phism and genetic diversity in lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker. Physi- ology and Molecular Biology of Plants, 20, 495 –500. https:// doi.org/10.1007/s12298-014-0253-3 Yang, X., Pang, H. B., Liu, B. L., Qiu, Z. J., Gao, Q., Wei, L., ... & Wang, Y. Z. (2012). Evolution of double positive autoregu- latory feedback loops in CYCLOIDEA2 clade genes is asso- ciated with the origin of floral zygomorphy. The Plant Cell, 24(5), 1834-1847. https://doi.org/10.1105/tpc.112.099457