Acta agriculturae Slovenica, 119/1, 1–11, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2716 Original research article / izvirni znanstveni članek Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers Mohammad ZABET 1, 2, Leyli ESMAEILZAEI 1, Ali IZANLOO 1, 3, Seyyed Hamid Reza RAMAZANI 1 Received June 04, 2022; accepted February 10, 2023. Delo je prispelo 4. junija 2022, sprejeto 10. februarja 2023 1 Department of Plant Production and Genetics, College of Agriculture, University of Birjand, Birjand, Iran 2 Corresponding author, e-mail: mzabet@birjand.ac.ir 3 The New Zealand Institute for Plant and Food Research Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers Abstract: To evaluate the genetic diversity of Iranian Nar- cissus genotypes (e.g., Shomal, Shastpar, Shahla, Yasuj, Shiraz-1, Shiraz-2, Kuchak-e-Atri, Dutch, Khosf-1, Khosf-2, Birjand, and Tabas), different RAPD and ISSR primers were examined at the Plant Breeding Laboratory of the Faculty of Agriculture, University of Birjand, Iran. In sum, RAPD and ISSR prim- ers produced 189 and 80 high-resolution bands. The average values of PIC, Ht, Hs, Nm, DST, FDT, NA, Ne, H, and I indi- ces were 0.287, 0.369, 0.089, 0.486, 0.279, 0.760, 1.952, 1.459, 0.282, 0.437 for RAPD markers and equal to 0.297, 0.380, 0.099, 0.524, 0.278, 0.732, 1.978, 1.495, 0.303, 0.467 for ISSR mark- ers, respectively. The analysis of molecular variance based on the RAPD and ISSR markers showed 23 and 13  % variations for intra-populations and 77  % and 87  % changes for inter- population variabilities, respectively. Cluster analysis based on the RAPD and ISSR markers grouped genotypes into four clus- ters. Based on RAPD and ISSR markers, the PCoA analysis also showed that the first three components justified equal to 96.9 % and 97.9 % of the total variance, respectively, indicating the dis- persion of the primers used. In general, it was concluded that the genetic diversity of narcissus species could be employed for breeding programs. Key words: cluster analysis; narcissus; genetic parameters; genetic diversity Genetska raznolikost in klasterska analiza genotipov dvo- barvnega narcisa (Narcissus tazetta L.) z uporabo RAPD in ISSR markerjev Izvleček: Za ovrednotenje genetske raznolikosti iran- skih genotipov narcisa (e.g., Shomal, Shastpar, Shahla, Yasuj, Shiraz-1, Shiraz-2, Kuchak-e-Atri, Dutch, Khosf-1, Khosf-2, Birjand, in Tabas), so bili preiskušeni različni RAPD in ISSR primerji v Plant Breeding Laboratory, Faculty of Agriculture, University of Birjand, Iran. Sumarno so RAPD in ISSR primerji dali 189 in 80 jasno razvidnih trakov. Poprečne vrednosti PIC, Ht, Hs, Nm, DST, FDT, NA, Ne, H in I indeksov so bile 0,287; 0,369; 0,089; 0,486; 0,279; 0,760; 1,952; 1,459; 0,282; 0;437 za RAPD markerje in 0,297; 0,380; 0,099; 0,524; 0,278; 0,732; 1,978; 1,495; 0,303; 0,467 za ISSR markerje. Analiza moleku- larne variance na osnovi RAPD in ISSR markerjev je pokazala 23 in 13 % variacij znotraj populacij in 77 % in 87 % sprememb med populacijami. Klasterska analiza na osnovi RAPD in ISSR markerjev je združila genotype v štiri skupine. Analiza glavnih komponent na osnovi RAPD in ISSR markerjev je pokazala, da so prve tri komponente razložile 96,9 % in 97,9 % celokupne spremenljivosti, kar kaže na razpršenost uporabljenih primer- jev. V splošnem lahko zaključimo, da bi se genetska raznolikost vrst narcisa lahko uporabila v žlahtniteljskih programih. Ključne besede: klasterska analiza; narcis; genetski para- metri; genetska raznolikost Acta agriculturae Slovenica, 119/1 – 20232 M. ZABET et al. 1 INTRODUCTION Narcissus (Narcissus tazetta L.), a member of the family of Amaryllidaceae, has many therapeutic and ornamental properties (Gotti et al., 2006). Narcissus is a beautiful plant of geophytic, monocotyledonous, and perennial plants whose genus has 65 species and 20,000 cultivars and hybrids (Saeedi, 2007). In general, the qual- ity of this flower, especially in terms of color, odor, and characteristics such as the number of sepals and petals, are primarily controlled by genetic factors but environ- mental factors, such as stress and plant nutrition, have re- markable impacts on these characteristics (Zadebagheri et al., 2011). At present, numerous researches are going on the agronomic characteristics of this plant in Iran, but there are few reports on the plant genetic diversity. Since the study and determination of the genetic diversity of a plant is the first step in the next breeding program, the purpose of this study was to determine the kinship, di- versity, and genetic structure of narcissus populations us- ing RAPD and ISSR molecular markers. Genetic resources can provide desirable genes and supply opportunities for breeders to produce new and desirable cultivars (Clegg, 1997; Bhandari et al., 2017). There are many methods to study the genetic diversity of plants. Among these ways, molecular markers (DNA- based techniques) are the most accurate and reliable method for determining and identifying genetic diver- sity and (along with multivariate statistical methods) has a significant potential for examining plants phylogenic, evolution, and genetic diversity (Pandey et al., 2008). Among the molecular markers developed in recent years, RAPD (Random Amplified Polymorphic DNA) marker is a simple PCR-based technique that was devel- oped by Williams et al. (1990) and Welsh & Mc-Clelland in 1990, and is based on the amplification of primer bind- ing regions in DNA genome (Welsh et al., 1991, Ezzat et al., 2016, Shi et al., 2018). In general, this method is widely used due to the lack of basic information needed to design and construct primers, the possibility of simul- taneously examining multiple loci in the sample genome, the lack of the need for a special probe in molecular anal- ysis (especially genetic diversity assessment) (Williams et al., 1990). This marker has been used well to study the genetic diversity of narcissus (Jiménez et al., 2017), soy- bean (Sharma et al., 2018), oil palm (Basyuni et al., 2018), tomato (Maske et al., 2018) genotypes and etc. ISSRs are semi-optional markers that are amplified by PCR in the presence of a primer that complements the target microsatellite. ISSR markers are useful in stud- ies on genetic diversity, genome mapping, gene tagging, phylogeny and evolution (Pradeep Reddy Reddy et al., 2002). Such multiplication does not require sequential information and leads to the production of multi-po- sition and highly polymorphic patterns. The ISSR frag- ments are 100- 3,000 bp length of DNA sequences that are located between two microsatellite regions with op- posite directions. Microsatellite-based primers are de- signed and used to amplify the DNA sequence between two ISSRs (Kumar et al., 2009). ISSR markers are potential markers in identifying genetic diversity compared to other markers and allow the study of the diversity of specific regions of the ge- nome at several locations simultaneously (Mengistu et al., 2004). ISSR markers has been used well for genetic diversity of cuscuta (Tajdoost et al., 2012), Persian vio- let (Asadi et al., 2016), narcissus (Jiménez et al., 2009), Solanum nigrum L. (Suganthi et al., 2018). Also, genetic diversity of different Turkish narcissus has been stud- ied using SRAP markers (Zeybekoğlu et al., 2019). A study using markers (ISSR) investigated the genetic di- versity of 31 narcissus species collected from 16 regions of Iran. The average percentage of polymorphic bands was 96.02 %. The highest resolution power (8.32), aver- age polymorphic information content (0.44) and marker index values (5.61) were observed for primers 811, 828 and 811, respectively. They stated that ISSR markers can be used as a diagnostic tool to evaluate genetic diversity in narcissus genotypes and reveal them (Zangeneh and Salehi, 2019). It should be noted that the Khosf (City of South Khorasan province) has a long historical tradition in nar- cissus cultivation and its area under cultivation has the highest area in the east of the country. The aim of this study was to investigate the genetic diversity of different narcissus ecotypes that were collected from different re- gions of Iran. It has also been the determination of the best genotype and its important traits for breeding pur- poses. 2 MATERIALS AND METHODS This experiment was carried out in the research farm and laboratory of plant breeding and biotechnol- ogy of the Faculty of Agriculture, University of Birjand, Iran during 2017 growing season. In this study, 12 dif- ferent genotypes of narcissus that been cultivated in Iran (consisted of Shomal, Shastpar, Shahla, Yasuj, Shiraz-1, Shiraz-2, Koochak-e-Atri, Dutch, Birjand, Khosf-1, Kho- saf-2, and Tabas) were collected and cultivated both in pots and in the field for relevant experiments. Leaf samples were taken after 60 days of planting in the four-leaf stage. The youngest leaves were selected for DNA extraction and stored in liquid nitrogen. The re- quired solutions are prepared in stock with higher accu- Acta agriculturae Slovenica, 119/1 – 2023 3 Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers racy. DNA extraction was performed by CTAB method (Doyle and Doyle, 1987). For this purpose, 0.5 g of young leaves was used. The quantity and quality of the extracted DNA were evaluated using two methods of nano drop and 1 % agarose gel electrophoresis. In this experiment, 27 RAPD primers and 11 ISSR primers were used. The primers were purchased in freeze-dried form from Sina- clon Company and diluted 1:10 using double-distilled water. The final concentration of primer was considered to be one μl in each 20 μl PCR reaction (Tables 1 & 2). The polymerase chain reaction was performed at a final volume of 20 μl (Table 3). 8 μl of the PCR prod- uct was stained with 2 μl of loading dye and 10 μl was poured into 1 % agarose gel (with 0.5X TBE buffer) wells. To better examine the bands, 5 μl of DNA size marker was injected into first the gel well. Samples were electro- phoresed at voltages 90 for one hour. Amplified fragments were observed and photographed by GelDoc under ul- traviolet light. Photos were scored using TL120 Lab Total software; was assigned to the presence of band, number 1and the absence of it, number zero. At first, according to geographical information, genotypes collected from different regions were divided into two groups (South Khorasan in one group and other genotypes in another group) and different statistical analyzes were performed. In this study, the genetic similarity index was em- ployed to determine genotype grouping, and cluster analysis, similarity coefficient and principal coordinate analysis were used to determine the distribution of prim- ers. These calculations were also performed using NT- SYS2.2 software. Molecular indices such as PIC (Sharma & Ghosh, 1955), population genetic index such as num- ber of allels (Na), number of effective allels (Ne) (Kimura & Crow, 1973), gene diversity index of Nei (H) (Nei, Primer Sequence Primer Sequence Primer Sequence RAPD R1 5’-TGCCGAGGTG-3’ R10 5’-AGGTGACCGT -3’ R19 5’-ACACCGATGG -3’ R2 5’-AGTCAGCCAC -3’ R11 5’- GTTGCGATCC -3’ R20 5’- CTTCTCGGAC -3’ R3 5’-AGGGGTCTTG -3’ R12 5’- GGAAACCCCT -3’ R21 5’-ACGGGACCTG -3’ R4 5’-GGTCCCTGAC -3’ R13 5’- TGGCGCACAC -3’ R22 5’-CCAGAACGGA -3’ R5 5’-GTGACGTAGG -3’ R14 5’-GGCACGCGTT -3’ R23 5’- GTGGCCGATG -3’ R6 5’-GGGTAACGCC -3’ R15 5’- GTTACGGACC -3’ R24 5’- ACGGAAGTGG -3’ R7 5’-GTGATCGCAG -3’ R16 5’- GGGCGACTAC -3’ R25 5’-GGACCCCTTAC -3’ R8 5’- CAATCGCCGT -3’ R17 5’-TCGCATCCAG -3’ R26 5’-TGAGTGGGTG -3’ R9 5’-TGGGCGATAG -3’ R18 5’-CTGGCGTTC -3’ R27 5’- GTTGCCAGCC -3’ ISSR I1 5’-CTCTCTCTCTCTCTCTRC-3’ I5 5 ’ -GAGAGAGAGAGAGA- GAC-3’ I9 5’-CTCTCTCTCTCTCTCTTG-3’ I2 5’-GAGAGAGAGAGAGAGAYG-3’ I6 5’-GAGAGAGAGAGAGAGA- YA-3’ I10 5’-CACACACACARY-3’ I3 5’-TCTCTCTCTCTCTCTCG-3’ I7 5’-GAGAGAGAGAGAGAGA- YC-3’ I11 5’-GACAGACAGACAGACA-3’ I4 5’-GAGAGAGAGAGAGAGYT-3’ I8 5’-ATCATCATCATCATC-3’ Table 1: RAPD and ISSR primers ISSR RAPD Step Tem.(oC) Time Cycle Num. (oC) Te μLm. Time Cycle Num. Initial denaturation 94 4min 1 95 4min 1 Denaturation 94 45s 40 94 1min 45 Annealing 45 5s 40 35 1min 45 Extension 72 55s 40 72 2min 45 Final extension 72 6min 1 72 6min 1 Table 2: The PCR reaction temperature program for the ISSR and RAPD markers Acta agriculturae Slovenica, 119/1 – 20234 M. ZABET et al. 1973), Shannon diversity index (I) (Lewontin, 1972), inter-population heterozygosity (Hs), intra-population heterozygosity (Dst), total heterozygosity (Ht), intra- population coefficient of variation (Gst) and fixation in- dex (Fst) (Lynch & Milligan, 1994) was calculated using POPGEN-1.31. Molecular analysis of variance was also performed with Genalex 6.1 software. 3 RESULTS AND DISCUSSION 3.1 DETERMINATION OF DNA QUANTITY AND QUALITY AND ANALYSIS OF MOLECULAR VARIANCE In this study, the amount of extracted DNA ranged from 83.5 to 565 ng μl-1, which was reduced to less than 12 ng after dilution. Also, in all samples the wavelength ratio of 260:280 was 1.90 to 2.04, which indicated that it was suitable. Results also showed that from 27 RAPD primers produced 189 bands (Figure 1), of which nine bands were single-shaped and 180 had polymorphic bands. An aver- age of seven bands were obtained per primer, so the used primers showed more than 95 % polymorphism. Out of a total of 11 ISSR primers, 80 bands with high-resolution were counted. Of the amplified bands, one band was single-shaped, and 79 bands were polymorphic. An av- erage of 7.27 bands were obtained per primer. Overall, the ISSR primers that were used showed 98 % polymor- phism. In general, RAPD and ISSR primers showed more than 95  % and 98  % polymorphism, respectively, and indicated the efficiency of primers in showing polymor- phism. Chehrazi (2007) reported that out of 14 RAPD markers used to study the genetic diversity of narcissus genotypes, 105 bands were amplified, of which 92 were polymorphic bands. Consistent with our findings, the average rate was 7.5 bands per primer, and the polymor- phism rate was equal to 96 %. Sargazi et al. (2016) studied the genetic diversity of fifteen ajowan populations using 25 RAPD primers and observed that all primers showed good polymorphism. In the study of genetic diversity of fennel using ISSR markers (Taheri et al., 2016), a total of 813 primers were reproduced from six primers, and the number of bands of each primer varied between 7-15, which is the average number of bands per primers con- sistent with this study. 3.2 GENETIC PARAMETERS The polymorphism data exhibit notable variations between individuals and populations and are important indicators to compare different primers in terms of their power to differentiate genotypes. According to the pre- sented results, it was found that the primers used had a relatively good PIC index, which indicates their accept- able efficiency in detecting and distinguishing genotypes (Table 4). In RAPD primers, the polymorphic content index differs from 0.15 to 0.47. The highest polymorphic content index (PIC) belonged to R13 (0.47), R1 (0.40), and R27 (0.40) primers, which showed their high ef- ficiency in differentiating these genotypes. The lowest PIC also belonged to R2 (0.16) and R3 (0.15) primers. The polymorphism ratio index differs from 2.25 to 11.00. The lowest value belonged to R19 (2.25) primer, and the highest amount was for the R6 (11.00) primer. Also, the lowest and the highest values of the marker index were obtained for R14 (0.27) and R13 (3.78) primers, respec- tively. The lowest and the highest values of resolving power belonged to R26 (0.44) and R10 (1.67) primers, re- spectively. The mean values of total heterozygosity, intra- population heterozygosity, gene flow (Nm), intra-popu- lation heterozygosity (Dst), and Fst index were achieved equal to 0.37, 0.09, 0.49, 0.28, and 0.76, respectively. Overall, the highest and the lowest values of the to- tal heterozygosity were gained equal to R13 (0.46) and R2 (0.20) primers, respectively. The lowest heterozygosity in- ter-populations were for primers R20 (0) and R10 (0.01), while the highest amount of heterozygosity inter-popula- tions was for R27 (0.22). The highest amount of gene flow belonged to R25 (1.14), and the lowest amount of gene flow was to R20 (0) and R10 (0.02). The highest heterozy- gosity intra-populations belonged to R10 and R7 (0.38), and the lowest was related to R2 (0.14). Primer R20 (1) Materials Reaction components The final concentration Double distilled water 6.5 µl ....... Master mix (Prepared from Yekta Tajhiz Azma Co.) 10 µl 2X Primer 1.5 µl 0.67 pm DNA 2.0 µl 20 ng µl-1 Table 3: Components of PCR reaction PCR master mix includes the following details: 0.25 U/μl Taq DNA Polymerase; 2x PCR buffer; 0.4 mM dNTPs; 3.2 mM MgCl2; 0.02 % bromophenol blue Acta agriculturae Slovenica, 119/1 – 2023 5 Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers had the highest Fst, and the lowest value with an average value of 0.50 was observed for R27. The maximum num- ber of effective alleles (Ne) calculated for primers R2, R3, R16, R17, R19, and R20 were 1.67, 1.67, 1.89, 1.91, 1.75, and 1.83, respectively, and for other markers, it was equal to 2.0. The lowest number of effective alleles (Ne) related to R19 and R3 (1.21), and the highest these were obtained for primers R27 (1.71), and the average number of effec- tive alleles (Ne) was 1.46. The highest gene diversity in- dex of Nei (H) and Shannon diversity index (I) belonged to R13 primer (0.46 and 0.65), and the lowest was related to R3 primer (0.15 and 0.25), and their average was 0.28 and 0.47, respectively (Table 4). Figure 1: Separation pattern of amplified bands of 12 narcissus genotypes using OPA09 primers Number Ht Hs Dst Fst Na Ne H I PIC EMR MI RP R1 0.38 0.16 0.22 0.58 2.00 1.79 0.42 0.60 0.40 8.00 3.19 1.08 R2 0.20 0.07 0.14 0.67 1.67 1.28 0.17 0.28 0.16 2.67 0.42 1.22 R3 0.27 0.03 0.24 0.90 1.67 1.21 0.15 0.25 0.15 4.00 0.62 1.37 R4 0.39 0.10 0.29 0.74 2.00 1.62 0.36 0.53 0.36 8.00 2.89 0.83 R5 0.43 0.08 0.35 0.81 2.00 1.30 0.23 0.37 0.19 6.00 1.67 0.67 R6 0.35 0.06 0.30 0.84 2.00 1.33 0.23 0.38 0.24 11.00 2.68 0.86 R7 0.43 0.04 0.38 0.89 2.00 1.59 0.35 0.52 0.31 5.00 1.55 1.13 R8 0.42 0.16 0.26 0.62 2.00 1.60 0.36 0.54 0.33 6.00 2.01 1.25 R9 0.40 0.11 0.29 0.72 2.00 1.57 0.34 0.51 0.36 6.00 2.24 0.89 R10 0.39 0.01 0.38 0.97 2.00 1.25 0.19 0.33 0.18 8.00 1.44 1.67 R11 0.36 0.14 0.22 0.60 2.00 1.45 0.29 0.45 0.29 5.00 1.43 0.83 R12 0.36 0.12 0.24 0.66 2.00 1.29 0.21 0.37 0.25 5.00 1.28 1.00 R13 0.46 0.14 0.33 0.71 2.00 1.86 0.46 0.65 0.47 8.00 3.78 1.00 R14 0.42 0.14 0.27 0.65 2.00 1.67 0.37 0.55 0.34 8.00 0.27 1.22 R15 0.33 0.06 0.27 0.80 2.00 1.46 0.28 0.44 0.30 6.00 1.80 0.89 R16 0.3 0.08 0.22 0.73 1.89 1.30 0.21 0.35 0.24 8.11 1.70 0.94 R17 0.33 0.04 0.29 0.88 1.91 1.34 0.23 0.37 0.25 9.09 2.31 0.86 R18 0.26 0.07 0.35 0.83 2.00 1.55 0.32 0.49 0.30 7.00 2.11 0.76 R19 0.29 0.06 0.23 0.81 1.75 1.21 0.16 0.27 0.28 2.25 0.41 1.58 R20 0.31 0.00 0.31 1.00 1.83 1.28 0.18 0.30 0.18 4.17 0.76 1.47 R21 0.40 0.06 0.33 0.83 2.00 1.47 0.28 0.44 0.28 7.00 1.97 1.08 R22 0.43 0.06 0.37 0.86 2.00 1.52 0.30 0.47 0.31 7.00 2.17 1.13 R23 0.41 0.10 0.30 0.74 2.00 1.41 0.27 0.43 0.26 8.00 2.11 0.92 R24 0.35 0.14 0.21 0.60 2.00 1.44 0.30 0.42 0.30 7.00 2.72 0.57 R25 0.37 0.10 0.26 0.71 2.00 1.41 0.27 0.42 0.29 9.00 2.62 0.71 R26 0.30 0.05 0.25 0.82 2.00 1.47 0.29 0.45 0.30 3.00 0.92 0.44 R27 0.45 0.22 0.22 0.50 2.00 1.71 0.39 0.57 0.40 6.00 2.40 0.92 Mean 0.37 0.09 0.28 0.76 1.95 1.46 0.28 0.47 0.29 1.90 0.64 1.00 Table 4: Genetic diversity parameters in narcissus genotypes with RAPD markers Acta agriculturae Slovenica, 119/1 – 20236 M. ZABET et al. In the ISSR, the polymorphic indices content (PIC) changed between 0.15 and 0.37. The highest PIC be- longed to primers I10 and I11 (0.37), which indicates its high efficiency in differentiating used genotypes. The lowest value belonged to the I2 primer (0.15). The poly- morphism ratio index was between 3 and 14, with the lowest value belonging to primer I2 and the highest to primer I1. The lowest marker index was related to primer I5 (0.08), and the marker index was related to primer I8 (3.28). The lowest resolution index was related to primer I8 (0.27), and the highest value was related to primer I7 (1.55). The highest total heterozygosity was for primer I6 (0.43), and the lowest was related to primer I9 (0.31). The lowest heterozygosity inter-populations were obtained for primer I7 (0), and the highest value was for primer I10 (0.16). The highest level of gene flow was obtained for primers I1 (0.96) and I11 (0.94), while the lowest level was related to I2 (0). The highest heterozygosity intra- populations belonged to primer I2 (0.37), and the lowest was related to primer I9 (0.17). The highest and the low- est value of Fst were in primer I2 (1) and primer I9 (0.55), respectively. The average number of alleles for 11 primers of ISSR was 1.98. The lowest number of effective alleles (Ne) related to I2 (1.18) and the highest these were ob- tained for primers I10 (1.81). The means values for Nei’s genetic diversity index (H) and Shannon diversity index (I) were 0.30 and 0.47, respectively. The highest value of Nei’s genetic diversity index (H) and Shannon diversity index (I) belonged to primer I10 (0.43 and 0.62), and the lowest of these were related to primer I2 (0.15 and 0.29) (Table 5). 3.3 DETERMINING SIMILARITIES BETWEEN GENOTYPES The results showed that in both markers, UN1 simi- larity coefficient is the best coefficient and UPGMA al- gorithm is the best clustering pattern due to having the highest correlation coefficient (Table 6). Chehrazi et al. (2007) reported a cophenetic correlation coefficient be- tween native and non-native narcissus genotypes was 0.97. Examination of similarity matrix in RAPD marker showed that the highest similarity was between Khosf-2 and Tabas genotypes (0.95). Genotypes such as Shomal with Shastpar, Shiraz-1 with Shiraz-2 and Birjand with Khosf-1 had high similarity (0.93). Shahla with Yasuj and Shahla with Shiraz-1 also had high similarity (0.92). Shastpar with Shahla and with Shiraz-1 and Yasuj with Shiraz-1 had a similarity in 0.91. Shahla with Shiraz-2, Yasuj with Shiraz-2, Shiraz-2 with Kuchak-e-Atri, Birjand with Khosf-2, and Khosf-1 with Khosf-2 had a relatively high similarity (0.90). The lowest similarity be- tween genotypes was belonged to Dutch genotype with other genotypes, so that the similarity coefficient of this genotype with Birjand (0.43), with Shahla (0.45) and with other genotypes was less than 0.50 (Table 7). In gen- eral, the similarity coefficients showed that the similar- ity between most genotypes except Dutch was more than 0.80 and there was a lot of similarity between genotypes Examination of similarity matrix of ISSR showed that the highest similarity coefficients were obtained be- tween Shomal and Yasuj genotypes (0.92), Shastopar with Shahla and Yasuj with Shiraz-1 (0.91) and Shomal with Shastopar, Shomal with Shiraz-1 and Yasuj with Shiraz-2 (0.90). The similarity between all genotypes except Dutch was relatively high and averaged above 0.80. The lowest Number Ht Hs Dst Fst Na Ne H I PIC EMR MI RP I1 0.37 0.14 0.23 0.62 2.00 1.42 0.28 0.45 0.35 14.00 0.85 0.80 I2 0.37 0.00 0.37 1.00 2.00 1.18 0.15 0.29 0.15 3.00 0.35 1.28 I3 0.35 0.11 0.24 0.70 2.00 1.40 0.27 0.43 0.30 5.00 1.49 0.70 I4 0.42 0.07 0.36 0.84 2.00 1.47 0.29 0.47 0.27 8.00 2.17 0.83 I5 0.41 0.10 0.31 0.75 2.00 1.47 0.30 0.46 0.31 10.00 0.08 1.00 I6 0.43 0.07 0.36 0.83 2.00 1.54 0.33 0.50 0.30 4.00 1.19 1.08 I7 0.33 0.06 0.27 0.81 1.83 1.46 0.28 0.43 0.24 4.16 1.02 1.55 I8 0.37 0.12 0.25 0.68 1.93 1.51 0.30 0.46 0.27 12.07 3.28 0.27 I9 0.31 0.14 0.17 0.55 2.00 1.56 0.33 0.50 0.34 4.00 1.35 0.96 I10 0.41 0.16 0.25 0.61 2.00 1.81 0.43 0.62 0.37 8.00 2.99 1.43 I11 0.37 0.13 0.25 0.66 2.00 1.62 0.36 0.53 0.37 5.00 0.83 1.07 Mean 0.38 0.10 0.28 0.73 1.98 1.49 0.30 0.47 0.30 7.02 2.15 1.09 Table 5: Genetic diversity parameters in narcissus genotypes with ISSR markers Acta agriculturae Slovenica, 119/1 – 2023 7 Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers Cophenetic coefficients RAPD ISSR UPGMA Single Complete UPGMA Single Complete SM 0.99** 0.98** 0.96** 0.96** 0.96** 0.65** J 0.99** 0.97** 0.94** 0.95** 0.94** 0.91** DICE 0.98** 0.98** 0.96** 0.96** 0.95** 0.93** UN1 0.99** 0.98** 0.97** 0.97** 0.60** 0.96** Table 6: Cophenetic correlation coefficient based on SIM, J, DICE, UNI in RAPD and ISSR Genotypes 1 2 3 4 5 6 7 8 9 10 11 12 1- Shomal 1 2-Shastpar 0.93 1 3-Shahla 0.88 0.91 1 4-Yasuj 0.87 0.87 0.92 1 5-Shiraz-1 0.88 0.91 0.92 0.91 1 6-Shiraz-2 0.87 0.88 0.90 0.90 0.93 1 7- Kuchak-e-atri 0.83 0.83 0.86 0.84 0.89 0.90 1 8-Dutch 0.49 0.49 0.45 0.49 0.48 0.47 0.50 1 9-Birjand 0.81 0.83 0.83 0.82 0.83 0.82 0.84 0.43 1 10-Khosf-1 0.81 0.80 0.86 0.83 0.84 0.84 0.82 0.49 0.93 1 11-Khosf-2 0.80 0.82 0.87 0.88 0.84 0.85 0.85 0.47 0. 90 0.90 1 12-Tabas 0.78 0.80 0.85 0.87 0.84 0.84 0.86 0.49 0.88 0.89 0.95 1 Table 7: Similarity matrix based on UN1 similarity coefficient for RAPD marker in different genotypes of narcissus Genotypes 1 2 3 4 5 6 7 8 9 10 11 12 1- Shomal 1.00 2-Shastpar 0.90 1.00 3-Shahla 0.86 0.91 1.00 4-Yasuj 0.92 0.88 0.87 1.00 5-Shiraz-1 0.90 0.86 0.85 0.91 1.00 6-Shiraz-2 0.83 0.79 0.82 0.90 0.87 1.00 7- Kuchak-e-atri 0.84 0.80 0.77 0.86 0.87 0.85 1.00 8-Dutch 0.51 0.60 0.59 0.51 0.50 0.47 0.48 1.00 9-Birjand 0.85 0.79 0.78 0.87 0.86 0.79 0.80 0.50 1.00 10-Khosf-1 0.85 0.79 0.82 0.87 0.84 0.84 0.82 0.44 0.87 1.00 11-Khosf-2 0.87 0.79 0.67 0.82 0.86 0.86 0.80 0.53 0.83 0.84 1.00 12-Tabas 0.84 0.83 0.83 0.84 0.87 0.83 0.84 0.46 0.83 0.88 0.88 1.00 Table 8: Similarity matrix based on UN1 similarity coefficient for ISSR marker in different genotypes of narcissus similarity coefficient was related to the Dutch with oth- ers, so that the similarity coefficient between the Dutch with Yasuj, Shastpar, Shahla, Kouchak-e-Atri, Shiraz-1, Shiraz-2, Birjand, Khosf-1, Khosf-2 and Tabas were es- timated as 0.51, 0.60, 0.59, 0.48, 0.50, 0.47, 0.50, 0.44, 0.53 and 0.46; respectively (Table 8). In general, it can be deduced from the similarity coefficients, there is a huge difference similarity between internal genotypes and in- ternal and external genotypes and led to its diversity. Acta agriculturae Slovenica, 119/1 – 20238 M. ZABET et al. 3.4 CLUSTER ANALYSIS AND PRINCIPAL COOR- DINATE ANALYSIS (PCOA) Based on the RAPD marker, the genotypes were grouped into four clusters (Figure 2). The first cluster was divided into two sub-clusters. The first sub-cluster included Shomal and Shastpar. The second sub-cluster, which also had two subgroups, included Shahla, Yasuj, Shiraz-1 and Shiraz-2. The Kuchak-e-atri was the only genotype of the second cluster. In the third cluster, there were two sub-clusters that Birjand and Khosf-1 were under the first cluster and Khosf-2 and Tabas were un- der the second cluster. The Dutch genotype was placed independently in the fourth cluster, which shows a clear difference from others. Based on the ISSR marker (Figure 3), the dendrogram divided the genotypes into four clusters. The first cluster was divided into three sub- clusters. The first sub-cluster included Shomal and Yasuj genotypes, the second sub-cluster included Shiraz-1, and the third sub-cluster included Shastpar and Shahla. The second cluster included two genotypes of Shiraz-2 and Kuchak-e-Atri. The third cluster had three sub-clusters. The first sub-cluster included Birjand genotype, the sec- ond sub-cluster included Khosf-2 and the third sub-clus- ter included Khosf-1 and Tabas genotypes. The Dutch genotype independently formed the fourth cluster. This result is consistent with the results of Zanganeh and Salehi (2019) that a foreign genotype formed a separate group independent of the rest. In general, several factors such as altering life cycles, environmental changes, climate change, and topography can affect the structure, diversity, and genetic similarity of different populations of a plant species in different ways (Sintayehu, 2018, Zhao et al., 2021). In addition, according to the principle of natural selection, plant populations in similar ecological conditions that grow in different geographical locations have more genetic similarities than plant populations grown in various eco- logical conditions. Our findings were in sequence with the principle, and the results showed a good relationship between genetic diversity and geographical diversity in most cases, and genetic diversity was followed by geo- graphical diversity. The findings indicate a logical rela- tionship between genetic diversity and geographical lo- cation. Similar results related to the relationship between geographical and climatic location with genetic diversity of different plant populations have been reported by oth- er researchers using RAPD and ISSR markers (Gharibi et al., 2011, Abou El-Nasr et al., 2013). In addition, the obtained results of RAPD marker in- dicated that the first three components explained 96.9 % of the total variance. In the meantime, the first, second, and third components were justified 87.6, 6.3, and 3.1 % of the total variance. The ISSR data also showed that the first three components explained 97.9 % of the total vari- ance, in which the first component justified 89.3 %, the second component equal to 5.7 % and the third compo- nent equal to 2.9 % of the total variance. In general, molecular markers allow detecting vari- ations and polymorphisms for specific regions of DNA among individuals of a population, and the lower per- centage of variance assigned to main factors indicates a more appropriate distribution of markers. It means that molecular markers have examined wider regions of the genome. Thus, they have targeted different parts of the genome that contain gene controlling various traits (Mohammadi & Prasanna, 2003, Marwal & Gaur, 2020). Therefore, it can be concluded that in the present study, Figure 2: Clustering of Narcissus genotypes based on RAPD markers. 1: Shomal, 2: Shastpar, 3: Shahla, 4: Yasuj, 5: Shiraz-1, 6: Shiraz-2, 7: Kuchak-e-atri, 8: Dutch, 9: Birjand, 10: Khosf-1, 11: Khosf-2, 12: Tabas Acta agriculturae Slovenica, 119/1 – 2023 9 Genetic variation and cluster analysis of Narcissus tazetta L. genotypes using RAPD and ISSR markers Figure 3: Clustering of Narcissus genotypes based on ISSR markers. 1: Shomal, 2: Shastpar, 3: Shahla, 4: Yasuj, 5: Shiraz-1, 6: Shi- raz-2, 7: Kuchak-e-atri, 8: Dutch, 9: Birjand, 10: Khosf-1, 11: Khosf-2, 12: Tabas RAPD and ISSR markers had good performance and were able to cover a large area of the genome. Hence, it can be concluded that the RAPD and ISSR markers examined in the present study had good performance and were able to cover a large area of the genome. These results are consistent with the results of Zanganeh and Salehi (2019). 4 CONCLUSIONS In general, based on different coefficients of simi- larity between genotypes, placement of genotypes in dif- ferent and distinct clusters and other information, it was been indicated the existence of genetic diversity between the studied genotypes. In most cases, there was a good relationship between genetic diversity and geographical diversity, therefore genetic diversity followed geographi- cal diversity. The study of genetic diversity of narcissus genotypes using 27 RAPD primers and 11 ISSR primers showed that these markers can be useful in identifying polymorphic regions and managing germplasm and can be used to study and determine the genetic distance and differences between narcissus genotypes. The results ob- tained from RAPD marker were highly consistent with ISSR and conformed the genotypes in clusters accord- ing to a distinct pattern. 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