Bangladesh J. Plant Taxon. 29(1): 31-41, 2022 (June) DOI: https://doi.org/10.3329/bjpt.v29i1.60447 © 2022 Bangladesh Association of Plant Taxonomists ASSESSMENT OF GENETIC VARIATION OF GENUS PARACARYUM (BORAGINACEAE) BY RAPD MARKERS DAN SHEN1* AND SOMAYEH ESFANDANI-BOZCHALOYI2 School of Design and Art, Xijing University, Xi 'an, Shaanxi, 710000, China Keywords: Gene flow; Endemism; Random Amplified Polymorphic DNA (RAPD). Abstract The present study reveals the genetic diversity of Iranian Paracaryum based on morphological and molecular characters of 12 species from 11 provinces of Iran. A total of 118 reproducible bands were generated by 10 of 30 random amplified polymorphic DNA (RAPD) primers, with an average of 11.8 bands per primer and 49% polymorphism. The largest number of effective alleles (Ne), Shannon Index (I) and genetic diversity (H) higher level of Shannon Index (I) and genetic diversity (H) were shown by Paracaryum persicum. Our data depicted the highest similarity between Paracaryum cyclhymenium and P. persicum and the lowest between P. sintenisii and P. bungei. P. bungei showed a relatively low level of genetic variation. Finally, the Neighbor Joining (NJ) trees based on RAPD markers data divided the populations into two different clusters, indicating their genetic difference, which is discussed in detail. Introduction The family Boraginaceae s.str consists of approximately 131 genera and 2,500 species, distributed throughout the temperate and subtropical regions of the world, but mainly distributed in dry, cliffy and sunny habitats of Eurasia, the Mediterranean region and western North America (Retief and Vanwyk, 1997). They are mainly annual, bi-annual or perennial herbs and shrubs, some trees and a few lianes (Retief and Vanwyk, 1997), with a high distribution in Iran. Cynoglossoideae Weigend. is the largest subfamily having about 900 species and 50 genera. Recent molecular studies have shown that a wide range of the previously recognized tribes belong to this subfamily (Chacón et al., 2016). The subtribe Cynogolossinae Dumort. (tribe Cynoglosseae W.D.J.Koch) is entirely restricted to the Old World, with a centre of diversity in western Asia and the Mediterranean (Chacón et al., 2016). The genus Paracaryum (DC.) Boiss. of the tribe Cynoglosseae of this family is herbaceous and includes approximately 67 species, mostly distributed in the Irano-Turanian phytogeographical region (Riedl, 1967). Paracaryum is a very complex genus from the point of view of taxonomy and nomenclature and includes 16 species, 12 of which occur in Iran (Riedl, 1967). This genus is characterized by anthers included in the corolla tube, ebracteate cymes, a four-lobed ovary, an obtuse five-lobed corolla with faucal scales, and winged nutlets. In the light of recent phylogenetic analyses based on rps16 and trnL-trnF DNA sequences, the classification of Paracaryum is uncertain within the Cynoglossum L. s.l. clade and the genus is not monophyletic. Amedi et al. (2020) determined meiotic chromosome numbers and meiotic behaviour of six populations belonging to four species of Paracaryum growing in Iran, namely P. modestum Boiss. & Hausskn. (2n = 2x = 24), P. persicum subsp. macrocarpum (2n = 2x = 24), P. undulatum (2n = 2x = 24) and P. rugulosum (2n = 2x = 24). All chromosome counts are consistent with a *Corresponding author: e-mail: shendan0515@126.com 1School of Design and Art, Xijing University, Xi 'an, Shaanxi, 710000, China 2Faculty Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran. https://doi.org/10.3329/bjpt.v29i1.60447 mailto:shendan0515@126.com 32 SHEN AND ESFANDANI-BOZCHALOYI proposed base number of x = 12. The fatty acid compositions of the fruits of ten Paracaryum taxa belonging to three different subgenera were investigated for chemotaxonomic allocation using gas chromatography. Among the twenty-two analysed fatty acids, oleic, linoleic and a-linolenic acids were the major fatty acids represented (Amedi et al., 2020). For a synthetic approach to the systematics of this family considering both phylogenetic and evolutionary aspects. and in most research fruit morphology has been used as the most important character. The present study has been carried out to evaluate the genetic diversity and relationships among the Iranian Paracaryum species using RAPD markers. This is the first study on the use of RAPD markers in the Paracaryum genus and aims at answering the following questions: 1) Is there infra and interspecific genetic diversity among Paracaryum species? 2) Is there any genetic distance among these species correlated with their geographical distribution? Materials and Methods Plant sampling A total of 116 individuals were sampled representing 15 distant populations representing 12 Paracaryum species in East Azerbaijan, Kermanshah, Esfahan, Tehran, Hamadan, Kurdistan, Khorasan, Kerman, Hormozgan, Semnan and Fars Provinces of Iran during July–August 2017– 2019 (Table 1). For morphometric and RAPD analysis, we used 116 plant accessions (up to twelve samples from each population) belonging to 15 different populations with different eco- geographic characteristics and were sampled and stored at -20°C till further use. More information about the geographical distribution of accessions are in Table 1 and Fig. 1. . 1. PCA plots of morphological characters revealing species delimitation in the Paracaryum species; sp1= P. cyclhymenium; sp2= Paracaryum persicum; sp3= Paracaryum platycalyx; sp4= Paracaryum rugulosum; sp5= Paracaryum sintenisii; sp6= Paracaryum strictum; sp7= Paracaryum undulatum; sp8= Paracaryum hirsutum; sp9= Paracaryum tenerum; sp10= Paracaryum bungei; sp11= Paracaryum salsum; sp12= Paracaryum intermedium. ASSESSMENT OF GENETIC VARIATION OF GENUS PARACARYUM 33 Table 1. Voucher details of Paracaryum species in this study from Iran. No Sp. Locality Latitude Longitude Altitude (m) Sp1 Paracaryum cyclhymenium (Boiss.) H. Riedl Tehran, Damavand Semnan, 20 km NW of Shahrud 38 ˚ 52'37̎ 47 ˚ 23' 92̎ 1144 Sp2 Paracaryum persicum (Boiss.) Boiss. subsp. persicum Kermanshah, Islamabad Tehran, road of Firozkuh 32°50̍ʹ03ʺ 51°24ʹ28ʺ 1990 Sp3 Paracaryum platycalyx Riedl Fars, 7km from Evaj to Lar 29◦20ʹ07̎ʺ 51° 52ʹ08ʺ 1610 Sp4 Paracaryum rugulosum (DC.) Boiss. Hamedan, 20 km S of Nahavand Azarbaiejan, 48 km from Tabriz to Marand 38 ˚ 52'373 47 ˚ 23' 92̎ 2234 Sp5 Paracaryum sintenisii Hausskn. ex Bornm. Azarbaiejan, Kaleiybar, Arasbaran 33° 57ʹ12ʺ 47° 57ʹ32ʺ 2500 Sp6 Paracaryum strictum (C. Koch) Boiss. Azarbaiejan, Arasbaran Hamedan, 20 km S of Nahavand 34 ˚ 52'373 48 ˚ 23' 92̎ 2200 Sp7 Paracaryum undulatum Boiss. Kordestan, Sanandaj Hamedan, Alvand 38 ˚ 52'373 47 ˚ 23' 92̎ 1144 Sp8 Paracaryum hirsutum (DC.) Boiss. Kermanshah, Islamabad 35°50ʹ03ʺ 51°24ʹ28ʺ 1700 Sp9 Paracaryum tenerum Kordestan, Sanandaj 36°14ʹ14ʺ 51°18ʹ07ʺ 1807 Sp10 Paracaryum bungei (Boiss.) Khatamsaz Ardestan, Taleghan; Bandar-Abbas; Esfahan, Ghamishleh, protected area, Kooh Dojdoon 32◦36ʹ93ʺ 51°27ʹ90ʺ 2500 Sp11 Paracaryum salsum (Boiss.) H.H. Hilger & D. Podlech Tehran, Shahrud –Bastan; Turan 37°07ʹ02ʺ 49°44ʹ32ʺ 48 Sp12 Paracaryum intermedium (Fresen.) Hilger & Podl. Khorassan, Kashmar-Darvaneh Hormozgan, Bandar-Abbas; 28◦57ʹ22ʺ 51°28ʹ31ʺ 430 Morphological studies One to twelve samples from each species were used for morphometric analysis. In total 14 morphological (10 qualitative, 4 quantitative) characters were studied. Data obtained were standardized (Mean= 0, variance = 1) and used to estimate Euclidean distance for clustering and ordination analyses (Podani, 2000). Calyx length, calyx width, corolla length, corolla shape, corolla colour, faucal appendages, nutlet shape, nutlet length, nutlet surface ornamentation, stamens position, style position, nutlet margin and disc, and sepal indumenta. DNA Extraction and RAPD Assay Fresh leaves were used randomly from one to twelve plants in each of the studied populations. These were dried with silica gel powder. To obtain genomic DNA, the CTAB-activated charcoal protocol was used Abeshu & Zewdu (2020). The quality of extracted DNA was examined by running on 0.8% agarose gel. A total of 25 decamer RAPD primers of Operon technology (Alameda, Canada) belonging to OPA, OPB, OPC, and OPD sets were used. Among them, ten primers with clear, enlarged, and rich polymorphism bands were chosen (Table 2). 34 SHEN AND ESFANDANI-BOZCHALOYI Data analyses Morphological studies Morphological characters (Mean = 0, Variance = 1) were first standardized and used to determine the Euclidean distance between taxa pairs (Podani, 2000). The ordination methods of UPGMA (Unweighted paired group using average) were used for grouping the plant specimens (Podani, 2000). To demonstrate morphological variation between populations. Molecular analyses The obtained RAPD bands were coded as binary characters (presence = 1, absence = 0) and used for the study of genetic diversity. Using two parameters, polymorphism information content (PIC) and marker index (MI), the discriminatory capacity of the primers used was evaluated to characterise the ability of each primer to detect polymorphic loci among the genotypes. Results and Discussion Species identification and interrelationship Morphometry: ANOVA showed substantial differences (P <0.01) between the studied species in quantitative morphological characteristics. PCA analysis was conducted to determine the most variable characters among the taxa analysed. It showed that over 80 % of the overall variance was composed of the first three variables. Characters such as nutlet shape, nutlet length, nutlet surface ornamentation, stamens position, and style position have shown the highest association (>0.7) in the first PCA axis with 58 per cent of the total variance. Characters affecting PCA axis 2 and 3 respectively were calyx length, calyx width, corolla length, corolla shape, and corolla colour. Different clustering and ordination methods produced similar results, and therefore, PCA plots of morphological characters are presented here (Fig. 2). Plant samples of each species were typically grouped and separate groups were formed. This finding indicates that the studied species belong to different groups based on their quantitative and qualitative morphological features. We did not find intermediate forms in the studied specimens. Fig. 2. Electrophoresis gel of studied ecotypes from DNA fragments produced by OPD-02 and OPA-06. sp1= P. cyclhymenium; sp2= P. persicum; sp3= P. platycalyx; sp4= P. rugulosum; sp5= P. sintenisii; sp6= P. strictum; sp7= P. undulatum; sp8= P. hirsutum; sp9= P. tenerum; sp10: P. bungei; sp11= P. salsum; sp12= P. intermedium. ASSESSMENT OF GENETIC VARIATION OF GENUS PARACARYUM 35 Species Identification and Genetic Diversity To study genetic relationships among Paracaryum species, ten RAPD primers were screened. All the primers generated reproducible polymorphic bands in all 12 Paracaryum species. Figure 3 shows an image of the amplification of the RAPD created by the OPD-02 and OPA-06 primer. In total, 114 amplified polymorphic bands were formed across 12 species of Paracaryum. The size of the amplified fragments ranged from 100 to 3000 bp. The highest and lowest number of polymorphic bands was 15 for OPC-04, OPD-05 and 7 for OPA-06, with an average of 11.4 polymorphic bands per primer. The PIC of the 10 RAPD primers ranged from 0.34 (OPD-03) to 0.56 (OPA-05) with an average of 0.49 per primer. MI of the primers ranged from 3.33 (OPD- 011) to 5.66 (OPC-04) with an average of 4.5 per primer. EMR of the RAPD primers ranged from 8.23 (OPC-04) to 12.55 (OPB-01) with an average of 11.08 per primer (Table 2). The primers with high EMR values were considered to be more informative in distinguishing the genotypes. Table 2. RAPD primers used for this study and the extent of polymorphism. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI OPA-05 5ʹ-AGGGGTCTTG-3ʹ 14 14 100.00% 0.56 5.86 10.55 4.77 OPA-06 5ʹ-GGTCCCTGAC-3ʹ 10 7 86.99% 0.43 4.51 9.43 3.85 OPB-01 5ʹ-GTTTCGCTCC-3ʹ 9 9 100.00% 0.54 5.34 12.55 4.44 OPB-02 5ʹ-TGATCCCTGG-3ʹ 12 12 100.00% 0.47 4.18 9.56 3.65 OPC-04 5'-CCGCATCTAC-3' 15 15 100.00% 0.55 5.23 8.23 5.66 OPD-02 5ʹ-GGACCCAACC-3ʹ 14 13 95.74% 0.47 4.66 8.56 4.67 OPD-03 5ʹ-GTCGCCGTCA-3ʹ 15 12 92.31% 0.34 4.21 8.60 3.55 OPD-05 5ʹ -TGAGCGGACA-3ʹ 13 13 100.00% 0.47 4.32 10.55 3.45 OPD-08 5ʹ-GTGTGCCCCA-3ʹ 10 9 89.89% 0.53 5.56 9.34 4.11 OPD-11 5ʹ-AGCGCCATTG-3ʹ 11 11 100.00% 0.39 4.25 11.19 3.33 Mean 12.8 11.4 96.78% 0.49 5.2 11.8 4.5 Total 128 114 TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: polymorphism index, EMR, effective multiplex ratio; MI, marker index; PIC, polymorphism information content for each of CBDP primers The genetic parameters were calculated for all the 12 Paracaryum species amplified with RAPD primers (Table 3). Unbiased expected heterozygosity (H) ranged from 0.12 (Paracaryum bungei) to 0.34 (Paracaryum persicum), with a mean of 0.19. A similar trend was observed for Shannon’s information index (I), with the highest value of 0.35 observed in P. persicum and the lowest value of 0.11 observed in P. bungei with a mean of 0.29. The observed number of alleles (Na) varied between 0.244 in P. hirsutum and 0.567 in P. intermedium. The effective number of alleles (Ne) ranged from 1.011 (P. strictum) to 1.099 (P. persicum). AMOVA test revealed substantial genetic variation (P = 0.01) among the studied species. It showed that 62% of the total variation was among species and 38% was within species (Table 4). In addition, genetic differentiation of these species was demonstrated by significant Nei’s GST (0.66, P = 0.001) and D_est values (0.348, P = 0.01). Compared to within species, these results revealed a greater distribution of genetic diversity among Paracaryum species. 36 SHEN AND ESFANDANI-BOZCHALOYI Fig. 3. NJ tree of RAPD data revealing species delimitation in the Paracaryum. Table 3. Genetic diversity parameters in the studied Paracaryum species. SP N Na Ne I He UHe %P Paracaryum cyclhymenium 5.000 0.455 1.077 0.277 0.34 0.22 55.05% P. persicum (Boiss.) Boiss. subsp. persicum 8.000 0.499 1.099 0.35 0.43 0.34 69.26% P.platycalyx 9.000 0.261 1.014 0.242 0.23 0.23 43.15% P. rugulosum 6.000 0.555 1.021 0.29 0.35 0.31 58.53% P. sintenisii 4.000 0.344 1.042 0.20 0.23 0.20 27.53% P. strictum 5.000 0.369 1.011 0.25 0.18 0.22 42.15% P. undulatum 9.000 0.261 1.014 0.242 0.33 0.23 43.15% P. hirsutum 6.000 0.244 1.032 0.26 0.23 0.18 55.53% P. tenerum 4.000 0.314 1.044 0.26 0.18 0.23 43.38% P. bungei 8.000 0.256 1.066 0.11 0.17 0.12 32.23% P. salsum 5.000 0.341 1.058 0.27 0.27 0.20 53.75% P. intermedium 3.000 0.567 1.062 0.29 0.224 0.213 44.73% N = number of samples, Na= number of different alleles; Ne = number of effective alleles, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism, populations. ASSESSMENT OF GENETIC VARIATION OF GENUS PARACARYUM 37 Two major clusters were formed in the NJ tree (Fig. 3). The first major cluster contained two sub-clusters. Five species namely, P. cyclhymenium, P. persicum, P. platycalyx, P. undulatum and P. hirsutum were separated from the rest of the species, joined the others with a great distance and comprised the first sub-cluster. The second sub-cluster comprised four species namely, P. rugulosum, P. sintenisii, P. strictum and P. tenerum. The second major cluster also comprised two sub-clusters: three species including P. bungei; P. salsum and P. intermedium were placed close to each other, while close genetic affinity between other species. Relationships obtained from RAPD data usually agree well with the relationship of species obtained from morphological data. This is supported by the parameters of AMOVA and the genetic diversity previously presented. The species are genetically well differentiated from each other. The species are well distinguished from each other genetically. These findings show that RAPD molecular markers can be used in the taxonomy of Paracaryum species. Table 4. Analysis of molecular variance (AMOVA) of the studied species. Source df SS MS Est. Var. % ΦPT Among Pops 33 1801.364 59.789 13.154 62% 62% Within Pops 142 214.443 4.777 3.888 38% Total 175 1955.777 16.060 100% df: degree of freedom; SS: sum of squared observations; MS: mean of squared observations; EV: estimated variance; ΦPT: proportion of the total genetic variance among individuals within an accession, (P < 0.001). Nei’s genetic identity and the genetic distance were determined among the studied species. The results show the highest degree of genetic similarity (0.908) between P. cyclhymenium and P. persicum. The lowest degree of genetic similarity was shown between P. sintenisii and P. bungei (0.711). Genetic diversity is a fundamental component of biodiversity and its conservation is essential for the long-term survival of any species in changing environments. Genetic diversity is non randomly distributed among different populations and is influenced by various factors such as geography, breeding systems, dispersal mechanisms, life span etc. Changes in environmental conditions often lead to variation in levels of genetic diversity among different populations, and under adverse circumstances, populations with low variability are generally considered less adapted (Ma, et al., 2021a; 2021b; Peng et al., 2021). Most authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (Ren et al., 2021). Experimental and field research has shown that habitat fragmentation and population decline have reduced the effective population size in the last decade. Similarly, most geneticists regard population size as a significant factor in preserving genetic variation. In fragmented populations, it is more vulnerable because of the loss of allelic richness and increased population differentiation via genetic drift (decreases heterozygosity and subsequent allele fixation) and inbreeding depression (increases homozygosity within populations). Awareness of genetic variability and diversity between and within different populations is therefore important for their conservation and management (Esfandani-Bozchaloyi et al., 2018a, 2018b, 2018c, 2017). In our study, data on the genetic diversity in the 12 taxa of Paracaryum are given in detail for the first time. The aim of the present study was to find diagnostic features to separate species of Paracaryum in Iran. Morphological characters are considered as a useful tool for the identification of the species, as indicated previously (Akcin, 2008). Also, fruits and seeds are known to be useful characters in the identification of Cynoglossum creticum Mill., C. officinale, C. montanum and C. glochidiatum (Akcin, 2008). However, due to variation in seed coat and fruit surface, two types of tuberculate and granulate, and two subtypes of granulate-punctuate and granulate-tuberculate were recognized in these species. The reticulate type of seed coat and detailed subtypes of reticulate 38 SHEN AND ESFANDANI-BOZCHALOYI types were determined based on the ornamentation of the seed coats (Akçin, 2008). In previous studies, the micro-morphology of seed and fruit was performed in several species and their importance in plant taxonomy was emphasized (Hou et al., 2021; Huang, et al., 2021; Jia, et al., 2020; Karasakal, et al., 2020a; 2020b; Khayatnezhad and Gholamin, 2020a; 2020b). Morphological studies of the studied Paracaryum species showed that both the quantitative (the ANOVA test result) and qualitative characters are well distinguished from each other (The PCA plot result). Furthermore, PCA analysis suggests that morphological characters, such as shape and size of leaves, size and indumenta of the calyx, corolla colour, corolla shape, wing and diameter of nutlets, the shape of nutlet and nutlet surface, may be used in the delimitation of species groups. Quantitative and qualitative characters were accounted for this morphological discrepancy. Paracaryum (Mattiastrum) modestum is cited as an unresolved name in http://www.theplantlist.org. The generic distinction (at least in the Iranian taxa) between Paracaryum and Mattiastrum (Boiss.) Brand is not clear-cut in some taxa. In the former, the margin of the nutlets is distinctly inrolled to form an aperture; whereas in Mattiastrum the margin of the nutlet or wing is flat or slightly inrolled and the aperture is not evident. Paracaryum modestum was transferred from Paracaryum to Mattiastrum. Genetic Structure and Gene Flow A primer's PIC and MI characteristics assist in assessing its usefulness in the study of genetic diversity. Sivaprakash et al. (2004) proposed that the ability to overcome genetic diversity by a marker technique could be more explicitly linked to the degree of polymorphism. In general, the PIC value between zero and 0.25 indicates a very low genetic diversity among genotypes, a mid- level of genetic diversity between 0.25 and 0.50, and a value of 0.50 indicates a high level of genetic diversity, between 0.25 to 0.50 shows a mid-level of genetic diversity and value ≥0.50 indicates a high level of genetic diversity (Khayatnezhad, and Gholamin, 2021; Guo et al., 2021; Das et al., 2021; Zhao et al., 2021). In this study, the RAPD primers’ PIC values ranged from 0.34 to 0.56, with a mean value of 0.49, indicating a moderate level ability of RAPD primers in determining genetic diversity among the Paracaryum species. Somewhat comparable but low PIC values have been reported with other markers like RAPD and AFLP in African plantain (Karasakal et al., 2020a, 2020b, Khayatnezhad and Gholamin, 2020), ISSR and RAPD in Salvia species AFLP in wheat and SCoT markers (Hou et al., 2021, Huang et al., 2021; Varamesh et al., 2014; Rajaei et al., 2020; Fataei et al., 2013, 2014; Sadigh et al., 2021). In CBDP markers were found to be more effective than SCoT markers about the average PIC which was higher. In our analysis, the RAPD markers were found to be successful in the estimating genetic diversity of Paracaryum species in terms of average percentage polymorphism (96.78%), average PIC value of RAPD markers (0.49), average MI (4.5) and average EMR of RAPD markers (11.8). However, various marker methods have been found to have a different resolution of the genome regions and the number of loci that cover the whole genome for genetic diversity estimation (Zheng et al., 2021, Zhu et al., 2021; Yin et al., 2021; Si et al., 2020, Wang et al., 2021; Paul et al., 2021; Wasana et al., 2021). According to Chacón et al. (2016), the phylogenetic analyses based on sequences from three cpDNA regions successfully resolved some major issues about the monophyly of the main tribes of Boraginaceae and provided more detailed insights into the evolution of the Cynoglosseae s.l. Detailed taxonomic and phylogenetic studies of Subtr. Cynoglossinae are required to resolve this complex group (Chacón et al., 2016). However, there is a whole range of segregate genera that have been proposed for Cynoglossum and their phylogenetic relationships are not at all resolved. Some of them may be monophyletic, but at present, all of them appear to be nested in Cynoglossum based on Chacón et al. (2016). http://www.theplantlist.org. ASSESSMENT OF GENETIC VARIATION OF GENUS PARACARYUM 39 Omphalodes Moench and Cynoglossum, were retrieved as either poly or paraphyletic, showing that the morphological characters used in traditional taxonomic classifications are highly homoplasious (Weigend et al. 2013). Although the polytomies obtained in Weigend et al. (2013) are here largely resolved, most nodes have remained unsupported, and Lindelofia, Mattiastrum, Microparacaryum, Paracaryum, Pardoglossum, Rindera, Solenanthus and Trachelanthus are retrieved as either para-, or polyphyletic and/or nested in Cynoglossum s.str. as they also suggested in Selvi et al. (2011). According to Ahmad et al. (2021) SRAP marker’s genetic structure revealed that despite the existence of limited gene flow, two distinct ecotypes were produced which may be the consequences of reproductive isolation caused by altitudinal gradient and different niches through parapatric speciation. The heterozygosity (H) and Shannon index (I) reflect diversity and differentiation among and within the germplasm collections, respectively and the higher the indices, the greater the genetic diversity. The degree of variability among Na, Ne, H and I indices using studied RAPD markers demonstrated a high level of genetic diversity among and within Paracaryum species. In conclusion, the findings of this study showed that the primers derived from RAPD were more effective than the other molecular markers in assessing the genetic diversity of the Paracaryum. 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Water Supply. (Manuscript received on 18 July, 2021; revised on 01 June, 2022)