Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 33-43, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1541 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Xiaoju Zhang, Li Bai, Somayeh Esfandani-Bozchaloyi (2022) Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae). Caryologia 75(2): 33-43. doi: 10.36253/caryologia-1541 Received: January 18, 2022 Accepted: May 24, 2022 Published: September 21, 2022 Copyright: © 2022 Xiaoju Zhang, Li Bai, Somayeh Esfandani-Bozchaloyi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) Xiaoju Zhang1, Li Bai2,*, Somayeh Esfandani-Bozchaloyi3 1 College of Humanities and Management, Xi’an Traffic Engineering Institute, Xi’an 710300, Shaanxi, China 2 Department of Mechanical and Electrical Technology, XiJing University, Xi’an 710123, Shaanxi, China 3 Department of Plant Sciences, university Shahid Beheshti, Iran *Corresponding author. E-mail: baili891066611@163.com Abstract. The genus Salicornia (Amaranthaceae) was established by Linnaeus. Com- monly known as ‘glassworts’, the species of the genus are articulated succulent herbs with cortical palisade, opposite decussate scale-leaves, thyrsoid cymes, flowers packed in cauline depressions and the diaspore composed of l-seeded utricle. Therefore, due to the importance of the plant species, we performed a combination of morpho- logical and molecular data analyses on this species. A total of 72 randomly collected plants from 8 natural populations in 2 provinces were evaluated using ISSR markers and morphological traits. Analysis of molecular variance (AMOVA) test revealed sig- nificant genetic difference among the studied populations, and also showed that 45% of total genetic variability was due to the diversity within the population, while 55% was due to the genetic differentiation among populations. A total number of 158 bands were detected by ISSR primers, of which 144 (89%) bands with an average of 14.4 bands per primer were polymorphic. The Percentage of Polymorphic Bands (PPB) ranged from 70% (ISSR-7) to 100% (ISSR-1, ISSR-4 and ISSR-5). The average Poly- morphic Information Content (PIC), Shannon’s Information index (I), and Number of effective alleles (Ne) were 0.49, 0.28, and 1.09, respectively. Keywords: genetic diversity, gene flow, genetic differentiation, Salicornia persica, Inter Simple Sequence Repeat (ISSR). INTRODUCTION Genetic diversity is a basic component of biodiversity and its conserva- tion is essential for survival of any species in the changing environments (Si et al. 2020; Liu et al. 2021). Most authors agree that genetic diversity is necessary to preserve the long-term evolutionary potential of a species (Peng et al. 2021; Ma et al. 2021). This is very important in fragmented popula- tions, because they are more vulnerable due to the loss of allelic richness and increased population differentiation by genetic drift (decreased heterozygo- sity and eventual fixation of alleles) and inbreeding depression (increased homozygosity within populations; Chen et al. 2021; Bi et al. 2021). Therefore, 34 Xiaoju Zhang, Li Bai understanding the genetic variability and diversity with- in and among different populations is crucial for their conservation and management (e.g., Esfandani-Bozch- aloyi et al., 2018a, 2018b, 2018c). The genus Salicornia (Amaranthaceae) was estab- lished by Linnaeus (1753). Commonly known as ‘glass- worts’, the species of the genus are articulated succulent herbs with cortical palisade, opposite decussate scale- leaves, thyrsoid cymes, flowers packed in cauline depres- sions and the diaspore composed of l-seeded utricle. Many species are green, but their foliage turns red in autumn. The hermaphrodite flowers are wind pollinated. The species inhabit saline habitats such asinland salt- marshes, saline seasonal river banks and tidal coastlines, but all tidal coasts and salines are not home to glass- worts. Salicornia species can generally tolerate immer- sion in salt water. They use the C 4 to take in carbon dioxide from the surrounding atmosphere. Salicornia has leafless stems with branches that resembles aspara- gus. The halophyte Salicornia supports soil microbial growth and boosts the TPHs degradation in saline oil- contaminated soils. Combining Salicornia and P.  aerugi- nosa accelerates TPH degradation and reduces saline oil- contaminated soils phytotoxicity (Ebadi et al. 2018). The first comprehensive account of family Cheno- podiaceae in Flora Iranica (Hedge et al. 1997) provides very useful and fundamental data on the arid flora of the Old World. The genus Salicornia is among the most diverse genera of the Salicornieae tribe. The genus cur- rently comprises 25 to 30 species (Kadereit et al. 2007). The taxonomy of the genus Salicornia is still far from satisfactory, although numerous species aggregates, spe- cies and microspecies have been described over the last 250 years. Frequently the name Salicornia europaea is used in a very broad sense to include most of the species of the genus. Additionally, the plants show a high level of phenotypic plasticity (Ingrouille and Pearson 1987). The salinity of their habitats fluctuates greatly due to dif- ferent factors - tidal cycles, evapotranspiration, precipi- tation and availability of fresh groundwater. This is the reason why Salicornia develops high physiological plas- ticity which causes phenotypic variation (Kadereit et al. 2007). Morphological distinction between the taxa is only possible when the plants are fresh, between flower- ing and fruiting (Gehu et al. 1979). Morphometric stud- ies using all phenotypic differences available, irrespec- tive of whether they have a genetic basis or not, could not reveal distinct taxa even on a small regional scale (Ingrouille and Pearson 1987). Salicornia plants tend to have phenotypic varia- tions depending on environmental conditions such as temperature, quality of soil, concentration of salt and population density. Ball and Akeroyd (1993) suggested that the specific limits of classification of the Salicornia plants based on morphological features, especially those of dried Salicornia plants, are obscure. To prove the rel- evance between the genotype and phenotype in Salicor- nia plants, genetic variability was analyzed by RAPD fingerprinting. The use of molecular markers is consid- ered to be the best for genetic diversity analysis since it has proved to be non-invasive in the sense that there are no negative effects on the stage of development, envi- ronment or management practices. Furthermore, these kinds of studies can be applied even on dead plants when the genomic DNA is extractable (Choudhury et al. 2001). Molecular markers play a significant role in the protection of biodiversity, identification of promising cultivars, quantitative trait loci (QTL) mapping, etc. Various PCR-based markers such as ISSR, SCoT, SRAP, etc. have been effectively used for the quantification of genetic diversity. Recent ISSR studies of natural popula- tions have demonstrated the hypervariable nature of the markers and their potential use for the population-level studies (Hultén and Fries 1986). In the present study, the ISSR markers and morphologic traits were used for the first time in Iran to analyze the genetic diversity in 72 Salicornia persica accessions belonging to 8 different populations. MATERIALS AND METHODS Plant materials For the morphometric and ISSR analyses, we used 72 plant accessions (four to twelve samples from each population) belonging to 8 different populations of Sali- cornia persica in Esfahan and Tehran Provinces of Iran during July-Agust 2018-2020 (Table 1). More informa- tion about the geographical distribution of the acces- sions are given in Table 1 and Fig. 1. The plant individu- als were identified morphologically using different litera- ture (Kadereit et al. 2007; Akhani 2003). DNA extraction and ISSR analysis Fresh leaves were randomly used from four to twelve samples for each of the studied populations. They dried by silica gel. The CTAB-activated charcoal protocol was used to extract genomic DNA (Esfandani-Bozchaloyi et al. 2019). For the ISSR analysis, 22 primers from the UBC (University of British Columbia) series were tested 35Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) for the DNA amplification. Ten primers were chosen for the ISSR analysis of genetic variability based on band reproducibility (Table 2). The PCR reactions were carried out in a 25μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 ng of genomic DNA, and 3 U of Taq DNA polymerase (Bioron, Germany). The amplifications’ reactions were performed in Techne thermocycler (Germany) with the following program: 5min initial denaturation step at 95°C, followed by 37 one-minute cycles at 95°C, 1 min at 50-56°C, and 1 min at 72°C. The reaction was com- pleted by the final 5-10 min extension step at 72°C. The amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. The fragment size was estimated using a 100bp molecu- lar size ladder (Fermentas, Germany). DATA ANALYSIS Morphological studies A total of 19 metric and 6 multistate characterers were used for measurements in different combinations (Table not included), modified from the character list detailed by Ingrouille and Pearson (1987). Of these 25 characters, 15 covered the overall vegetative morpholo- gy, and 10 were characteristics of the fertile spike, fertile spike segments and flowers. Though vegetative morphol- ogy may be partly uninformative due to the wide phe- notypic plasticity, both vegetative and fertile spike char- acteristics were used, because some vegetative traits have been shown useful in separating populations and taxa at least in single cases (Ingrouille and Pearson 1987). The data obtained were standardized (mean= 0, variance = 1) and used to estimate the Euclidean distance for cluster- ing and ordination analyses (Podani 2000). To group the plant specimens, The UPGMA (unweighted pair group with arithmetic mean), Ward’s (Minimum spherical traits), and MDS (multidimensional scaling) ordination methods were used (Podani 2000). PAST software ver- sion 2.17 (Hammer et al. 2012) was used for the multi- variate statistical analyses of the morphological data. Molecular analysis The ISSR profiles obtained for each sample were scored as binary traits. The discriminating capability of the used primers was evaluated by means of two param- Table 1. Voucher details and diversity within Iranian populations and subspecies of Salicornia persica in this study. No Subspecies Locality Pop1 subsp. persica Akhani Esfahan,the river of Zayanderud at Varzaneh Pop2 subsp. persica Akhani Esfahan,Nain, it is 70km to Varzaneh Pop3 subsp. persica Akhani Fars, Tashk lake Pop4 subsp. persica Akhani Esfahan, northern coasts of Batlaq-e Gavkhooni Pop5 subsp. rudshurensis Akhani Tehran ;60 km west of Tehran, 25 km SE of Karaj Pop6 subsp. rudshurensis Akhani Tehran: ca. 60 km W Tehran, Mardabad salt flats, along Rude Shur Pop7 subsp. rudshurensis Akhani Tehran, Karaj located in 25 km NW Rude Shur Pop8 subsp. rudshurensis Akhani Tehran ; Rude-Shur River, which is located 40 km west of Tehran Figure 1. Distribution map of studied populations of Salicornia per- sica in Iran. 36 Xiaoju Zhang, Li Bai eters, polymorphism information content (PIC) and marker index (MI), to characterize the capacity of each primer to detect polymorphic loci among the genotypes. The number of polymorphic bands (NPB) was calcu- lated for each primer. The parameters like Nei’s genetic diversity (H), Shannon’s information index (I), number of effective alleles, and percentage of polymorphism (P% = number of polymorphic loci/number of total loci) were determined (Weising et al. 2005; Freeland et al. 2011). Shannon’s index was calculated by the following formula: H’ = -Σpiln pi. Rp is defined per primer as: Rp = ∑ Ib, where “Ib” is the band informativeness, which takes the values of 1-(2x [0.5-p]), and “p” is the propor- tion of each genotype containing the band. The percent- age of polymorphic loci, the mean loci by accession and by population, UHe, H’ and PCA were calculated by GenAlEx 6.4 software (Peakall and Smouse 2006). Nei’s genetic distance among the populations was used for Neighbor Joining (NJ) clustering and Neighbor-Net net- working (Freeland et al. 2011). Mantel test checked the correlation between geographical and genetic distances of the studied populations (Podani 2000). The analy- ses were done by PAST ver. 2.17 (Hammer et al. 2012), DARwin ver. 5 (2012), and SplitsTree4 V4.13.1 (2013) software. AMOVA (Analysis of molecular variance) test (with 1000 permutations) implemented in GenAlex 6.4 (Peakall and Smouse 2006) and Nei’s GST analy- sis implemented in GenoDive ver.2 (2013) were used to show the genetic difference of the populations. Moreo- ver, the populations’ genetic differentiation was studied by G(ST)est = standardized measure of genetic differen- tiation (Hedrick 2005), and Dest = Jost measure of differ- entiation. To assess the population structure of the Salicornia persica accessions, a heuristic method based on the Bayes- ian clustering algorithms was utilized. The clustering method based on the Bayesian model implemented in the STRUCTURE software (Falush et al., 2007) was used on the same data set to better detect the population substruc- tures. This clustering method is based on an algorithm that assigns genotypes to homogeneous groups based on the number of clusters (K). Assuming Hardy-Weinberg and linkage equilibrium within the clusters, the software estimates allele frequencies in each cluster and population membership for each individual (Pritchard et al. 2000). The number of potential subpopulations varied from two to ten, and their contribution to the genotypes of the accessions was calculated based on 50,000 iteration burn- ins and 100,000 iteration sampling periods. The most probable number (K) of subpopulations was identified fol- lowing Evanno et al. (2005). In K-Means clustering, two summary statistics, pseudo-F and Bayesian Information Criterion (BIC), provide the best fit for k. Gene flow (Nm) was calculated using POPGENE (version 1.31) software. Gene flow was estimated indirectly using the following formula: Nm = 0.25(1 - FST)/FST. In order to test for a correlation between pair-wise genetic distances (FST) and geographical distances (in km) among the populations, Mantel test was performed using Tools for Population Genetic Analysis (TFPGA; Miller, 1997) (computing 999 permutations). This approach considers an equal amount of gene flow among all populations. Population assign- ment test based on maximum likelihood was performed in GenoDive ver. 2 (2013). The presence of shared alleles was determined by drawing the reticulogram network based on the least square method by DARwin ver 5 (2012). Table 2. Details about the banding pattern revealed by ISSR primers. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI ISSR-1 DBDACACACACACACACA 10 10 100.00% 0.28 5.11 ISSR-2 GGATGGATGGATGGAT 9 7 93.00% 0.38 6.41 ISSR-3 GACAGACAGACAGACA 24 20 87.00% 0.56 4.34 ISSR-4 AGAGAGAGAGAGAGAGYT 10 10 100.00% 0.49 3.88 ISSR-5 ACACACACACACACACC 15 15 100.00% 0.41 5.66 ISSR-6 GAGAGAGAGAGAGAGARC 11 9 94.00% 0.25 4.99 ISSR-7 CTCTCTCTCTCTCTCTG 13 7 70.00% 0.64 4.21 ISSR-8 CACACACACACACACAG 13 9 82.00% 0.32 4.32 ISSR-9 GTGTGTGTGTGTGTGTYG 12 10 93.00% 0.25 6.56 ISSR-10 CACACACACACACACARG 25 21 91.00% 0.57 4.11 Average 15.8 14.4 89.00% 0.49 5.12 Total 158 144 Note: TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: poly- morphism index, PIC, polymorphism information content for each of ISSR primers. 37Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) RESULTS Morphometry The morphological evaluation revealed considerable variations among the accessions for spike characteris- tics. Based on the botanical traits, 41 out of 72 evaluated accessions were identified as subsp. persica and 31 acces- sions as subsp. rudshurensis (Fig. 1). ANOVA showed significant differences (P <0.01) in quantitative morphological traits among the popula- tions under study. In order to determine the most vari- able traits among the taxa studied, the PCA analysis was performed. It revealed that the first three factors com- prised over 76% of the total variations. In the first PCA axis with 52% of total variations, such traits as Length of visible part of central flower of cyme, 2nd fertile seg- ment; Width of 2nd fertile segment at base showed the highest correlation (>0.7). Length of 2nd fertile segment measured over the central; Length of spike and Number of fertile segments on longest were the traits influencing the PCA axes 2 and 3, respectively. Different clustering and ordination methods produced similar results and, therefore, the PCA plot of morphological traits are pre- sented here (Fig. 2). In general, the PCA plot of the stud- ied populations did entirely delimit the studied popula- tions and revealed that the plants in these populations are not intermixed. Populations’ genetic diversity In the present study, 10 out of 22 selected ISSR primers amplified 158 clear discernible bands, of which 144 (80 %) were polymorphic, showing the high dis- criminative and resolving power of the used ISSRs in the studied germplasm. The total number of bands per primer ranged from 9 (ISSR-2) to 25 (ISSR-10), with an average of 15.8. The number of polymorphic bands per primer varied from 7 (ISSR-2, ISSR-7) to 21 (ISSR-10), with an average of 14.4. The band sizes of the amplified products were found between 100 and 3,000 bp. To char- acterize the capacity of each primer to detect polymor- phism and to evaluate the discriminating capability of each primer in the studied germplasm, various diversity indices such as the highest percentage of polymorphic bands, Ne, I and, PIC were calculated. The highest per- centage of polymorphic bands was produced by primers ISSR-1, ISSR-4 and ISSR-5 (100%), while primer ISSR-7 produced the lowest percentage of polymorphic bands (70%). The PIC values across all primers averaged 0.49. ISSR-7 showed the highest (0.64) and ISSR-6, ISSR-9 the lowest (0.25) PIC value, respectively (Table 2). The genetic diversity parameters determined in 8 geographical populations of Salicornia persica are pre- sented in Table 3. The highest value of polymorphism percentage (52.15%) was observed in Esfahan, northern coasts of Batlaq-e Gavkhooni (population No. 4, subsp. persica), which shows a high value for the genetic diver- sity (0.38) and Shannon’s information index (0.45). The population of Tehran; Rude-Shur River, which is located 40 km west of Tehran (No. 8, subsp. rudshurensis) has the lowest value for the percentage of polymorphism (15.91%) and the lowest value for Shannon’s information index (0.17) and He (0.18). Populations’ genetic differentiation AMOVA (PhiPT = 0.88, P = 0.0010) revealed signifi- cant difference among the studied populations (Table 4). It also revealed that 45% of total genetic variations was due to the diversity within the population and 55% was due to the genetic differentiation among the popula- tions. The pairwise comparison of Nei’s genetic identity among the studied populations Salicornia persica (Table Table 3. Genetic diversity parameters in the studied Salicornia persica populations. SP N Na Ne I He UHe %P Pop1 16.000 0.892 1.168 0.321 0.251 0.265 34.63% Pop2 6.000 0.344 1.035 0.31 0.23 0.25 40.53% Pop3 11.000 0.441 1.036 0.33 0.25 0.27 42.53% Pop4 8.000 0.247 1.021 0.45 0.38 0.33 52.15% Pop5 5.000 0.290 1.024 0.23 0.25 0.18 24.30% Pop6 10.000 0.452 1.089 0.29 0.22 0.25 45.05% Pop7 10.000 0.333 1.006 0.222 0.22 0.22 33.23% Pop8 9.000 1.247 1.275 0.171 0.184 0.142 15.91% Abbreviations: N = number of samples, Na= number of different alleles; Ne = number of effective alleles, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism, populations. 38 Xiaoju Zhang, Li Bai 5) showed a higher genetic similarity (0.93) between the populations of Esfahan, Nain, it is 70km to Varzaneh (pop. No. 2) and Fars, Tashk lake (pop. No. 3), while the lowest genetic similarity value (0.712) occurred between Esfahan, the river of Zayanderud at Varzaneh (pop. No. 1) and Tehran, Karaj located in 25 km NW Rude Shur (pop. No. 7). Table 4. Analysis of molecular variance (AMOVA) of the studied Salicornia persica. Source df SS MS Est. Var. % ΦPT Among Pops 56 1221.364 52.789 21.154 55% 55% Within Pops 120 114.443 12.905 12.905 45% Total 176 1365.807 33.060 100% df: degree of freedom; SS: sum of squared observations; MS: mean of squared observations; EV: estimated variance; ΦPT: proportion of the total genetic variance among individuals within an accession, (P < 0.001). Table 5. Pairwise Population Matrix of Nei Unbiased Genetic Identity. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 1.000 pop1 0.833 1.000 pop2 0.810 0.933 1.000 pop3 0.875 0.873 0.830 1.000 pop4 0.818 0.896 0.874 0.812 1.000 pop5 0.852 0.858 0.844 0.838 0.884 1.000 pop6 0.712 0.846 0.800 0.796 0.881 0.794 1.000 pop7 0.779 0.855 0.809 0.794 0.874 0.752 0.862 1.000 pop8 Figure 2. PCA plot of Salicornia persica populations based on morphological traits. 39Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) Populations’ genetic affinity Two major clusters were formed in the UPGMA tree (Fig. 3). The first major cluster contained two sub- clusters: the population of Esfahan, Nain, it is 70km to Varzaneh; Fars, Tashk lake and Esfahan, northern coasts of Batlaq-e Gavkhooni (pop. No. 2,3,4, subsp. persica) is distinct and remains separate from the other popula- tions with a great distance and comprises the first sub- cluster. The second sub-cluster was formed by the other populations from subsp. rudshurensis, (pop. No. 5-8) which showed close genetic affinity. The second major cluster contained only subsp. persica, which separated from the other studied populations and joined the oth- ers with a great distance. These results show that the plant specimens of each studied subspecies were grouped together, indicating that the subspecies are delimited based on the ISSR molecular markers. Therefore, this result confirms our morphology results. The Nm analy- sis by Popgene software also produced mean Nm= 0.17, which is considered a very low value of gene flow among the studied species. Mantel test after 5000 permutations produced significant correlation between genetic dis- tance and geographical distance in these populations (r = 0.33, P = 0.001). Populations’ genetic structure K = 2 reveal the presence of 2 genetic groups. Simi- lar results were obtained by Evanno test performed on STRUCTURE analysis, which produced a major peak at k = 2. Both analyses revealed that Salicornia persica populations show genetic stratification. The STRUC- TURE plot based on k = 2 revealed the genetic differ- ence in population of Esfahan and Fars province (pop. No. 1-4; subsp. persica) (red colored) with other popula- tions. However, it showed genetic affinity between pop- ulations 5-8 of Tehran province (subsp. rudshurensis) (green colored) Figure 3. UPGMA plot of populations in Salicornia persica populations based on ISSR data (Population numbers according to Table 1). 40 Xiaoju Zhang, Li Bai The mean Nm = 0.17 was obtained for all ISSR loci, which indicates the low amount of gene flow among the populations, and supports the genetic stratification as indicated by K-Means and STRUCTURE analyses. How- ever, the reticulogram obtained based on the least square method (Figure 4) revealed some amount of shared alleles among populations 1 and 2, and populations 3 and 8. This result agrees with the grouping obtained with UPGMA tree, as the populations were placed close to each other. As evidenced by STRUCTURE plot based on the admixture model, the shared alleles comprise a very limited part of the genomes in the populations, and all the results consistently show a high degree of genetic stratification within Salicornia persica populations. DISCUSSION The present study revealed interesting data about the genetic variability, genetic stratification, and morpholog- ical divergence in the central parts of Iran. The genetic diversity is of fundamental importance in the continu- ity of a species, as it is used to bring about the necessary adaptation to cope with the changes in the environment (Wang et al. 2021; Yin et al. 2021; Zhao et al. 2021). The degree of genetic variability within a species highly cor- relates with its reproduction mode. The higher degree of open pollination/cross breeding brings about a higher level of genetic variability in the studied taxon (Jia et al. 2020; Shi et al. 2021; Zheng et al. 2021; Zhu et al. 2021). The PIC and MI characteristics of a primer help to Figure 4. Reticulogram of Salicornia persica populations based on least squares method analysis of ISSR data (Population numbers accord- ing to Table 1). 41Population Differentiation and Gene Flow of Salicornia persica Akhani (Chenopodiaceae) determine its effectiveness in the genetic diversity anal- ysis. Sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic diversity may be more directly related to the degree of polymorphism. Generally, the PIC value between 0 and 0.25 imply a very low genetic diversity among genotypes, the value between 0.25 and 0.50 shows a mid-level genetic diver- sity, and the value ≥0.50 suggests a high level of genetic diversity. In this research, the ISSR primers’ PIC values ranged from 0.25 to 0.64, with a mean value of 0.49, which indicated the high capability of ISSR primers for determining the genetic diversity among the Salicornia persica accessions. Papini et al. (2004) found that diploid and tetra- ploid accessions of Salicornia resolved as sister clades. The study was based on ITS sequences of twelve sam- ples of Salicornia (all but one from Italy) representing four species (three tetraploid, one diploid). The presence of RAPD polymorphic bands in the populations stud- ied indicates the presence of genetic polymorphism in these populations. Moreover, the occurrence of specific bands/loci only in some of the populations illustrates the occurrence of unique insertion/deletion in DNA mate- rial of these genotypes. As the range of species in Salicornia is imperfectly known, it is rather premature to evaluate their plant geographical importance. However, based on present data two species groups can be distinguished: The first are Central and South-Central Iranian species includ- ing S. persica subsp. persica, S. persica subsp. rudshuren- sis, S. perspolitana, S. iranica and S. x tashkensis and the second group consisted only of S. sinus-persica which is endemic around the Persian Gulf. The Central and South-Central Iran possess several endemic species of desert and arid flora with remarkable phytogeographic importance. Furthermore the area is part of the Zagros Mountains which is known as a very important plant diversity center in SW Asia (Akhani, 2008). Sagane et al. (2003) conducted a study in Japan on identification of Salicornia population through morphological and RAPD fingerprinting. They observed variations in plant length, segment number, length and number of branches, and incidence of the secondary branches etc. on the basis of genotype based on the RAPD marker they identified five groups in three selected populations. The genetic diversity of 102 individuals of S. per- sica (15 populations) were studied using 10 Start Codon Targeted (SCoT) markers (Liu & Esfandani-Bozchaloyi 2022). Their result showed high polymorphic bands (94.18%), polymorphic information content (0.27), and allele number (1.38) showed SCoT as a reliable marker system for genetic analysis of this species. According to Chatrenoor & Akhani (2021) an integrated morpho‐ molecular study of Salicornia (Amaranthaceae‐Che- nopodiaceae) in Iran proves Irano‐Turanian region the major center of diversity of annual glasswort spe- cies. Their results (1) confirm the efficiency of plastid sequences comparing to ETS sequences for clarif y- ing species-level phylogeny of Salicornia; (2) identify the S.  persica clade as a monophyletic Irano-Turanian endemic lineage; (3) recognize nine origins of the Irano- Turanian Salicornia based on nuclear ETS sequences; (4) approve the monophyly of tetraploid species using plas- tid sequences. Gohil and Pandya (2006) conducted study to find out the degree and the nature of genetic diver- gence among Salicornia brachiata (Roxb.) genotypes. Gohil and Pandya (2006) found a significant difference amongst the salicornia genotypes for all the phenological characters, (like height, canopy, main branch, segment, spike length, spike/branch and seed yield) indicating high genetic variability present in the population. The genotypes under study were grouped into five clusters, indicating wide diversity in the material for majority of the characters. Previous results from molecular stud- ies imply near 100% inbreeding in Salicornia, which certainly contributes greatly to the taxonomic difficul- ties in the group because of inbreeding lines with min- ute but fixed phenotypic differences (Noble et al., 1992). The other study using RAPD technique showed correla- tions between DNA polymorphism and geographical distribution in S. ramosissima. According to Kadereit et al. (2007), the main reason for the taxonomic confu- sion are the young age of the extant lineages, the ram- pant dispersal of Salicornia which has led to widespread genotypes with high phenotypic plasticity. This is the reason why Salicornia plants have different names in dif- ferent regions, and morphological parallelism resulted in the fact that different genotypes have the same name in one region. Anita K. Badlani, (2011) was undertak- en to assess the genetic diversity among germplasm of Salicornia collected from 11 different locations using RAPD and ISSR marker system. This study will pro- vide the genetic back ground of S. brachiata populations and extent of molecular diversity existing among them. The characterized diversity and identified polymorphic markers can be a good source of plant genetic resources and can be further exploited for genetic improvement of the species through marker assisted breeding. In conclusion, the results of this study showed that to evaluate the genetic diversity of Salicornia persica, the primers derived from ISSR were more effective than the other molecular markers. 42 Xiaoju Zhang, Li Bai REFERENCES Akhani, H. 2003. Salicornia persica Akhani (Chenopodi- aceae), a remarkable new species from Central Iran. Linz. Biol. Beit. 35: 607-612. Akhani, H., 2008. Taxonomic revision of the genus Sali- cornia L. (Chenopodiaceae) in Central and Southern Iran. Pak. 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Genetic Diversity And Relationships Among Salvia Species By Issr Markers; Genetika-Belgrade, 53(2): 559-574. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 2 - 2022 Firenze University Press Cytogenetic Studies of Six Species in Family Araceae from Thailand Piyaporn Saensouk1, Surapon Saensouk2,*, Rattanavalee Senavongse2 Effect of Ag Nanoparticles on Morphological and Physio-biochemical Traits of the Medicinal Plant Stevia Rebaudiana Sherzad R. Abdull, Sahar H. Rashid*, Bakhtiar S. Ghafoor, Barzan S. 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