Assessment of variability among molecular and morphological characters of several wild Rutaceae species

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Thirteen stem and leaves morphological characters and 12 ISSR primers were used to evaluate 17 Rutaceae accessions. The results shows that morphological and molecular assessments confirmed different accessions grouping. Biplot analysis based on morphological characters revealed 3 groups based on the character similarity and 1 distinct group consisting Lunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp ., and M. minutum with a weak relationship of the observed characters. While the ISSR study divided the accessions into 3 clusters, which include L. acidissima in a distinct cluster. The second cluster composed of Clausena sp, Melicope sp. and Fragara sp. The third cluster was divided into two sub-clusters. The first sub-cluster included M. trichopetala , M. minutum , M. latifolia , L. amara , A. marmelos , Z. ovalifolium , G. pentaphyla , Lunasia sp, Z. rhetsa and M. exotica . While, F. lucida , P. littorale and E. suaveolens are the member of the second sub-cluster. The study has demonstrated the effectiveness of ISSR primers in differentiating species up to the subfamily level and it is crucial to prioritize further research to enhance the specificity of morphological characters. Rutaceae species genetic variability morphological and molecular markers relationship Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Rutaceae is a large plant family, and predominantly located in tropical, and subtropical area. The family was reported to have 162 genera with composed of 2085 species (Groppo et al. 2022 ). The species member may increase along with the discovery of new species (Aiyakool and Vajrodaya 2023 ; Fernandez-Hilario and Arteaga 2017 ). Tropical America, southern Africa (especially in the Cape Province, with the tribe Diosmeae), and Australasia were reported to be the main region of centers of diversity and the order Sapindales being the largest family in number of species (Appelhans et al. 2021 ; Groppo et al. 2012 ). Citrus L., a speciose genus of Asian origin, is widely cultivated for their edible fruits (oranges, lemons, tangerines, and grapefruits) and other plant organs for food condiments. While other group in Rutaceae family, are well known as important sources of aromatic oils (Cimik 2021 ; Liaqat et al. 2018 ; Silva et al. 2017 ), drugs (Adamska-Szewczyk et al. 2016 ; Musa et al. 2022 ; Mutinda et al. 2022 ), ornamentals (Licciardello et al. 2022 ) and for other purposes. Recently, there has been a substantial increase in the use of both morphological as well as biochemical, cytological or even molecular markers for the assessment of genetic diversity and fingerprinting of various plant species (Devi et al. 2022 ). Assessing genetic diversity is crucial, not only for crop improvement but also for efficient management and conservation of germplasm resources. Morphological studies are expected to bring to light morphovariants. Molecular studies are essential in detecting genetic variations among genotypes, which helps select diverse parents to successfully carry out a new crossing program. Additionally, the molecular data in conjunction with data generated on morphological variability of current taxa will also be helpful in establishing inter- and intra-population phylogenetic relationships (Zarei et al. 2021 ). Up to this moment, the ordinal position of Rutaceae family was referred to Engler subfamilial classification based on characters of the gynoecium, especially fruit type and the recognized the systematic of Rutaceae into seven subfamillies, i.e. Rhabdodendroideae, Aurantioideae (Citroideae), Flindersioideae, Spathelioideae, Dictyolomatoideae, Rutoideae, and Toddalioideae (Groppo et al. 2022 ). Several recent studies however, have cast doubt on his proposal based on phytochemical and molecular studies (Appelhans et al. 2021 ; Chase et al. 1999 ). The species under Rutaceae family also has undergone frequent interspecific hybridization among the wild or even with cultivated populations which leads to varying basic chromosome count, occurrence of polyploids and variation in morphology under different environmental conditions (Santos et al. 2022 ; Stace and Armstrong 1992 ). Futher study at molecular level to confirm morpho-variability in wild species Rutaceae is needed to establish a comprehensive characterization and relationship of existing diversity in wild species Rutaceae germplasm (Ouédraogo et al. 2019 ). The information is important to analyze the mechanism how the genetic diversity is maintained, also that underlying genetic mechanism leading to speciation and evolution and further use for breeding purpose (Sharma et al. 2012 ). For endangered species like Pleiospermium littorale (Miq.) Tanaka (syn. Limnocitrus littoralis (Miq.) Swingle) (IUCN, 1998), the study are needed to investigate their potential economic values and conservation strategies of the respected species (Nguyen et al. 2022 ). The paper investigate the molecular and morphological characters among wild Rutaceae species that naturally grown in Indonesia. 2. MATERIALS AND METHODS The plant materials were 17 accessions from Rutaceae family, i.e Aegle marmelos, Lunasia amara, Zanthoxylum rhetsa, Micromelum minutum, Zanthoxylum ovalifolium, Fragara sp., Limonia acidissima, Melicope sp., Clausena sp., Euodia suaveolens, Melicope trichopetalla, Glycosmis pentaphyla, Melicope latifolia, Murraya exotica, Lunasia sp., Ferroniella lucida and Pleiospermium littorale . The plants are a part of a core collection of Purwodadi Botanical Garden, located at Pasuruan, East Java, Indonesia. 2.1 Diversity and differentiation of morphological traits The studied Rutaceae species reached at least 10 years old and undergone complete vegetative and reproductive growth cycles. The observation was based on citrus descriptor list from IPGRI (1991) and carried out on at least 10 plant part samples. The observations were carried out during the early wet season of 2022–2023. The assessment covered quantitative and qualitative parameters, as presented in Table 1 . Table 1 Plant characteristics used for morphological observation No. Morphological characters Character category 1. Stem surface 1 = smooth, 2 = grooved and ridged 2. Density of branches 3 = sparse, 5 = medium, 7 = dense 3. Branch angle (attachment to main trunk) 3 = narrow, 5 = medium, 7 = wide 4. Spine density on adult tree (not on sucker) 0 = absent, 3 = low, 5 = medium, 7 = high 5. Shoot tip color 1 = green, 2 = purple 6. Shoot tip surface 1 = glabrous, 2 = intermediate, 3 = pubescent 7. Vegetative life cylce 1 = evergreen, 2 = deciduous, 3 = semi-persistent 8. Leaf division 1 = simple, 2 = bifoliate, 3 = trofoliate, 4 = pentafoliate 9. Length of petiole relative to length of leaf lamina 1 = sessile (petiole absent), 2 = brevipetiolate (petiole shorter than leaf lamina), 3 = longipetiolate (petiole longer than or same length as leaf lamina) 10. Leaf lamina shape 1 = elliptic, 2 = ovate, 3 = obovate, 4 = lanceolate, 5 = orbicular, 6 = obcordate 11. Leaf lamina margin 1 = crenate, 2 = dentate, 3 = entire, 4 = sinuate 12. Leaf apex 1 = attenuate, 2 = acuminate, 3 = acute, 4 = obtuse, 5 = rounded, 6 = emarginate 13. Absense/presense of petiole wings 0 = absent, 1 = present 2.2 Assessment of genetic variability 2.2.1 DNA isolation and purification The method of DNA extraction, isolation, and quantification was following Plant Genomic DNA Kit manual ( https://en.tiangen.com/upload/file/20220509/20220509165908_43410.pdf ). Young leaf samples (100g) were ground using mortar in 700 µl 65°C pre-heated GP1 (containing 0.1% β-Mercaptoethanol (β-ME)). 700 µl chloroform was added to the mixture, mixed by inverting the tube several times, and centrifuged for 5 min at 12,000 rpm (~ 13,400 × g). The supernatants were pipetted to a new tube, were added 700 µl Buffer GP2, mix by inverting the tube several times. All of the mixture was pipetted into the Spin Column CB3 (the Spin Column CB3 was placed in the Collection Tube first). the CB3 lid was closed and centrifuged for 30 s at 12,000 rpm (~ 13,400 × g). the filtrate was Discarded and the Spin Column CB3 was placed into the Collection Tube. 500 µl Buffer GD was added and centrifuged at 12000 rpm (~ 13,400 × g) for 30 s then the filtrate was discarded. 600 µl buffer PW (Ensure was added to the Spin Column CB3, and centrifuged for 30 s at 12,000 rpm (~ 13,400 × g), the flow-through was discarded. This step was Repeated twice. the Spin Column CB3 was placed in the Collection Tube, centrifuged for 2 min at 12,000 rpm (~ 13,400 × g), and the flow-through was discarded. The lid of CB3 was opened and stayed at room temperature for a while to dry the membrane completely. Spin Column CB3 was placed in a new centrifuge tube, and 50–200 µl Buffer TE was added directly onto the CB3 membrane, incubated for 2–5 min at room temperature (15–30°C), and then centrifuged for 2 min at 12,000 rpm (~ 13,400 × g) to elute 2.2.2 DNA amplification and separation DNA amplified using MyTaq™ HS Red Mix (Meredian Bioscience). 50 ng DNA sample of each accession was amplified in PCR (Thermocycler-Biometra) at the specific annealing temperature for each primer. The primers were UBC807, UBC810, UBC813, UBC816, UBC821, UBC824, UBC830, UBC843, UBC853, UBC873, UBC886 and UBC891 (Table 2 ). PCR amplification with ISSR marker was started with the denaturation cycle at 95ºC for 1 minute, followed by 35 denaturation cycles at 95ºC for 15 seconds, temperature annealing for 15 seconds with temperature extension up to 72°C for 10 seconds. The PCR cycle was terminated by 1 cycle temperature extension at 72°C for 10 minutes. The products of amplification were separated in 2.0% agarose gel containing gelred (10 mg/l) in 0.5 x TBE solution for 50 minutes at 100 volts The presence of an amplified product (fragment) was identified and biodocumented. Table 2 List of ISSR primers used in the study No Primers Sequences Annealing temperature (°C) 1. UBC807 (AG)8T 50.4 2. UBC810 (GA)8T 50.4 3. UBC813 (CT)8T 50.4 4. UBC816 (CA)8T 50.4 5. UBC821 (GT)8T 50.4 6. UBC824 (TC)8G 52 7. UBC830 (TG)8G 52.8 8. UBC843 (CT)8RA 52 9. UBC853 (TC)8RT 52 10 UBC873 (GACA)4 52 11. UBC886 VDV(CT)7 52 12 UBC891 HVH(GT)7 52 2.3 Statistical analysis The data obtained from morphological and molecular studies were subjected to cluster analysis based on UPGMA (Unweighted Pair Group Method with Arithmetic mean) to evaluate the similarity/dissimilarity among the studied populations. Matrix similarity was computed based on Dice’s coefficient Past 4.04, and the cluster analysis was performed for grouping accessions based on Dice’s coefficient according to the UPGMA method. Redundancy analysis was performed on both morphological and molecular datasets separately for analyzing the morphologically and genetically structured populations using the Biodiversity R package. ISSR bands were treated as binary characters and coded accordingly (presence = 1, absence = 0) from each primer. The Number of Scored Bands (NSB), the Number of Polymorphic Bands (NPB), and the Polymorphism Information Content (PIC) were calculated for each primer, in addition to the Marker Index (MI), Effective Multiplex Ratio (EMR), and Resolving Power (RP). PIC for the dominant markers was calculated according to Nei ( 1973 ). $$PIC=1-\sum {{P}_{i}}^{2}$$ where = Pi represents the frequency of its accession. To calculate EMR and MI, the following formula was used. EMR = p x pf where = p is the number of polymorphic bands of a primer and pf is the frequency of polymorphic bands (Powell et al., 1996 ). MI = EMR x PIC where = EMR is the effective multiplex ratio of a marker, and PIC is the polymorphism information content of a primer. Resolving power (Rp) was assessed according to the following formula. Rp = Ʃ Ib where = Ib is the band informativeness with Ib = 1 – [2 x (0.5-p)] and p is the proportion of accessions containing the polymorphic band. All calculations were processed according to (Hancı 2022 ). 3. RESULTS 3.1 Morphological features of Rutaceae species The morphological characteristics of the studied Rutaceae wild species were varied in terms of stem surface, density of banches, branch angle and trunk surface. The variations are also observed in terms of leaf division, leaf lamina shape, leaf lamina margin, leaf apex and relative petiole lenght to lenght of leaf lamina as presented in Fig. 1 . While all species have similar features of shoot tip color and surface, vegetative life cycle, and the absence of petiole wings. Pinciple Component Analysis (PCA) was used to analyze genetic diversity in Rutaceae based on 9 morphological characters and determine their influence on the studied Rutaceae accessions. The PCA aimed to identify the variables contributing to species diversity. The analysis of 9 parameters on the studied Rutaceae accessions resulted in four principle components (PC). PC 1 (F1), PC2 (F2), and PC 3 (F3) contributed to 29.33%, 21.059%, and 15.505% to the diversity, respectively, with a cumulative diversity value of 65.894% (Fig. 2 ). The variables in these PC determine the diversity in the studied Rutaceae accessions. Positive values in the results of PCA indicate a significant contribution to diversity, while negative values indicate not significant contribution to diversity (Gupta et al. 2023 ; Talekar et al. 2022 ). The observed variables in PC 1 (F1) are the main determinants of the diversity of the 17 Rutaceae accessions studied if the characteristic vector value is > 0.5, namely: length of petiole relative to length of leaf lamina and leaf apex. The main determining observation variable in PC 2 (F2) is branch angle, and in PC 3 (F3) is density of branches (Table 3 ). Table 3 Eigen vector values of 9 principle component based on morphological characters of the studied Rutaceae accessions. F1 F2 F3 F4 F5 F6 F7 F8 F9 Stem surface 0.284 0.346 -0.365 -0.383 0.042 -0.273 -0.641 -0.157 -0.091 Density of branches 0.142 -0.271 0.629 0.027 -0.464 -0.074 -0.344 -0.401 -0.106 Branch angle -0.048 0.548 -0.226 0.448 -0.366 0.020 0.246 -0.438 -0.243 Spine density 0.346 0.327 0.178 -0.034 -0.130 0.759 -0.156 0.343 -0.078 Leaf division 0.120 0.475 0.421 -0.437 0.106 -0.153 0.391 -0.186 0.410 Length of petiole relative to length of leaf lamina 0.515 -0.139 -0.193 0.432 -0.029 -0.020 -0.066 -0.086 0.692 Leaf lamina shape -0.151 0.304 0.394 0.500 0.587 -0.122 -0.341 0.037 -0.035 Leaf lamina margin -0.467 0.243 0.035 0.058 -0.509 -0.205 -0.243 0.465 0.378 Leaf apex 0.506 0.053 0.132 0.139 -0.128 -0.513 0.233 0.496 -0.353 The biplot analysis identified the group of each accession based on their common characteristics. The biplot analysis of the studied Rutacecae accessions revealed that first component accounting for 29.33% and the second component for 21.06% of the total diversity (Fig. 3 ). In quadrant 1, three accessions namely Z. ovalifolium, L. acidissima , and F. lucida shared their common features on leaf division, stem surface, spine density, and leaf apex. Six accessions, A. marmelos, Fragara sp., Melicope sp., M. trichopetala, G. pentaphyla and M. exotica showed similarities in terms of branch angle, leaf lamina shape, and leaf lamina margin and were grouped into Quandrant II. In Quadrant IV, P. littorale and Z. ovalifolium shared their common characteristics in density of branches and length of petiole relative to length of leaf lamina. No common parameter could be the basis of grouping in Quadrant III indicating a weak influence of the characters on the other six accessions, Lunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp ., and M. minutum . 3.2 Genetic diversity of Rutaceae species Table 4 displays the results of ISSR primers amplification, including the number of polymorphic bands (NPB), percentage of polymorphic bands (PPB), Polymorphism Information Content (PIC), Marker Index (MI), Effective Multiplex Ratio (EMR), and Resolving Power (RP). All 76 bands detected by 12 primers were found to be polymorphic. The primers with the highest number of bands were recorded at UBC807 and UBC886, with 11 and 10 bands, respectively. The UBC853 primer had the lowest number of bands, with only 3 detected bands. Table 4 Polymorphism percentage, total bands, PIC, MI, EMR, and RP of ISSR markers No Primers NPB PPB (%) PIC MI EMR RP 1. UBC807 11 100 0.43 4.75 3.47 6.94 2. UBC810 6 100 0.45 2.71 1.18 4.12 3. UBC813 4 100 0.42 1.66 2.06 2.35 4. UBC816 5 100 0.42 2.08 1.47 2.94 5. UBC821 5 100 0.31 1.53 0.94 1.88 6. UBC824 4 100 0.38 1.50 1.00 2.00 7. UBC830 5 100 0.26 1.30 0.76 1.53 8. UBC843 7 100 0.28 1.96 1.18 2.35 9. UBC853 3 100 0.38 1.14 0.76 1.53 10 UBC873 8 100 0.42 3.37 2.41 4.83 11. UBC886 10 100 0.48 4.82 4.06 8.12 12. UBC891 8 100 0.38 3.06 2.06 4.12 Mean 6.33 100 0.38 2.49 1.78 3.56 Remarks : NPN = Number of Polymorphic Bands, PPB = Percentage of Polymorphic Bands, PIC = Polymorphism Information Content, MI = Marker Index, EMR = Effective Multiplex Ratio, RP = Resolving Power The average PIC value for all primers was 0.38, with the highest value was detected in UBC886 (0.48) and the lowest value was attributed to UBC830 (0.26). The average MI was 2.49, with the largest MI associated with UBC886 (4.82) and the smallest value belonging to UBC830 (1.30). The average EMR was 1.79, with UBC807 having the highest EMR (3.47) and UBC830 and UBC853 having the lowest EMR (0.76). The average RP was 3.56, with the largest RP attributed to UBC807 (6.94) and the lowest value belonging to UBC830 and UBC853 (1.53). Table 5 shows a similarity matrix based on Dice's coefficient for accessions. The values for similarity ranged from 0.00 to 0.56. The highest similarity value of 0.56 was observed between A. marmelos and L. amara . On the other hand, the least similarity value of 0.00 was observed between L. acidissima and Clausena sp. Tabel 5. Similarity matrix between Rutaceae family based on ISSR primers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 1.00 2 0.28 1.00 3 0.36 0.32 1.00 4 0.43 0.36 0.33 1.00 5 0.52 0.22 0.26 0.47 1.00 6 0.46 0.12 0.27 0.31 0.33 1.00 7 0.42 0.18 0.11 0.31 0.50 0.48 1.00 8 0.37 0.35 0.28 0.39 0.44 0.31 0.42 1.00 9 0.35 0.27 0.25 0.38 0.43 0.29 0.41 0.36 1.00 10 0.56 0.22 0.31 0.38 0.48 0.33 0.50 0.48 0.31 1.00 11 0.43 0.36 0.19 0.46 0.33 0.35 0.35 0.35 0.37 0.38 1.00 12 0.39 0.25 0.40 0.33 0.35 0.27 0.32 0.40 0.34 0.35 0.21 1.00 13 0.43 0.49 0.35 0.50 0.51 0.16 0.37 0.44 0.42 0.39 0.37 0.38 1.00 14 0.25 0.31 0.28 0.27 0.25 0.26 0.26 0.32 0.29 0.25 0.19 0.39 0.34 1.00 15 0.47 0.36 0.28 0.36 0.47 0.36 0.44 0.39 0.25 0.55 0.36 0.23 0.42 0.11 1.00 16 0.13 0.11 0.00 0.14 0.19 0.07 0.13 0.17 0.18 0.13 0.17 0.21 0.12 0.00 0.21 1.00 17 0.42 0.23 0.30 0.26 0.28 0.36 0.29 0.46 0.34 0.39 0.20 0.42 0.48 0.26 0.41 0.05 1.00 Based on Dice’s coefficient, the UPGMA hierarchical clustering was constructed to group the studied Rutaceace accessions and three distinctive clusters were identified as presented in Fig. 4 . The first cluster consisted of L. acidissima only. The second cluster included Clausena sp, Melicope sp. and Fragara sp. The third cluster was divided into two sub-clusters. The first sub-cluster included M. trichopetala , M. minutum , M. latifolia , L. amara , A. marmelos , Z. ovalifolium , G. pentaphyla , Lunasia sp, Z. rhetsa and M. exotica . The second sub-cluster included F. lucida , P. littorale and E. suaveolens . 4. DISCUSSION Analysis of genetic diversity can assist in reliable classification of collections required for crop improvement. Recent studies revealed that molecular markers and morphological analysis could supplement each other in plant classification and establishing the distinctness among varieties, variety identification and genetic diversity analysis (Jain et al. 2017 ). The morphological analysis is employed in the first step of plant identification and has become a valuable source of information for agronomic and breeding programs. The study usually used as the foremost tools before continually using other methods. The marker also comprehends in the determination of the species and other category under the species (Tolangara et al. 2020 ). The study of morphological characteristics of Rutaceae accessions revealed that length of petiole relative to length of leaf lamina, leaf apex, branch angle and density of branches are the main factor in differentiating the accessions in PC’s. These differentiating characters were also confirmed by Kandowangko and Febriyanti ( 2023 ) under the genus Citrus. Branch angle and density of branches are related with plant architecture and affected by both genetic and environmental influences. The environmental factors as external factors have a significant impact on these characteristics along with the increase of plant ages. Trees may dynamically adjust their shape to cope with mechanical stress as the surrounding environment changes over time (Tsugawa et al. 2022 ). Based on biplot analysis, three accessions namely Z. ovalifolium, L. acidissima , and F. lucida shared their common features on leaf division, stem surface, spine density, and leaf apex in quadrant 1. According to Pavani et al. ( 2015 ), Z. ovalifolium has brownish stem with 8–12 smooth leaflets. The leaflet has elliptic-abovate shape with rounded to abruptly acuminate apex. These characteristics are also commonly found in L. accidisima , in that the bark is fissured and scaly with the sharp spines the zig-zag twigs pattern and densely in younger branches. The leaves of L. accidissima are dark-green, leathery, often minutely toothed, blunt or notched at the apex (Murrinie et al. 2017 ). F. lucida was also reported to have these similar characteristics, since formerly genus Feroniella was considered allied to Limonia L. due to its tough pericarp. Recently, the difference between Limonia and Feroniella was revealed on flower structure (Mabberley 2010 ), which are not included in this study. Six accessions, i.e. A. marmelos, Fragara sp., Melicope sp., M. trichopetala, G. pentaphylla and M. exotica were grouped based on their similarities on branch angle, leaf lamina shape, and leaf lamina margin. A. marmelos is a a spiny tree with deciduous trifoliate leaves. According to several reports, morphological variations on the leaf color, shape, surface and margin were existed under this species (Vasava et al. 2018 ). Several variants of A. marmelos in India have light to dark green leaves, lanceolate to ovate leaf shape, smooth to rough leaf surface and crenulate to crenate leaf margin (Singh et al. 2024 ). Genus Melicope has lanceolate leaf shape with long, medium to wide angle banches from the main trunk as the common features within the group (Appelhans et al. 2018 ). G. pentaphylla has a compound leaf comprised of 5 to 9 foliates. The leaf has elliptic to lanceolate shapes, alternate, with entire and unlobed margin (Sundrarajoo et al. 2022 ). The morphological variations were also reported among the species under the genus Glycosmis, in terms of number of leaflets, number of ovary within the locule and fruit shape (Yasir et al. 2018 ). Similar to A. marmelos, Fragara sp., Melicope sp., M. trichopetala, G. pentaphylla , M. exotica was also reported to have elliptic to obovate leaves with entire leaf margin. The variations in leaf were commonly found in the number of foliates, size, thickness and leaf color (Mou et al. 2021 ). In Quadrant IV, P. littorale and Z. ovalifolium shared their common characteristics in density of branches and length of petiole relative to length of leaf lamina. Both accessions have very short petiole compared to leaf lamina with medium to dense branches. The obvious differences between these species were on the leaf type. P. littorale has simple leaf with emarginate or rounded laef apex while Z. ovalifolium shows trifoliate leaves with obtuse or acuminate leaf apex (Pavani et al. 2015 ). Quadrant III has six accessions, Lunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp ., and M. minutum with no common basis parameter of grouping. The morphological features used in the study were limited to leaf and stem characteristics which were unable or insufficient to become the basis of plant grouping or differentiation. Appelhans et al. ( 2021 ) suggested to use reproductive plant parts like flower and its organs to discriminate several genera in Rutaceae family, which are very difficult to obtain during the study. The absent of character basis on quadrant III group might also be due to the plant plasticity. Plant phenotype presents a variation in their morphology either naturally or in connection with local adaptations (Duminil and Di Michele 2009 ). Alternatively some species can be morphologically very similar and may be falled into the same group despite the fact that they represent separate taxonomic entities (Pocovi et al. 2020 ). The 12 primers of ISSR markers used in the study successfully detected genetic variation in the Rutaceae family with high polymorphism. The statistical analysis employed to elucidate the degree of polymorphism confirmed the PIC values averaged of 0,38 and ranged from 0.26 to 0.48 that were categorized as informative and sufficient for differentiation of the studied genotype groups (Al-Mousa et al. 2023 ; Sabreena et al. 2021). The effectiveness of the marker system and the methodological approaches were also evaluated by employing additional indicators such as EMR, MI and RP. EMR is the number of polymorphic fragments detected per assay and depended on polymorphic loci. MI is primer capacity to detect polymorphic loci among different genotypes and RP is the ability of primers to distinguish or dicriminatiry efficiency of primers among genotypes (Kumar and Agrawal 2019). The frequency of polymorphic loci was varied from primer to primer (Venkatesan et al. 2021 ). In present study, the primers were found to have polymorphic loci ranging from 3 to 11. The highest frequency of polymorphic loci was observed in UBC886 with 10 and UBC807 with 11 polymorphic bands. These indicates that both primers are the most effective and reliable among the employed primers in distinguishing the studied genotypes The constructed cluster based on ISSR markers took the studied Rutaceae accessions into three clusters (Fig. 4 ) and most of the tested species were found to belong to the Rutodeae subfamily (Groppo et al. 2012 ). These grouping were then updated and divided the Rutodeae into two sub-family groups, namely the Amyridoideae sub-family and the Aurantiodeae sub-family (Morton and Telmer 2014 ). Lunasia, Zanthoxylum and Melicope belong to the Amyridoideae subfamily (Groppo et al. 2022 ), but in this particular study these three species were mixed with the Aurantiodeae species. These indicated that the ISSR primers used in this study is capable in differentiating species up to the Rutodeae subfamily. The phylogenetic trees obtained from 12 ISSR markers and morphological data of 17 accessions of Rutaceae family were found to be different. In most cases actually, molecular markers and morphological characteristics have inadequate correlation. Morphological data is based on observable phenotypic appearance of organisms, quantitative in nature, controlled by multiple genes and influenced by both genetic and environmental factors. On the other hands, molecular data derived from genetic markers (such as ISSR, SSR, and SCoT markers) that are generally located in the non-coding regions of genome, act as “neutral” markers, not associated with biological function and are less influenced by environmental factors (Hong et al. 2021 ). Second, molecular markers such as ISSR can reveal hidden genetic variations that may not be apparent from morphological traits alone. Morphological traits can be influenced by convergent or divergent evolution, which can lead to discrepancies between morphological and molecular data (Li et al. 2023 ). Furthermore, when hybridization for instans, the event can blur species boundaries and affect phylogenetic relationships, as they lead to genetic exchange and introgression of alleles. Molecular markers are somehow better equipped to detect introgression than morphological traits (Kwiatek et al. 2021 ). Based on these facts, theoretically, it is difficult to search ISSR markers which are highly associated with morphological characteristics. 5. CONCLUSION The study of genetic diversity based on morphological and molecular characters on 17 wild Rutaceae accessions revealed different species grouping. Biplot analysis based on stem and leaves characters put the accessions into 4 groups, which included Lunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp ., and M. minutum on the group with weak characters relationships. The molecular assessment using 12 ISSR primers divided the Rutaceae accessions into 3 clusters, involving L. acidissima in a distinct group. The second cluster included Clausena sp, Melicope sp. and Fragara sp. and the third was divided into two sub-clusters, involving M. trichopetala , M. minutum , M. latifolia , L. amara , A. marmelos , Z. ovalifolium , G. pentaphyla , Lunasia sp, Z. rhetsa and M. exotica in the first sub-cluster and F. lucida , P. littorale and E. suaveolens in the second sub-cluster. Declarations Disclosure statement : The authors report no conflict of interests. Finacial support statement : The authors report no significant financial support for this work that could have influenced its outcome Author contribution statement : KB designed the study, and prepared the research material, sample observation, data analysis, writing the manuscript. CM, NDY and II conducted the research, experiment resource, sample observation. SM and FY conducted data analysis, sample observation, writing the manuscript. HMY prepared the research material, gathered the primary and secondary data and supported the research. 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Corrêa): Importance, biology, propagation, and future perspectives, Trees - Structure and Function. Springer Berlin Heidelberg. https://doi.org/10.1007/s00468-018-1754-4 Venkatesan J, Ramu V, Sethuraman T, Sivagnanam C, Doss G (2021) Molecular marker for characterization of traditional and hybrid derivatives of Eleusine coracana (L.) using ISSR marker. J Genet Eng Biotechnol 19: 1–12. https://doi.org/10.1186/s43141-021-00277-1 Yasir M, Tripathi MK, Singh P, Shrivastava R (2018) The genus Glycosmis [Rutaceae]: A comprehensive review on its phytochemical and pharmacological perspectives. Nat Prod J 9: 98–124. https://doi.org/10.2174/2210315508666180622121212 Zarei A, Rezaei A, Esmailpour M, Ebrahimi A (2021) A comparative assessment of morphological and molecular characterization among three Ziziphus species. Physiol Mol Biol Plants 27: 1007–1025. https://doi.org/10.1007/s12298-021-01000-7 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4624814","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323228188,"identity":"4519f1b0-8190-4c28-81ff-a30560183bcb","order_by":0,"name":"Kurniawan Budiarto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACCQkgTgCx2BuAhIEFkVoeADEDzwGQFgnitEiCtUDsIkKL5OwewxsJNffq+CWfX93wo0CCgb+9OwGvFmmZM8YWCceKJSRn55Td7AE6TOLM2Q14tchJ5JhJJLAlSBjczkm7wQPUYiCRS4yWf0AtN8+k3fxDjBZpkJbENqCWG+zHbhNli+ScY8UWiX0JkjN7cthuyxhI8BD0i8Tt5o03f3xL4OdnP/7s5ps/NnL87b34tSABHgMwSaxyEGB/QIrqUTAKRsEoGEEAAI6lRBjlApTDAAAAAElFTkSuQmCC","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":true,"prefix":"","firstName":"Kurniawan","middleName":"","lastName":"Budiarto","suffix":""},{"id":323228189,"identity":"ab15dd00-b356-4403-aca7-280accadf274","order_by":1,"name":"Chaireni Martasari","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Chaireni","middleName":"","lastName":"Martasari","suffix":""},{"id":323228190,"identity":"94be39d4-12f7-4b04-8912-dd0c374af343","order_by":2,"name":"Shofiyatul Mas’udah","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Shofiyatul","middleName":"","lastName":"Mas’udah","suffix":""},{"id":323228191,"identity":"4a7034e2-19ea-4925-b239-a10a72a06041","order_by":3,"name":"Nina Dwi Yulia","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"Dwi","lastName":"Yulia","suffix":""},{"id":323228192,"identity":"365c9729-9a3b-4f4e-a16e-4d50c1057235","order_by":4,"name":"Farida Yulianti","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Farida","middleName":"","lastName":"Yulianti","suffix":""},{"id":323228193,"identity":"f6828301-5bfe-4633-b8d6-eb8da464acfa","order_by":5,"name":"Hadi Mokhamad Yusuf","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Hadi","middleName":"Mokhamad","lastName":"Yusuf","suffix":""},{"id":323228194,"identity":"9985c561-eedf-495f-8d3f-7aafaf8a4013","order_by":6,"name":"Imro’ah Ikarini","email":"","orcid":"","institution":"National Agency for Research and Innovation (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Imro’ah","middleName":"","lastName":"Ikarini","suffix":""},{"id":323228195,"identity":"49cd1f7e-204c-4154-858d-d94faab0f193","order_by":7,"name":"Adi Setiawan","email":"","orcid":"","institution":"University of Brawijaya","correspondingAuthor":false,"prefix":"","firstName":"Adi","middleName":"","lastName":"Setiawan","suffix":""}],"badges":[],"createdAt":"2024-06-23 10:19:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4624814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4624814/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60604169,"identity":"ef376b8f-88b4-4fc4-8a87-5e03cd650206","added_by":"auto","created_at":"2024-07-18 16:32:25","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":716560,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological leaf features of the studied Rutaceae species; (a) \u003cem\u003eA. marmelos\u003c/em\u003e, (b) \u003cem\u003eL. amara\u003c/em\u003e, (c) \u003cem\u003eZ. rhetsa\u003c/em\u003e, (d) \u003cem\u003eM. minutum\u003c/em\u003e, (e) \u003cem\u003eZ. ovalifolium\u003c/em\u003e, (f) \u003cem\u003eFragara \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e, (g) \u003cem\u003eL. acidissima\u003c/em\u003e, (h) \u003cem\u003eMelicope \u003c/em\u003esp., (i) \u003cem\u003eClausena \u003c/em\u003esp\u003cem\u003e.\u003c/em\u003e, (j) \u003cem\u003eE. suaveolens\u003c/em\u003e, (k) \u003cem\u003eM. trichopetala\u003c/em\u003e, (l) \u003cem\u003eG. pentaphyla\u003c/em\u003e, (m) \u003cem\u003eM. latifolia, \u003c/em\u003e(n) \u003cem\u003eM. exotica, \u003c/em\u003e(o) \u003cem\u003eLunasia \u003c/em\u003esp\u003cem\u003e., \u003c/em\u003e(p) \u003cem\u003eF. lucida \u003c/em\u003eand (q) \u003cem\u003eP. littorale \u003c/em\u003e(\u003cem\u003eL. littoralis\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4624814/v1/9b01353fbf156ca3b997eec0.jpeg"},{"id":60604166,"identity":"3d593707-cd5a-44d9-b3fd-76a50e0cc6d8","added_by":"auto","created_at":"2024-07-18 16:32:25","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":135353,"visible":true,"origin":"","legend":"\u003cp\u003eA scree plot of eigenvalues and cumulative variability for the nine Principal Components (F) from the PCA analysis on the morphological characters of the studied Rutaceae accessions.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4624814/v1/8f78d9e4a59ebfdfe9e1cdd5.jpeg"},{"id":60605004,"identity":"be92a161-eac4-46e4-bd89-145b781b2761","added_by":"auto","created_at":"2024-07-18 16:40:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162753,"visible":true,"origin":"","legend":"\u003cp\u003eScatter biplot 17 of the studied Rutaceae species and the active moprhological characters\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4624814/v1/e261d0d5745a00d82824524d.png"},{"id":60604168,"identity":"9ad21fa9-3424-4399-87d4-1f7d80d875f1","added_by":"auto","created_at":"2024-07-18 16:32:25","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":251904,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of 17 accessions of Rutaceae family revealed by 12 ISSR primers\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4624814/v1/360d91bbf986931d5e594740.jpeg"},{"id":83924020,"identity":"1c811446-1250-451d-859e-d9b9c9592007","added_by":"auto","created_at":"2025-06-04 14:13:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2328186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4624814/v1/1f97cd51-3354-479c-b775-fd6326c9eb8d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of variability among molecular and morphological characters of several wild Rutaceae species","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eRutaceae is a large plant family, and predominantly located in tropical, and subtropical area. The family was reported to have 162 genera with composed of 2085 species (Groppo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The species member may increase along with the discovery of new species (Aiyakool and Vajrodaya \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fernandez-Hilario and Arteaga \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Tropical America, southern Africa (especially in the Cape Province, with the tribe Diosmeae), and Australasia were reported to be the main region of centers of diversity and the order Sapindales being the largest family in number of species (Appelhans et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Groppo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Citrus L., a speciose genus of Asian origin, is widely cultivated for their edible fruits (oranges, lemons, tangerines, and grapefruits) and other plant organs for food condiments. While other group in Rutaceae family, are well known as important sources of aromatic oils (Cimik \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liaqat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), drugs (Adamska-Szewczyk et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Musa et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mutinda et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), ornamentals (Licciardello et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and for other purposes.\u003c/p\u003e \u003cp\u003eRecently, there has been a substantial increase in the use of both morphological as well as biochemical, cytological or even molecular markers for the assessment of genetic diversity and fingerprinting of various plant species (Devi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Assessing genetic diversity is crucial, not only for crop improvement but also for efficient management and conservation of germplasm resources. Morphological studies are expected to bring to light morphovariants. Molecular studies are essential in detecting genetic variations among genotypes, which helps select diverse parents to successfully carry out a new crossing program. Additionally, the molecular data in conjunction with data generated on morphological variability of current taxa will also be helpful in establishing inter- and intra-population phylogenetic relationships (Zarei et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUp to this moment, the ordinal position of Rutaceae family was referred to Engler subfamilial classification based on characters of the gynoecium, especially fruit type and the recognized the systematic of Rutaceae into seven subfamillies, i.e. Rhabdodendroideae, Aurantioideae (Citroideae), Flindersioideae, Spathelioideae, Dictyolomatoideae, Rutoideae, and Toddalioideae (Groppo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Several recent studies however, have cast doubt on his proposal based on phytochemical and molecular studies (Appelhans et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chase et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The species under Rutaceae family also has undergone frequent interspecific hybridization among the wild or even with cultivated populations which leads to varying basic chromosome count, occurrence of polyploids and variation in morphology under different environmental conditions (Santos et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Stace and Armstrong \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFuther study at molecular level to confirm morpho-variability in wild species Rutaceae is needed to establish a comprehensive characterization and relationship of existing diversity in wild species Rutaceae germplasm (Ou\u0026eacute;draogo et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The information is important to analyze the mechanism how the genetic diversity is maintained, also that underlying genetic mechanism leading to speciation and evolution and further use for breeding purpose (Sharma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For endangered species like \u003cem\u003ePleiospermium littorale\u003c/em\u003e (Miq.) Tanaka (syn. \u003cem\u003eLimnocitrus littoralis\u003c/em\u003e (Miq.) Swingle) (IUCN, 1998), the study are needed to investigate their potential economic values and conservation strategies of the respected species (Nguyen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The paper investigate the molecular and morphological characters among wild Rutaceae species that naturally grown in Indonesia.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":" \u003cp\u003eThe plant materials were 17 accessions from Rutaceae family, i.e \u003cem\u003eAegle marmelos, Lunasia amara, Zanthoxylum rhetsa, Micromelum minutum, Zanthoxylum ovalifolium, Fragara\u003c/em\u003e sp., \u003cem\u003eLimonia acidissima, Melicope\u003c/em\u003e sp., \u003cem\u003eClausena\u003c/em\u003e sp., \u003cem\u003eEuodia suaveolens, Melicope trichopetalla, Glycosmis pentaphyla, Melicope latifolia, Murraya exotica, Lunasia\u003c/em\u003e sp., \u003cem\u003eFerroniella lucida\u003c/em\u003e and \u003cem\u003ePleiospermium littorale\u003c/em\u003e. The plants are a part of a core collection of Purwodadi Botanical Garden, located at Pasuruan, East Java, Indonesia.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Diversity and differentiation of morphological traits\u003c/h2\u003e \u003cp\u003eThe studied Rutaceae species reached at least 10 years old and undergone complete vegetative and reproductive growth cycles. The observation was based on citrus descriptor list from IPGRI (1991) and carried out on at least 10 plant part samples. The observations were carried out during the early wet season of 2022\u0026ndash;2023. The assessment covered quantitative and qualitative parameters, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlant characteristics used for morphological observation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorphological characters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCharacter category\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStem surface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;smooth, 2\u0026thinsp;=\u0026thinsp;grooved and ridged\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDensity of branches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026thinsp;=\u0026thinsp;sparse, 5\u0026thinsp;=\u0026thinsp;medium, 7\u0026thinsp;=\u0026thinsp;dense\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBranch angle (attachment to main trunk)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026thinsp;=\u0026thinsp;narrow, 5 = medium, 7\u0026thinsp;=\u0026thinsp;wide\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpine density on adult tree (not on sucker)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;=\u0026thinsp;absent, 3\u0026thinsp;=\u0026thinsp;low, 5 = medium, 7\u0026thinsp;=\u0026thinsp;high\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShoot tip color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;green, 2\u0026thinsp;=\u0026thinsp;purple\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShoot tip surface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;glabrous, 2\u0026thinsp;=\u0026thinsp;intermediate, 3\u0026thinsp;=\u0026thinsp;pubescent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVegetative life cylce\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;evergreen, 2\u0026thinsp;=\u0026thinsp;deciduous, 3\u0026thinsp;=\u0026thinsp;semi-persistent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf division\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;simple, 2\u0026thinsp;=\u0026thinsp;bifoliate, 3\u0026thinsp;=\u0026thinsp;trofoliate, 4\u0026thinsp;=\u0026thinsp;pentafoliate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength of petiole relative to length of leaf lamina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;sessile (petiole absent), 2\u0026thinsp;=\u0026thinsp;brevipetiolate (petiole shorter than leaf lamina), 3 = longipetiolate (petiole longer than or same length as leaf lamina)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf lamina shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;elliptic, 2\u0026thinsp;=\u0026thinsp;ovate, 3\u0026thinsp;=\u0026thinsp;obovate, 4\u0026thinsp;=\u0026thinsp;lanceolate, 5\u0026thinsp;=\u0026thinsp;orbicular, 6\u0026thinsp;=\u0026thinsp;obcordate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf lamina margin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;crenate, 2\u0026thinsp;=\u0026thinsp;dentate, 3\u0026thinsp;=\u0026thinsp;entire, 4\u0026thinsp;=\u0026thinsp;sinuate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf apex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;=\u0026thinsp;attenuate, 2\u0026thinsp;=\u0026thinsp;acuminate, 3\u0026thinsp;=\u0026thinsp;acute, 4\u0026thinsp;=\u0026thinsp;obtuse, 5\u0026thinsp;=\u0026thinsp;rounded, 6\u0026thinsp;=\u0026thinsp;emarginate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsense/presense of petiole wings\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026thinsp;=\u0026thinsp;absent, 1\u0026thinsp;=\u0026thinsp;present\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Assessment of genetic variability\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 DNA isolation and purification\u003c/h2\u003e \u003cp\u003eThe method of DNA extraction, isolation, and quantification was following Plant Genomic DNA Kit manual (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://en.tiangen.com/upload/file/20220509/20220509165908_43410.pdf\u003c/span\u003e\u003cspan address=\"https://en.tiangen.com/upload/file/20220509/20220509165908_43410.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Young leaf samples (100g) were ground using mortar in 700 \u0026micro;l 65\u0026deg;C pre-heated GP1 (containing 0.1% β-Mercaptoethanol (β-ME)). 700 \u0026micro;l chloroform was added to the mixture, mixed by inverting the tube several times, and centrifuged for 5 min at 12,000 rpm (~\u0026thinsp;13,400 \u0026times; g). The supernatants were pipetted to a new tube, were added 700 \u0026micro;l Buffer GP2, mix by inverting the tube several times. All of the mixture was pipetted into the Spin Column CB3 (the Spin Column CB3 was placed in the Collection Tube first). the CB3 lid was closed and centrifuged for 30 s at 12,000 rpm (~\u0026thinsp;13,400 \u0026times; g). the filtrate was Discarded and the Spin Column CB3 was placed into the Collection Tube. 500 \u0026micro;l Buffer GD was added and centrifuged at 12000 rpm (~\u0026thinsp;13,400 \u0026times; g) for 30 s then the filtrate was discarded. 600 \u0026micro;l buffer PW (Ensure was added to the Spin Column CB3, and centrifuged for 30 s at 12,000 rpm (~\u0026thinsp;13,400 \u0026times; g), the flow-through was discarded. This step was Repeated twice. the Spin Column CB3 was placed in the Collection Tube, centrifuged for 2 min at 12,000 rpm (~\u0026thinsp;13,400 \u0026times; g), and the flow-through was discarded. The lid of CB3 was opened and stayed at room temperature for a while to dry the membrane completely. Spin Column CB3 was placed in a new centrifuge tube, and 50\u0026ndash;200 \u0026micro;l Buffer TE was added directly onto the CB3 membrane, incubated for 2\u0026ndash;5 min at room temperature (15\u0026ndash;30\u0026deg;C), and then centrifuged for 2 min at 12,000 rpm (~\u0026thinsp;13,400 \u0026times; g) to elute\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 DNA amplification and separation\u003c/h2\u003e \u003cp\u003eDNA amplified using MyTaq\u0026trade; HS Red Mix (Meredian Bioscience). 50 ng DNA sample of each accession was amplified in PCR (Thermocycler-Biometra) at the specific annealing temperature for each primer. The primers were UBC807, UBC810, UBC813, UBC816, UBC821, UBC824, UBC830, UBC843, UBC853, UBC873, UBC886 and UBC891 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCR amplification with ISSR marker was started with the denaturation cycle at 95\u0026ordm;C for 1 minute, followed by 35 denaturation cycles at 95\u0026ordm;C for 15 seconds, temperature annealing for 15 seconds with temperature extension up to 72\u0026deg;C for 10 seconds. The PCR cycle was terminated by 1 cycle temperature extension at 72\u0026deg;C for 10 minutes. The products of amplification were separated in 2.0% agarose gel containing gelred (10 mg/l) in 0.5 x TBE solution for 50 minutes at 100 volts The presence of an amplified product (fragment) was identified and biodocumented.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of ISSR primers used in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnnealing temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(AG)8T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(GA)8T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CA)8T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(GT)8T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(TC)8G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(TG)8G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8RA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(TC)8RT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(GACA)4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVDV(CT)7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHVH(GT)7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data obtained from morphological and molecular studies were subjected to cluster analysis based on UPGMA (Unweighted Pair Group Method with Arithmetic mean) to evaluate the similarity/dissimilarity among the studied populations. Matrix similarity was computed based on Dice\u0026rsquo;s coefficient Past 4.04, and the cluster analysis was performed for grouping accessions based on Dice\u0026rsquo;s coefficient according to the UPGMA method. Redundancy analysis was performed on both morphological and molecular datasets separately for analyzing the morphologically and genetically structured populations using the Biodiversity R package.\u003c/p\u003e \u003cp\u003eISSR bands were treated as binary characters and coded accordingly (presence\u0026thinsp;=\u0026thinsp;1, absence\u0026thinsp;=\u0026thinsp;0) from each primer. The Number of Scored Bands (NSB), the Number of Polymorphic Bands (NPB), and the Polymorphism Information Content (PIC) were calculated for each primer, in addition to the Marker Index (MI), Effective Multiplex Ratio (EMR), and Resolving Power (RP). PIC for the dominant markers was calculated according to Nei (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1973\u003c/span\u003e).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$PIC=1-\\sum {{P}_{i}}^{2}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere =\u0026thinsp;Pi represents the frequency of its accession.\u003c/p\u003e \u003cp\u003eTo calculate EMR and MI, the following formula was used.\u003c/p\u003e \u003cp\u003eEMR\u0026thinsp;=\u0026thinsp;p x pf\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere =\u0026thinsp;p is the number of polymorphic bands of a primer and pf is the frequency of polymorphic bands (Powell et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eMI\u0026thinsp;=\u0026thinsp;EMR x PIC\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere =\u0026thinsp;EMR is the effective multiplex ratio of a marker, and PIC is the polymorphism information content of a primer.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eResolving power (Rp) was assessed according to the following formula.\u003c/p\u003e \u003cp\u003eRp = Ʃ Ib\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere =\u0026thinsp;Ib is the band informativeness with Ib\u0026thinsp;=\u0026thinsp;1 \u0026ndash; [2 x (0.5-p)] and p is the proportion of accessions containing the polymorphic band. All calculations were processed according to (Hancı \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Morphological features of Rutaceae species\u003c/h2\u003e \u003cp\u003eThe morphological characteristics of the studied Rutaceae wild species were varied in terms of stem surface, density of banches, branch angle and trunk surface. The variations are also observed in terms of leaf division, leaf lamina shape, leaf lamina margin, leaf apex and relative petiole lenght to lenght of leaf lamina as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. While all species have similar features of shoot tip color and surface, vegetative life cycle, and the absence of petiole wings.\u003c/p\u003e \u003cp\u003ePinciple Component Analysis (PCA) was used to analyze genetic diversity in Rutaceae based on 9 morphological characters and determine their influence on the studied Rutaceae accessions. The PCA aimed to identify the variables contributing to species diversity. The analysis of 9 parameters on the studied Rutaceae accessions resulted in four principle components (PC). PC 1 (F1), PC2 (F2), and PC 3 (F3) contributed to 29.33%, 21.059%, and 15.505% to the diversity, respectively, with a cumulative diversity value of 65.894% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The variables in these PC determine the diversity in the studied Rutaceae accessions. Positive values in the results of PCA indicate a significant contribution to diversity, while negative values indicate not significant contribution to diversity (Gupta et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Talekar et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The observed variables in PC 1 (F1) are the main determinants of the diversity of the 17 Rutaceae accessions studied if the characteristic vector value is \u0026gt;\u0026thinsp;0.5, namely: length of petiole relative to length of leaf lamina and leaf apex. The main determining observation variable in PC 2 (F2) is branch angle, and in PC 3 (F3) is density of branches (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEigen vector values of 9 principle component based on morphological characters of the studied Rutaceae accessions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF9\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStem surface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDensity of branches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.629\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBranch angle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.548\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.243\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpine density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf division\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.391\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength of petiole relative to length of leaf lamina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.515\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf lamina shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.587\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.341\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf lamina margin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.378\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf apex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.506\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe biplot analysis identified the group of each accession based on their common characteristics. The biplot analysis of the studied Rutacecae accessions revealed that first component accounting for 29.33% and the second component for 21.06% of the total diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In quadrant 1, three accessions namely \u003cem\u003eZ. ovalifolium, L. acidissima\u003c/em\u003e, and \u003cem\u003eF. lucida\u003c/em\u003e shared their common features on leaf division, stem surface, spine density, and leaf apex. Six accessions, \u003cem\u003eA. marmelos, Fragara\u003c/em\u003e sp., \u003cem\u003eMelicope\u003c/em\u003e sp., \u003cem\u003eM. trichopetala, G. pentaphyla\u003c/em\u003e and \u003cem\u003eM. exotica\u003c/em\u003e showed similarities in terms of branch angle, leaf lamina shape, and leaf lamina margin and were grouped into Quandrant II. In Quadrant IV, \u003cem\u003eP. littorale\u003c/em\u003e and \u003cem\u003eZ. ovalifolium\u003c/em\u003e shared their common characteristics in density of branches and length of petiole relative to length of leaf lamina. No common parameter could be the basis of grouping in Quadrant III indicating a weak influence of the characters on the other six accessions, \u003cem\u003eLunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp\u003c/em\u003e., and \u003cem\u003eM. minutum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Genetic diversity of Rutaceae species\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays the results of ISSR primers amplification, including the number of polymorphic bands (NPB), percentage of polymorphic bands (PPB), Polymorphism Information Content (PIC), Marker Index (MI), Effective Multiplex Ratio (EMR), and Resolving Power (RP). All 76 bands detected by 12 primers were found to be polymorphic. The primers with the highest number of bands were recorded at UBC807 and UBC886, with 11 and 10 bands, respectively. The UBC853 primer had the lowest number of bands, with only 3 detected bands.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePolymorphism percentage, total bands, PIC, MI, EMR, and RP of ISSR markers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNPB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePPB (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePIC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eRemarks : NPN\u0026thinsp;=\u0026thinsp;Number of Polymorphic Bands, PPB\u0026thinsp;=\u0026thinsp;Percentage of Polymorphic Bands, PIC\u0026thinsp;=\u0026thinsp;Polymorphism Information Content, MI\u0026thinsp;=\u0026thinsp;Marker Index, EMR\u0026thinsp;=\u0026thinsp;Effective Multiplex Ratio, RP\u0026thinsp;=\u0026thinsp;Resolving Power\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe average PIC value for all primers was 0.38, with the highest value was detected in UBC886 (0.48) and the lowest value was attributed to UBC830 (0.26). The average MI was 2.49, with the largest MI associated with UBC886 (4.82) and the smallest value belonging to UBC830 (1.30). The average EMR was 1.79, with UBC807 having the highest EMR (3.47) and UBC830 and UBC853 having the lowest EMR (0.76). The average RP was 3.56, with the largest RP attributed to UBC807 (6.94) and the lowest value belonging to UBC830 and UBC853 (1.53).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eshows a similarity matrix based on Dice's coefficient for accessions. The values for similarity ranged from 0.00 to 0.56. The highest similarity value of 0.56 was observed between \u003cem\u003eA. marmelos\u003c/em\u003e and \u003cem\u003eL. amara\u003c/em\u003e. On the other hand, the least similarity value of 0.00 was observed between \u003cem\u003eL. acidissima\u003c/em\u003e and \u003cem\u003eClausena\u003c/em\u003e sp. Tabel 5. Similarity matrix between Rutaceae family based on ISSR primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"18\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c18\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on Dice\u0026rsquo;s coefficient, the UPGMA hierarchical clustering was constructed to group the studied Rutaceace accessions and three distinctive clusters were identified as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The first cluster consisted of \u003cem\u003eL. acidissima\u003c/em\u003e only. The second cluster included \u003cem\u003eClausena\u003c/em\u003e sp, \u003cem\u003eMelicope\u003c/em\u003e sp. and \u003cem\u003eFragara\u003c/em\u003e sp. The third cluster was divided into two sub-clusters. The first sub-cluster included \u003cem\u003eM. trichopetala\u003c/em\u003e, \u003cem\u003eM. minutum\u003c/em\u003e, \u003cem\u003eM. latifolia\u003c/em\u003e, \u003cem\u003eL. amara\u003c/em\u003e, \u003cem\u003eA. marmelos\u003c/em\u003e, \u003cem\u003eZ. ovalifolium\u003c/em\u003e, \u003cem\u003eG. pentaphyla\u003c/em\u003e, \u003cem\u003eLunasia\u003c/em\u003e sp, \u003cem\u003eZ. rhetsa\u003c/em\u003e and \u003cem\u003eM. exotica\u003c/em\u003e. The second sub-cluster included \u003cem\u003eF. lucida\u003c/em\u003e, \u003cem\u003eP. littorale\u003c/em\u003e and \u003cem\u003eE. suaveolens\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eAnalysis of genetic diversity can assist in reliable classification of collections required for crop improvement. Recent studies revealed that molecular markers and morphological analysis could supplement each other in plant classification and establishing the distinctness among varieties, variety identification and genetic diversity analysis (Jain et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The morphological analysis is employed in the first step of plant identification and has become a valuable source of information for agronomic and breeding programs. The study usually used as the foremost tools before continually using other methods. The marker also comprehends in the determination of the species and other category under the species (Tolangara et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The study of morphological characteristics of Rutaceae accessions revealed that length of petiole relative to length of leaf lamina, leaf apex, branch angle and density of branches are the main factor in differentiating the accessions in PC\u0026rsquo;s. These differentiating characters were also confirmed by Kandowangko and Febriyanti (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) under the genus Citrus. Branch angle and density of branches are related with plant architecture and affected by both genetic and environmental influences. The environmental factors as external factors have a significant impact on these characteristics along with the increase of plant ages. Trees may dynamically adjust their shape to cope with mechanical stress as the surrounding environment changes over time (Tsugawa et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on biplot analysis, three accessions namely \u003cem\u003eZ. ovalifolium, L. acidissima\u003c/em\u003e, and \u003cem\u003eF. lucida\u003c/em\u003e shared their common features on leaf division, stem surface, spine density, and leaf apex in quadrant 1. According to Pavani et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), Z. \u003cem\u003eovalifolium\u003c/em\u003e has brownish stem with 8\u0026ndash;12 smooth leaflets. The leaflet has elliptic-abovate shape with rounded to abruptly acuminate apex. These characteristics are also commonly found in \u003cem\u003eL. accidisima\u003c/em\u003e, in that the bark is fissured and scaly with the sharp spines the zig-zag twigs pattern and densely in younger branches. The leaves of \u003cem\u003eL. accidissima\u003c/em\u003e are dark-green, leathery, often minutely toothed, blunt or notched at the apex (Murrinie et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eF. lucida\u003c/em\u003e was also reported to have these similar characteristics, since formerly genus Feroniella was considered allied to \u003cem\u003eLimonia\u003c/em\u003e L. due to its tough pericarp. Recently, the difference between Limonia and Feroniella was revealed on flower structure (Mabberley \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which are not included in this study.\u003c/p\u003e \u003cp\u003eSix accessions, i.e. \u003cem\u003eA. marmelos, Fragara\u003c/em\u003e sp., \u003cem\u003eMelicope\u003c/em\u003e sp., \u003cem\u003eM. trichopetala, G. pentaphylla\u003c/em\u003e and \u003cem\u003eM. exotica\u003c/em\u003e were grouped based on their similarities on branch angle, leaf lamina shape, and leaf lamina margin. \u003cem\u003eA. marmelos\u003c/em\u003e is a a spiny tree with deciduous trifoliate leaves. According to several reports, morphological variations on the leaf color, shape, surface and margin were existed under this species (Vasava et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several variants of \u003cem\u003eA. marmelos\u003c/em\u003e in India have light to dark green leaves, lanceolate to ovate leaf shape, smooth to rough leaf surface and crenulate to crenate leaf margin (Singh et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Genus Melicope has lanceolate leaf shape with long, medium to wide angle banches from the main trunk as the common features within the group (Appelhans et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eG. pentaphylla\u003c/em\u003e has a compound leaf comprised of 5 to 9 foliates. The leaf has elliptic to lanceolate shapes, alternate, with entire and unlobed margin (Sundrarajoo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The morphological variations were also reported among the species under the genus Glycosmis, in terms of number of leaflets, number of ovary within the locule and fruit shape (Yasir et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similar to \u003cem\u003eA. marmelos, Fragara\u003c/em\u003e sp., \u003cem\u003eMelicope\u003c/em\u003e sp., \u003cem\u003eM. trichopetala, G. pentaphylla\u003c/em\u003e, \u003cem\u003eM. exotica\u003c/em\u003e was also reported to have elliptic to obovate leaves with entire leaf margin. The variations in leaf were commonly found in the number of foliates, size, thickness and leaf color (Mou et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Quadrant IV, \u003cem\u003eP. littorale\u003c/em\u003e and \u003cem\u003eZ. ovalifolium\u003c/em\u003e shared their common characteristics in density of branches and length of petiole relative to length of leaf lamina. Both accessions have very short petiole compared to leaf lamina with medium to dense branches. The obvious differences between these species were on the leaf type. \u003cem\u003eP. littorale\u003c/em\u003e has simple leaf with emarginate or rounded laef apex while \u003cem\u003eZ. ovalifolium\u003c/em\u003e shows trifoliate leaves with obtuse or acuminate leaf apex (Pavani et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eQuadrant III has six accessions, \u003cem\u003eLunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp\u003c/em\u003e., and \u003cem\u003eM. minutum\u003c/em\u003e with no common basis parameter of grouping. The morphological features used in the study were limited to leaf and stem characteristics which were unable or insufficient to become the basis of plant grouping or differentiation. Appelhans et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) suggested to use reproductive plant parts like flower and its organs to discriminate several genera in Rutaceae family, which are very difficult to obtain during the study. The absent of character basis on quadrant III group might also be due to the plant plasticity. Plant phenotype presents a variation in their morphology either naturally or in connection with local adaptations (Duminil and Di Michele \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Alternatively some species can be morphologically very similar and may be falled into the same group despite the fact that they represent separate taxonomic entities (Pocovi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 12 primers of ISSR markers used in the study successfully detected genetic variation in the Rutaceae family with high polymorphism. The statistical analysis employed to elucidate the degree of polymorphism confirmed the PIC values averaged of 0,38 and ranged from 0.26 to 0.48 that were categorized as informative and sufficient for differentiation of the studied genotype groups (Al-Mousa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sabreena et al. 2021). The effectiveness of the marker system and the methodological approaches were also evaluated by employing additional indicators such as EMR, MI and RP. EMR is the number of polymorphic fragments detected per assay and depended on polymorphic loci. MI is primer capacity to detect polymorphic loci among different genotypes and RP is the ability of primers to distinguish or dicriminatiry efficiency of primers among genotypes (Kumar and Agrawal 2019). The frequency of polymorphic loci was varied from primer to primer (Venkatesan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In present study, the primers were found to have polymorphic loci ranging from 3 to 11. The highest frequency of polymorphic loci was observed in UBC886 with 10 and UBC807 with 11 polymorphic bands. These indicates that both primers are the most effective and reliable among the employed primers in distinguishing the studied genotypes\u003c/p\u003e \u003cp\u003eThe constructed cluster based on ISSR markers took the studied Rutaceae accessions into three clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and most of the tested species were found to belong to the Rutodeae subfamily (Groppo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These grouping were then updated and divided the Rutodeae into two sub-family groups, namely the Amyridoideae sub-family and the Aurantiodeae sub-family (Morton and Telmer \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Lunasia, Zanthoxylum and Melicope belong to the Amyridoideae subfamily (Groppo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but in this particular study these three species were mixed with the Aurantiodeae species. These indicated that the ISSR primers used in this study is capable in differentiating species up to the Rutodeae subfamily.\u003c/p\u003e \u003cp\u003eThe phylogenetic trees obtained from 12 ISSR markers and morphological data of 17 accessions of Rutaceae family were found to be different. In most cases actually, molecular markers and morphological characteristics have inadequate correlation. Morphological data is based on observable phenotypic appearance of organisms, quantitative in nature, controlled by multiple genes and influenced by both genetic and environmental factors. On the other hands, molecular data derived from genetic markers (such as ISSR, SSR, and SCoT markers) that are generally located in the non-coding regions of genome, act as \u0026ldquo;neutral\u0026rdquo; markers, not associated with biological function and are less influenced by environmental factors (Hong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Second, molecular markers such as ISSR can reveal hidden genetic variations that may not be apparent from morphological traits alone. Morphological traits can be influenced by convergent or divergent evolution, which can lead to discrepancies between morphological and molecular data (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, when hybridization for instans, the event can blur species boundaries and affect phylogenetic relationships, as they lead to genetic exchange and introgression of alleles. Molecular markers are somehow better equipped to detect introgression than morphological traits (Kwiatek et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Based on these facts, theoretically, it is difficult to search ISSR markers which are highly associated with morphological characteristics.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe study of genetic diversity based on morphological and molecular characters on 17 wild Rutaceae accessions revealed different species grouping. Biplot analysis based on stem and leaves characters put the accessions into 4 groups, which included \u003cem\u003eLunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp\u003c/em\u003e., and \u003cem\u003eM. minutum\u003c/em\u003e on the group with weak characters relationships. The molecular assessment using 12 ISSR primers divided the Rutaceae accessions into 3 clusters, involving \u003cem\u003eL. acidissima\u003c/em\u003e in a distinct group. The second cluster included \u003cem\u003eClausena\u003c/em\u003e sp, \u003cem\u003eMelicope\u003c/em\u003e sp. and \u003cem\u003eFragara\u003c/em\u003e sp. and the third was divided into two sub-clusters, involving \u003cem\u003eM. trichopetala\u003c/em\u003e, \u003cem\u003eM. minutum\u003c/em\u003e, \u003cem\u003eM. latifolia\u003c/em\u003e, \u003cem\u003eL. amara\u003c/em\u003e, \u003cem\u003eA. marmelos\u003c/em\u003e, \u003cem\u003eZ. ovalifolium\u003c/em\u003e, \u003cem\u003eG. pentaphyla\u003c/em\u003e, \u003cem\u003eLunasia\u003c/em\u003e sp, \u003cem\u003eZ. rhetsa\u003c/em\u003e and \u003cem\u003eM. exotica\u003c/em\u003e in the first sub-cluster and \u003cem\u003eF. lucida\u003c/em\u003e, \u003cem\u003eP. littorale\u003c/em\u003e and \u003cem\u003eE. suaveolens\u003c/em\u003e in the second sub-cluster.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDisclosure statement : The authors report no conflict of interests.\u003c/p\u003e\n\u003cp\u003eFinacial support statement : The authors report no significant financial support for this work that could have influenced its outcome\u003c/p\u003e\n\u003cp\u003eAuthor contribution statement\u003cstrong\u003e\u0026nbsp;:\u0026nbsp;\u003c/strong\u003eKB designed the study, and prepared the research material, sample observation, data analysis, writing the manuscript. CM, NDY and II conducted the research, experiment resource, sample observation. SM and FY conducted data analysis, sample observation, writing the manuscript. HMY prepared the research material, gathered the primary and secondary data and supported the research. AS prepared the research material and review the manuscript.\u003c/p\u003e\n\u003cp\u003eData availability statement : The authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamska-Szewczyk A, Glowniak K, Baj T (2016) Furochinoline alkaloids in plants from Rutaceae family - A review. 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Physiol Mol Biol Plants 27: 1007\u0026ndash;1025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12298-021-01000-7\u003c/span\u003e\u003cspan address=\"10.1007/s12298-021-01000-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rutaceae species, genetic variability, morphological and molecular markers, relationship","lastPublishedDoi":"10.21203/rs.3.rs-4624814/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4624814/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMorphological and molecular assessments were carried out to confirm the genetic variability and relationship among wild Rutaceae species. Thirteen stem and leaves morphological characters and 12 ISSR primers were used to evaluate 17 Rutaceae accessions. The results shows that morphological and molecular assessments confirmed different accessions grouping. Biplot analysis based on morphological characters revealed 3 groups based on the character similarity and 1 distinct group consisting \u003cem\u003eLunasia sp., M. latifolia, L. amara, E. suaveolens, Clausena sp\u003c/em\u003e., and \u003cem\u003eM. minutum\u003c/em\u003e with a weak relationship of the observed characters. While the ISSR study divided the accessions into 3 clusters, which include \u003cem\u003eL. acidissima\u003c/em\u003e in a distinct cluster. The second cluster composed of \u003cem\u003eClausena\u003c/em\u003e sp, \u003cem\u003eMelicope\u003c/em\u003e sp. and \u003cem\u003eFragara\u003c/em\u003e sp. The third cluster was divided into two sub-clusters. The first sub-cluster included \u003cem\u003eM. trichopetala\u003c/em\u003e, \u003cem\u003eM. minutum\u003c/em\u003e, \u003cem\u003eM. latifolia\u003c/em\u003e, \u003cem\u003eL. amara\u003c/em\u003e, \u003cem\u003eA. marmelos\u003c/em\u003e, \u003cem\u003eZ. ovalifolium\u003c/em\u003e, \u003cem\u003eG. pentaphyla\u003c/em\u003e, \u003cem\u003eLunasia\u003c/em\u003e sp, \u003cem\u003eZ. rhetsa\u003c/em\u003e and \u003cem\u003eM. exotica\u003c/em\u003e. While, \u003cem\u003eF. lucida\u003c/em\u003e, \u003cem\u003eP. littorale\u003c/em\u003e and \u003cem\u003eE. suaveolens\u003c/em\u003e are the member of the second sub-cluster. The study has demonstrated the effectiveness of ISSR primers in differentiating species up to the subfamily level and it is crucial to prioritize further research to enhance the specificity of morphological characters.\u003c/p\u003e","manuscriptTitle":"Assessment of variability among molecular and morphological characters of several wild Rutaceae species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 16:32:20","doi":"10.21203/rs.3.rs-4624814/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13e90b97-1cbe-4027-9083-e190c923919f","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-04T14:05:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 16:32:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4624814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4624814","identity":"rs-4624814","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}