Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 59-69, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1493 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Zhu Lin, Hamed Khodayari (2022) Genetic Characterization of Salicor- nia persica Akhani (Chenopodiaceae) Assessed Using Random Amplified Polymorphic DNA. Caryologia 75(2): 59-69. doi: 10.36253/caryologia-1493 Received: November 19, 2021 Accepted: July 06, 2022 Published: September 21, 2022 Copyright: © 2022 Zhu Lin, Hamed Khodayari. 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 Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, 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. Genetic Characterization of Salicornia persica Akhani (Chenopodiaceae) Assessed Using Random Amplified Polymorphic DNA Zhu Lin1,*, Hamed Khodayari2 1 Sias University, Xin Zheng City, Henan Province, 451150, China 2 Department of Biology, Faculty of Science, Lorestan University, 5Km Khorramabad toward Tehran, Khorramabad, Iran *Corresponding author. E-mail: masxiongg@163.com Abstract. Salicornia is a wild and annual (biennial, in some species) halophytic plant from Chenopodiaceae family that grows near salt marshes and salted wetlands or the vicinity of coastal areas in Asia, North America, and the Middle East. It is also grown in semi-arid areas of Iran such as Isfahan, Fars, and Yazd provinces. Genetic variabil- ity and populations, structure were studied in 10 geographical populations of Salicor- nia persica. Genetic diversity parameters were determined in these populations. 10 of 20 random amplified polymorphic DNA (RAPD) primers produced 146 reproducible bands with average of 14.6 bands per primer and 88.67% of polymorphism. OPA10 primer showed the highest number of effective allele (Ne), Shannon index (I) and genetic diversity (H). The highest values of genetic diversity in RAPD markers were obtained in Esfahan, Nain, it is 70km to Varzaneh populations. WARD trees of RAPD data grouped the populations in two different clusters/groups, indicating their genetic difference which is discussed in details. The results of this study showed that the level of genetic variation in Salicornia persica is relatively low. WARD-based dendrogram showed a close relationship between members of Esfahan, Varzaneh and Esfahan, the river of Zayanderud at Varzaneh while the Yazd, Meybud and Yazd, Nádüshan protect- ed population differ the most from the other populations. Principal component analy- sis, however, showed some minor differences with WARD-based dendrograms. Keywords: gene flow, Salicornia persica, Random Amplified Polymorphic DNA (RAPD). INTRODUCTION Genetic diversity is one aspect of biological diversity that is extreme- ly important for conservation strategies, especially in rare and narrowly endemic species (Wang et al., 2021; Yin et al., 2021; Zhao et al., 2021). Most of the authors agree that genetic diversity is necessary to preserve the long- term evolutionary potential of a species (jia et al. 2020; Shi et al., 2021; Zheng et al., 2021; Zhu et al., 2021). Salicornia is a wild and annual (biennial, in some species) halophytic plant from Chenopodiaceae family that grows near salt marshes and salted 60 Zhu Lin, Hamed Khodayari wetlands or the vicinity of coastal areas in Asia, North America, and the Middle East. It is also grown in semi- arid areas of Iran such as Isfahan, Fars, and Yazd prov- inces (Akhani, 2003). Salicornia varieties are often mul- tipurpose (Al-Oudat and Qadir, 2011; si et al, 2020) and it is very rich in vitamins, minerals and highly unsatu- rated oils. Salicornioideae species are also rich in dietary fiber and many bioactive substances, such as phytoster- ols, polysaccharides, and phenolic compounds mainly flavonoids and phenolic acids (Choi et al., 2014). Sali- cornia, commonly called Saloni is a member of Cheno- podiaceae (Amaranthaceae). A family that constitute among it vegetables like spinach and beets etc. Salicornia is a halophyte which tolerates extreme saline conditions and grows along the coastlines and feeds on salt water. It is a leaf less annual succulent salt marsh halophyte considered to be a potential alternative crop of seawa- ter agriculture, due to its economic potential (Glenn et al. 1991). The Salicornia species are small, usually less than 30 cm tall, succulent herbs with a jointed horizon- tal main stem and erect lateral branches. The leaves are small and scale-like and as such the plant may appear leafless. Many species are green, but their foliage turns red in autumn. The hermaphrodite flowers are wind pollinated, and the fruit is small and succulent and contains a single seed. Salicornia species can generally tolerate immersion in salt water. Salicornia has leafless stems with branches that resembles asparagus, hence the plants other common name sea asparagus. Common names for the genus include glasswort, pickleweed, and marsh samphire. According to a molecular phylogenetic study by Kadereit et al. (2006), Salicornia is monophyl- etic and nested within the morphologically and ecologi- cally closely related Sarcocornia. Salicornia and Sarco- cornia differ from all other Salicornioideae by seeds that lack perisperm (Shepherd et al., 2005). Salicornia split from Sarcocornia during the Middle Miocene (14.2– 9.4 mya), but its extant lineages started to diversify only in the Early Pleistocene (1.4–1.8 mya; Kadereit et al., 2006; Liu et al., 2021). According to Zhang and Bai (2022) A total of 72 randomly collected plants from 8 natural populations Salicornia persica Akhani in 2 provinces were evaluated using ISSR markers and morphological traits. Salicornia persica is frequent along several salt marshes, river salt margins, and salty river estuar- ies in the Provinces Esfahan, Fars and Yazd. However, populations in these areas are threatened because many important wetlands (e.g., Gavkhooni salt swamp) and rivers (e.g., Zayandeh Rud) are drying out. observations around Tashk lake show that the species is grazed by goats. The species is characterized by having ascending habit, verticillate inflorescence branches, and reversed pentagonal central flowers that are truncated at the apex and reach to the upper segments. The molecular markers are extensively used in germplasm characteri- zation, fingerprinting, genetic analysis, linkage map- ping, and molecular breeding. RAPD (Random Ampli- fied Polymorphic DNA) analysis using PCR in associa- tion with short primers of arbitrary sequence has been demonstrated to be sensitive in detecting variation among individuals. The advantages of this technique are: a) a large number of samples can be quickly and economically analyzed using only micro-quantities of material; b) the DNA amplicons are independent from the ontogenetic expression; and c) many genom- ic regions can be sampled with a potentially unlim- ited number of markers (Peng et al., 2021; Ma et al., 2021 Esfandani- Bozchaloyi et al., 2017a, b, c, d). We have no information on genetic variability, gene flow and genetic structure of Salicornia persica populations. Moreover, due to extensive morphological variability of this species in the country, there is possibility of having infra-specific taxonomic forms in this species. There- fore, we carried out population genetic analysis and morphometric study of 10 geographical populations for the first time in the country. MATERIALS AND METHODS Plant materials A total of 60 individuals were sampled represent- ing 10 natural populations of Salicornia persica in Esfa- han, Fars and Yazd Provinces of Iran during May-Agust 2016-2020 (Table 1, Fig. 1). According to previous refer- ences, all the population were identified (Kadereit et al., 2006; Akhani 2003). Morphological studies A total of 19 metric and 6 multistate characterers were used for measurements in different combinations (Table 2), modified from the character list detailed by Ingrouille and Pearson (1987). Of these 25 characters, 15 covered the overall vegetative morphology, and 10 were characteristics of the fertile spike, fertile spike segments and f lowers. Though vegetative morphology may be partly uninformative due to the wide phenotypic plastic- ity, both vegetative and fertile spike characteristics were used, because some vegetative traits have been shown useful in separating populations and taxa at least in sin- gle cases (Ingrouille and Pearson 1987, ). 61Genetic Characterization of Salicornia persica Assessed Using Random Amplified Polymorphic DNA DNA extraction and RAPD assay Fresh leaves were used randomly from 5-10 plants in each of the studied populations. These were dried by silica gel powder. CTAB activated charcoal protocol was used to extract genomic DNA (Esfandani- Bozch- aloyi et al., 2019). The quality of extracted DNA was examined by running on 0.8% agarose gel. 22 decamer RAPD primers of Operon technology (Alameda, Cana- da) belonging to OPA, OPB, OPC, OPH, OPI, OPM sets were used in this study. PCR reactions were carried 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 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 94°C, followed by 40 cycles of 1min at 94°C; 1 min at 52-57°C and 2 min at 72°C. The reaction was completed by final exten- sion step of 7-10 min 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 by using a 100 bp molecular size ladder (Fermentas, Germany). DATA ANALYSES Morphological studies Morphological characters were first standardized (Mean = 0, Variance = 1) and used to establish Euclid- ean distance among pairs of taxa (PODANI, 2000). For grouping of the plant specimens, The UPGMA (Unweighted paired group using average) and Ward (Minimum spherical characters) as well as ordination methods of MDS (Multidimensional scaling) and PCoA Table 1. Location and herbarium accession numbers of the studied populations of Salicornia persica in Iran. Pop. no Locality 1 Esfahan, Varzaneh 2 Esfahan, the river of Zayanderud at Varzaneh 3 Esfahan, Nain, it is 70km to Varzaneh 4 Esfahan; Varzaneh to Kashan 5 Fars, Tashk lake 6 Fars, Lake Bakhtegan 7 Fars, Abadeh Tashk 8 Yazd, Kavire Marvast 9 Yazd, Meybud 10 Yazd, Nádüshan Table 2. Evaluated morphological characters for populations of Sali- cornia persica 1. Height of plant from rooting point to apex, mm 2. Number of sterile internodes below the terminal spike 3. Number of 1st order branches 4. Number of 2nd order branches 5. Number of 3rd order branches 6. Number of 4th order branches 7. Length of longest basal 1st order branch, mm 8. Number of sterile internodes on longest basal 1st order branch 9. Number of fertile segments on main axis of longest basal 1st order branch 10. Branching angle of longest basal 1st order branch, 11. Branching habit of longest basal 1st order branch, 12. Length of longest (regular) ultimate 1st order branch, mm 13. Number of fertile segments on longest (regular) ultimate 1st order branch 14. Branching angle of longest (regular) ultimate 1st order 15.Branching habit of longest (regular) ultimate 1st order 16. Length of spike, mm 17. Number of fertile segments 18. Length of 2nd fertile segment measured over the central flower of cyme, mm 19. Height of triangular apex of 2nd fertile segment, mm 20. Width of 2nd fertile segment at base, mm 21. Maximum width of 2nd fertile segment, mm 22. Place of the widest point of 2nd fertile segment, 23. Length of visible part of central flower of cyme, 2nd fertile segment, mm 24. Width of central flower of cyme, 2nd fertile segment, mm 25. Place of the widest point of central flower of 2nd Figure 1. Map of distribtion of populations Salicornia persica. 62 Zhu Lin, Hamed Khodayari (Principal coordinate analysis) were used (Podani, 2000). ANOVA (Analysis of variance) were performed to show morphological difference among the populations while, PCA (Principal components analysis) biplot was used to identify the most variable morphological characters among the studied populations (Podani 2000). PAST version 2.17 (Hammer et al., 2012) was used for multi- variate statistical analyses of morphological data. Random Amplified Polymorphic DNA (RAPD) analysis RAPD bands obtained were treated as binary char- acters and coded accordingly (presence =1, absence = 0). Parameter like Nei’s gene diversity (H), Shannon infor- mation index (I), number of effective alleles, and per- centage 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 cal- culated by the formula: H’ = -Σpiln pi. Rp is defined per primer as: Rp = ∑ Ib, were “Ib” is the band informative- ness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. The percentage of polymorphic loci, the mean loci by acces- sion and by population, UHe, H’ and PCA were calculat- ed by GenAlEx 6.4 software (Peakall and Smouse 2006). Nei’s genetic distance among 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). These analy- ses were done by PAST ver. 2.17 (Hammer et al. 2012), DARwin ver. 5 (2012) software. AMOVA (Analysis of molecular variance) test (with 1000 permutations) as implemented in GenAlex 6.4 (Peakall and Smouse 2006) were used to show genetic difference of the populations. The genetic structure of populations was studied by Bayesian based model STRUCTURE analysis (Pritchard et al. 2000; Chen et al. 2021 ; bi et al. 2021 ), and maxi- mum likelihood-based method of K-Means clustering of GenoDive ver. 2. (2013). For STRUCTURE analysis, data were scored as dominant markers . The Evanno test was performed on STRUCTURE result to determine prop- er number of K by using delta K value (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 was determined by (i) Calculating Nm an estimate of gene flow from Gst by PopGene ver. 1.32 (1997) as: Nm = 0.5(1 - Gst)/Gst. This approach consid- ers equal amount of gene flow among all populations. (ii) Population assignment test based on maximum like- lihood as performed in Genodive ver. 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). RESULTS Morphometric analyses In present study 60 plant samples were collected from 10 geographical populations. ANOVA test revealed significant difference in quantitative morphological characters among the studied populations (P < 0.05). Clustering and PCA plot of Salicornia persica popula- tions based on morphological characters produced simi- lar results therefore only PCA plot is presented and dis- cussed (Fig. 2). The result showed morphological differ- ence/ divergence among most of the studied populations. This morphological difference was due to quantitative characters only. For example, character (Length of long- est basal 1st order branch), separated population No. 1-4, character (Length of longest (regular) ultimate 1st order branch) separated population No. 8, while character Length of 2nd fertile segment measured over the central flower of cyme, separated populations 5 and 6,7 from the other populations. RAPD analysis Out of 20 RAPD primers used, 10 primers produced reproducible bands. In total, 146 RAPD bands (loci) were obtained with average of 14.6 bands per primer and only 10 bands ranging in size from 100– 2000 bp were common in all 10 populations studied and 129 bands were polymorph. T he mea n percentage of poly mor phism was 88.67%. R APD primers including OPB-05, OPA-13, produced the highest polymorphism (85-100%), while OPA-10, OPC-01 produced the lowest percentage of polymorphism (69-77%). The mean value of effective alleles, Shannon index and genetic diversity of RAPD loci studied were 1.06, 0.20 and 0.25, respectively, while OPA-10 primers showed the highest number of effec- tive alleles (1.098), Shannon index (0.377) and genetic diversity (0.34) (Table 3). Among the primers used OPA-10 produced the highest number of bands (24), while primers OPA-04 produced the lowest number of bands (7). The Primer OPA-04 also produced the highest number of polymor- phic bands (22). The primer OPC-01 produced 8 specific bands, primer OPH-07 produced 6 specific bands, while some 63Genetic Characterization of Salicornia persica Assessed Using Random Amplified Polymorphic DNA other primers produced 4 and 3 specific bands and the primers OPA-04, OPM-10 and OPA-10 produced 1 spe- cific band. Some of the populations showed the presence of specific bands, for example only Esfahan, the river of Zayanderud at Varzaneh population had band 1100 bp of RAPD primer OPA-10, band 900 bp of the primer OPM-10 and band 730 bp of the primer OPA-04. The Fars, Lake Bakhtegan population showed specific bands of 420 bp of the primer OPB-05, while Esfahan, Nain, it is 70km to Varzaneh population was the only popu- lation having band 150 bp of the primer OPA-09, band 635 bp of the primer OPC-01 and 220 bp of OPH-07. Yazd, Kavire Marvast population had the band of 370 bp of the primer OPA-13. Some bands were present in all except one population, for example bands 930 bp of the Figure 2. PCA plot of morphological data in Salicornia persica populations studied; 1. Esfahan, Varzaneh ; 2. Esfahan, the river of Zay- anderud at Varzaneh; 3. Esfahan, Nain, it is 70km to Varzaneh; 4. Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Abadeh Tashk.8. Yazd, Kavire Marvast .9. Yazd, Meybud. 10. Yazd, Nádüshan. Table 3. Genetic diversity parameters in the studied populations of Salicornia persica (N = number of samples, Ne = number of effective alleles, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism, popula- tions). RAPD Loci Total band Na Ne I He UHe %P OPB05 19 0.201 1.00 0.29 0.21 0.22 42.23% OPA04 7 0.341 1.058 0.24 0.20 0.20 53.75% OPA10 24 0.455 1.098 0.377 0.34 0.32 55.05% OPM10 10 0.499 1.067 0.24 0.23 0.24 49.26% OPH07 11 0.555 1.020 0.22 0.24 0.28 43.53% OPA13 15 0.431 1.088 0.29 0.25 0.25 41.53% OPA05 20 0.255 1.021 0.23 0.28 0.22 47.15% OPC01 11 0.724 1.067 0.143 0.095 0.180 38.74% OPI12 16 1.115 1.003 0.257 0.251 0.280 52.57% OPA09 12 1.215 1.075 0.157 0.271 0.270 40.57% Mean 14.6 0.77 1.05 0.28 0.25 0.25 49.13% 64 Zhu Lin, Hamed Khodayari primer OPA-05 and 870 bp of OPM-10 were absent only in Yazd, Kavire Marvast population. Population genetic differentiation AMOVA (PhiPT = 0.66, P = 0.010), and Gst analysis (0.279, p = 0.001) revealed significant difference among the studied populations (Table 4). It also revealed that, 41% of total genetic variability was due to within pop- ulation diversity and 59% was due to among popula- tion genetic differentiation. Pairwise AMOVA produced significant difference among the studied populations. Moreover, we got high values for Hedrick standardized fixation index after 999 permutation (G’st = 0.279, P = 0.001) and Jost, differentiation index (D-est = 0.777, P = 0.001). These results indicate that the geographical popu- lations of Salicornia persica are genetically differentiated from each other. Populations, genetic affinity UPGMA tree and Neighbor-Net network produced similar results therefore only Neighbor-Net network is presented and discussed (Fig. 3). We have almost com- plete separation of the studied population in the net- work, supporting AMOVA result. The populations 3 and 4 are distinct and stand separate from the other popu- lations with great distance. The populations 1 and 2, as well as populations 6 and 7 show closer genetic affinity and are placed close to each other. Nei’s genetic identity and genetic distance for RAPD data determined among the Salicornia persica are giv- en in Table 5. The value of genetic identity varied from 0.552 between Esfahan, Varzaneh and Yazd, Meybud to 0.918 between Esfahan, Nain, it is 70km to Varzaneh and Esfahan; Varzaneh to Kashan. The mean value of genetic identity for RAPD markers was 0.75. WARD and NJ dendrograms of RAPD data pro- duced similar results supported by PCA ordination plot (Fig. 2). The Cophenetic correlation of WARD tree was higher (r = 0.95) and therefore, it is discussed below. In general, two major clusters were obtained. The popula- tions of Esfahan, Varzaneh ; 2. Esfahan, the river of Zayanderud at Varzaneh; 3. Esfahan, Nain, it is 70km Table 4. Analysis of molecular variance (AMOVA) of the studied species. Source df SS MS Est. Var. % ΦPT Among Pops 18 226.576 22.327 19.082 59% 59% Within Pops 12 104.767 18.530 21.530 41% Total 30 330.342 40.613 100% Figure 3. Neighbor-Net of RAPD data in Salicornia persica popula- tions studied. 1. Esfahan, Varzaneh ; 2. Esfahan, the river of Zay- anderud at Varzaneh; 3. Esfahan, Nain, it is 70km to Varzaneh; 4. Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Abadeh Tashk.8. Yazd, Kavire Marvast .9. Yazd, Meybud. 10. Yazd, Nádüshan. Table 5. Nei’s genetic identity (above diagonal) for RAPD data among Salicornia persica. pop1 pop2 pop3 pop4 pop5 pop6 pop7 pop8 pop9 pop10 1.000 pop1 0.882 1.000 pop2 0.838 0.755 1.000 pop3 0.883 0.683 0.918 1.000 pop4 0.594 0.591 0.714 0.741 1.000 pop5 0.719 0.591 0.808 0.871 0.750 1.000 pop6 0.652 0.737 0.764 0.735 0.609 0.794 1.000 pop7 0.706 0.573 0.837 0.744 0.621 0.784 0.724 1.000 pop8 0.552 0.737 0.764 0.735 0.609 0.729 0.875 0.868 1.000 pop9 0.534 0.581 0.714 0.641 0.621 0.639 0.661 0.635 0.829 1.000 pop10 65Genetic Characterization of Salicornia persica Assessed Using Random Amplified Polymorphic DNA to Varzaneh; 4. Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Abadeh Tashk.8. Yazd, Kavire Marvast protected formed the first major cluster, out of which, Esfahan, Varzaneh and Esfa- han, the river of Zayanderud at Varzaneh show more RAPD similarity and the Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Aba- deh Tashk.8. Yazd, Kavire Marvast protected population differ the most from the other populations. Two other populations form the second major clus- ter, which, Yazd, Meybud and Yazd, Nádüshan showed close genetic affinity. Mantel test after 5000 permuta- tions produced significant correlation between genetic distance and geographical distance in these populations (r = 0.55, P = 0.001). Therefore, the populations that are geographically more distant have less amount of gene flow, and we have isolation by distance (IBD) in the Sali- cornia persica. Populations genetic structure K = 2 reveal the presence of 2 genetic group. Simi- lar result was obtained by Evanno test performed on STRUCTURE analysis which produced a major peak at k = 2 (Fig. 5). Both these analyses revealed that Salicor- nia persica populations show genetic stratification. STRUCTURE plot based on k = 2, revealed genetic affinity between populations 1: Esfahan, Varzaneh; 2. Esfahan, the river of Zayanderud at Varzaneh; 3. Esfa- han, Nain, it is 70km to Varzaneh; (red colored), also population 9: Yazd, Meybud. 10. Yazd, Nádüshan (green colored), as well as populations 5 and 7 (green colored). The mean Nm = 0.33 was obtained for all RAPD loci, which indicates low amount of gene flow among the pop- ulations and supports genetic stratification as indicated by K-Means and STRUCTURE analyses. Population assignment test also agreed with Nm result and could not identify significant gene flow among these popula- tions. However, reticulogram obtained based on the least square method (Figure not included), revealed some amount of shared alleles among populations 7 and 8, and between 10 and 6 and 7, also between 8, and 9. This result is in agreement with grouping we obtained with PCA plot, as these populations were placed close to each other. As evidenced by STRUCTURE plot based on admixture model, these shared alleles comprise very limited part of the genomes in these populations and all these results are in agreement in showing high degree of genetic stratifica- tion within Salicornia persica populations. DISCUSSION Nature is having a hard time where human activi- ties, global environmental changes, habitat loss and spe- cies extinction often lead to a loss of biodiversity. For example, habitat fragmentation and population decline could reduce the effective population size and threaten the viability of the target species (Falk and Holsinger, 1991; Paul et al. 2021; Salami et al. 2021; Wasana et al. 2021). Many biologists argue that establish correct con- servation strategies minimizing biodiversity lossand a good example is conserve geographically-rare spe- cies (Esfandani-Bozchaloyi et al., 2018a, 2018b, 2918c, 2018d). Programs to conserve rare and endemic plants should take into account the use of molecular mark- ers because can contribute to the setting of conserva- tion priorities (Frankham et al., 2004). Unfortunately, limited information is available regarding the popula- tion genetics of rare, endemic, threatened or endangered species. Endemic (and rare) plants with narrow distri- Figure 5. STRUCTURE plot of RAPD data in Salicornia persica populations studied. 1. Esfahan, Varzaneh ; 2. Esfahan, the river of Zayanderud at Varzaneh; 3. Esfahan, Nain, it is 70km to Varzaneh; 4. Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Abadeh Tashk.8. Yazd, Kavire Marvast .9. Yazd, Meybud. 10. Yazd, Nádüshan. Figure 4. NJ tree of RAPD data in Salicornia persica populations studied. 1. Esfahan, Varzaneh ; 2. Esfahan, the river of Zayanderud at Varzaneh; 3. Esfahan, Nain, it is 70km to Varzaneh; 4. Esfahan; Varzaneh to Kashan, 5. Fars, Tashk lake 6. Fars, Lake Bakhtegan; 7. Fars, Abadeh Tashk.8. Yazd, Kavire Marvast .9. Yazd, Meybud. 10. Yazd, Nádüshan. 66 Zhu Lin, Hamed Khodayari bution range have been analyzed traditionally within the framework of the theoretical predictions of small populations. In these taxa, the lowest population genet- ic diversity levels are expected, and many study cases confirm such predictions (Cole, 2003). Our study of the genetic structure of Salicornia persica populations has important implications for the conservation and man- agement of this narrowly distributed. Molecular analyses 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. Taxonomic studies of the genus by Moss (1954), Duval-Jouve (1868), Scott (1977), Ball (1964) and oth- ers Ball and Tutin (1959), Ball and Brown (1970) and the recent revision of Eurasian Salicornias by Kadereit and her school (Kadereit et al.,2012), are notable. There are three regional studies of Salicornia that were based on molecular data (Papini et al., 2004; Murakeözy et al., 2007;). 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 material of these genotypes. As stated before, there were bands which occurred in some of the populations but were absent in the others. Since, even single base change at the primer annealing site is manifested as appear- ance or disappearance of RAPD and ISSR bands, these bands may indicate the occurrence of genetic changes in the genome of the populations through the loss or rearrangement of some of their nucleotides (Smith et al. 1996; Dadzie et al. 2021; Fikirie et al. 2020; Hindersah & Kalay 2021; Hindersah et al. 2021; Mieso & Befa 2020). Both RAPD and morphological trees obtained are in general agreement separating the populations studied in two groups, indicating their genetic difference, which is also partly supported by ISSR analysis. Diversity Study in Salicornia species In field growing plants morphological variations can be observed in growth pattern, plant canopy and num- ber of other features. Genetic diversity of the parental lines is good indicator of progeny performance. Success through hybridization and subsequent selection depends primarily on the selection of parents having high genet- ic variability for various agronomic traits. Sanane 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. Gohil and Pandya (2006) conducted study to find out the degree and the nature of genetic divergence among Salicornia brachia- ta (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 mate- rial for majority of the characters. Zhang and Bai (2022) studied in Iran on population differentiation and gene flow of Salicornia persica using ISSR markers and mor- phological traits. Analysis of molecular variance (AMO- VA) test revealed significant 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 differ- entiation among populations. Previous results from molecular studies imply near 100% inbreeding in Salicornia, which certainly contrib- utes greatly to the taxonomic difficulties in the group because of inbreeding lines with minute but fixed phe- notypic differences (Noble et al., 1992). DNA polymor- phism was detected among the three Spanish popula- tions of Salicornia using Random Amplification of Pol- ymorphic DNA (RAPD) approach (Luque et al., 1995). The other study using RAPD technique showed cor- relations between DNA polymorphism and geographi- cal distribution in S. ramosissima (Kruger et al., 2002). According to Kadereit et al. (2007), the main reason for the taxonomic confusion are the young age of the extant lineages, the rampant dispersal of Salicornia which has led to widespread genotypes with high phenotypic plas- ticity. This is the reason why Salicornia plants have dif- ferent names in different regions, and morphological parallelism resulted in the fact that different genotypes have the same name in one region. Anita K. Badlani, (2011) was undertaken to assess the genetic diversity among germplasm of Salicornia collected from 11 dif- ferent locations using RAPD and ISSR marker system. 67Genetic Characterization of Salicornia persica Assessed Using Random Amplified Polymorphic DNA This study will provide 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. CONCLUSIONS This study proved the potential of RAPD marker analyses in the identification and evaluation of genetic relationships between populations of Salicornia persica. This is the first report about the application of these techniques to estimate the genetic variation and genetic characterization of Salicornia persica accessions. 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