EVALUATION OF GENETIC DIVERSITY AND MORPHOLOGICAL VARIABILITY IN STELLARIA MEDIA (L.) VILL. USING RAPD MARKER

Stellaria media (L.) Vill., known under the name of chickweed, is an annual medicinal plant in the family Caryophyllaceae. This species is distributed in the north regions of Iran. In the present study, the Random Amplified Polymorphic DNA (RAPD) technique was used to estimate infraspecific variation in different populations of Stellaria media. Samples from 11 populations were collected from different regions of the country during spring 2018. For RAPD investigations, populations were divided into three geographical regions. This study provides important data on the genetic diversity, population structure and morphological characteristics of Stellaria media. The AMOVA and Gst analyses showed that the populations of this species are genetically differentiated. Nm analysis revealed very low value of genetic diversity among the studied population and mantel test indicated isolation by distance occurred among them. The studied populations of S. media are differentiated in morphological characteristics and genetic content. Introduction Genetic diversity, one of the basic levels of biodiversity, determines the evolutionary potential of a taxon or population to adapt to variable environmental conditions (Morton, 2005). Genetic diversity arises from different factors such as mutation or gene flow, while events, for example, genetic drift and directional selection, can decrease the diversity. The family Caryophyllaceae comprised of about 81 genera and 2600 species (Bittrich, 1993; Ullah et al., 2019; Esfandani– Bozchaloyi et al., 2017a,b,c,d; 2018a,b,c; Esfandani -Bozchaloyi and Sheidai 2018). Stellaria L. (Caryophyllaceae, Alsinoideae) includes both annual and perennial herbaceous plants that are widely distributed in the temperate zones of Europe and Asia (Lu and Rabeler, 2001; Keshavarzi and Esfandani–Bozchaloyi, 2014a, b; Ullah et al., 2019) and about 120 species with worldwide distribution, mainly in the north temperate zone (Morton, 2005; Ullah et al., 2018). In Flora Iranica, this genus has nine species under two sections: sect. Pseudalsine Boiss. consists of one species S. alsinoides Boiss. & Buhse and sect. Stellaria of six species viz., S. holostea L., S. persica Boiss., S. graminea L., S. nemorum L., S. media (L.) Vill., and S. pallida (Dumort.) Pire (Rechinger, 1988). The main center of diversification for Stellaria is Eurasia, with a center of distribution in the mountains of central Asia. Some species are also cosmopolitan (Bittrich, 1993; Ullah et al., 2018). There are limited chromosome records for Stellaria in the world. Basic chromosome numbers of x=10, 11, 12 and 13 have been reported for the genus (Federov, 1969 1974; Moore, 1973; Goldblatt, 1981). Stellaria media, known as chickweed, are annual and with slender stems, they have hairs on one side of the stem. The leaves are linear or oval, smooth or minutely, 13 to 17 × *Corresponding author, email: sh.mehri2000@gmail.com


Introduction
Genetic diversity, one of the basic levels of biodiversity, determines the evolutionary potential of a taxon or population to adapt to variable environmental conditions (Morton, 2005). Genetic diversity arises from different factors such as mutation or gene flow, while events, for example, genetic drift and directional selection, can decrease the diversity. The family Caryophyllaceae comprised of about 81 genera and 2600 species (Bittrich, 1993;Ullah et al., 2019;Esfandani-Bozchaloyi et al., 2017a,b,c,d;2018a,b,c;Esfandani -Bozchaloyi and Sheidai 2018). Stellaria L. (Caryophyllaceae, Alsinoideae) includes both annual and perennial herbaceous plants that are widely distributed in the temperate zones of Europe and Asia (Lu and Rabeler, 2001;Keshavarzi and Esfandani-Bozchaloyi, 2014a, b;Ullah et al., 2019) and about 120 species with worldwide distribution, mainly in the north temperate zone (Morton, 2005;Ullah et al., 2018). In Flora Iranica, this genus has nine species under two sections: sect. Pseudalsine Boiss. consists of one species S. alsinoides Boiss. & Buhse and sect. Stellaria of six species viz., S. holostea L., S. persica Boiss., S. graminea L., S. nemorum L., S. media (L.) Vill., and S. pallida (Dumort.) Pire (Rechinger, 1988). The main center of diversification for Stellaria is Eurasia, with a center of distribution in the mountains of central Asia. Some species are also cosmopolitan (Bittrich, 1993;Ullah et al., 2018).
There are limited chromosome records for Stellaria in the world. Basic chromosome numbers of x=10, 11, 12 and 13 have been reported for the genus (Federov, 1969(Federov, 1974Moore, 1973;Goldblatt, 1981). Stellaria media, known as chickweed, are annual and with slender stems, they have hairs on one side of the stem. The leaves are linear or oval, smooth or minutely, 13 to 17 × 1.5 to 7 mm. Flowers are hermaphrodite. Sepals prominently 4 to 6-nerved. The number of stigmas and stamens are 3 each. S. media is common in waste places, open areas, lawns, meadows, and widely distributed to temperate regions of Europe, Asia and Northern America. S. pallida is distributed in the all regions of Iran. This plant is edible and nutritious and considered to be a herbal remedy and useful in folk medicine. S. pallida is very similar to S. media. Stellaria media has some medicinal properties. This species has been used as to soothe severe itchiness even where all other remedies have failed (Slavokhotova et al., 2011). It is considered for rheumatic pains, skin diseases, and period pain as well as for bronchitis and arthritis (Slavokhotova et al., 2011). Rani et al. (2012) have studied some stem and leaf anatomical features through the pharmacognostical study for quality control of S. media. Arora and Sharma (2012) did pharmacognostical and phytochemical studies of S. media and showed the presence of epidermis, palisade cells, trichomes and vascular bundles in leaf. S. media possesses significant chemicals known as saponins, which can cause poisoning in cattle (Haragan, 1991). Many studies have been done on taxonomy, pollen morphology, phylogeny, seed micromorphology, anatomy, trichome and cytology of Stellaria species (Esfandani-Bozchaloyi and Keshavarzi, 2014;Keshavarzi and Esfandani-Bozchaloyi, 2014 a, b;Ullah et al. 2018). However, genetic diversity of Stellaria species have been reported only in a few studies (Verkleij et al., 1980;Chinnappa and Morton, 1984), and outcrossing or inbreeding, genetic structure, genetic variability within/between populations and ecological adaptation in Stellaria of Iran have not been investigated yet.
The molecular markers are extensively used in germplasm characterization, fingerprinting, genetic analysis, linkage mapping, and molecular breeding. RAPD (Random Amplified Polymorphic DNA) analysis using PCR in association 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 genomic regions can be sampled with a potentially unlimited number of markers (Ellis and Burke, 2007;Esfandani-Bozchaloyi et al., 2017a,b,c,d). The present investigation has been carried out to evaluate the genetic diversity and relationships among 11 geographical populations of S. media using RAPD markers. This is the first study on the use of RAPD markers in S. media; Therefore, we performed molecular study of 110 collected specimens of 11 geographical populations.

Materials and Methods
Morphological studies 110 plant sample were selected from eleven populations located in three provinces of Iran. Identification of S. media species were based on the descriptions provided by Flora Iranica (Rechinger, 1988). The sampling sites are provided in Table 1 and Fig. 1. Voucher specimens were deposited at the herbarium of Islamic Azad University, Science and Research Branch, Tehran, Iran (IAUH).

DNA extraction
Fresh leaves were used randomly from one to twelve 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-Bozchaloyi et al., 2018a,b,c). The quality of extracted DNA was examined by running on 0.8% agarose gel. 25 decamer RAPD primers of Operon technology (Alameda, Canada) belonging to OPA, OPB, OPC, OPD sets were used in this study. 10 primers with clear, enlarged, and rich polymorphism bands were chosen. PCR were carried out in 25 μl reactions containing 20 ng of template DNA, 0.3 mM dNTPs, 1μM primers, 1.0 μl of 20×PCR buffer (Cinnagen, Iran), 1.8 mM of MgCl 2 and 5 units of Taq polymerase (Cinnagen, Iran). The amplification was carried out, with programmed as initial pre-denaturation at 95°C for 5 min followed by 36 cycles of denaturation at 94°C for 45 s, annealing at temperature (52-55°C) for 40 s, and extension at 72°C for 1min. A final 5 min. extension at 72°C followed the completion of 38 cycles.

Morphological studies
For morphological studies, 43 morphological characters including 16 qualitative and 26 quantitative characters were studied following Attar et al. (2019, Table 2). Morphological traits were standardized (Mean = 0, Variance = 1) and used to estimate Euclidean distance for ordination analyses (Podani, 2000). PCA (Principal Components Analysis) biplot and MDS (Multidimensional Scaling) were applied for grouping and identifying the most variable morphological traits among the populations (Podani, 2000). We used PAST version 2.17 (Hammer et al., 2012) for multivariate statistical analyses.
For AMOVA (Analysis of molecular variance), we used GenAlex 6.4 software (Peakall and Smouse 2006;Meirmans and Van Tienderen, 2004) to determine the genetic differentiation of the species and Nei , sGst analysis in GenoDive ver.2 (2013) (Hedrick, 2005;Jost, 2008) were used to reveal genetic distance of the species. First data were scored as dominant markers so we used from STRUCTURE analysis for estimation of the parameters that is related to gene flow among studied population. Burn-in = 10000, and 10 runs were performed for estimation of the relationship between the genetic structures and geographical distance. Maximum likelihood method and Bayesian Information Criterion (BIC) was studied by structure analysis (Falush and Stephens 2007;Evanno et al., 2005;Meirmans, 2012). Gene flow was determined by calculating Nm from Gst by PopGene ver. 1.32, 1997 (Pritchard et al., 2000).

RESULTS AND DISCUSSION
In this study 11 populations of Stellaria media were selected from northern regions of Iran. Genetic diversity parameters revealed that the highest percent of genetic polymorphism (70%) and gene diversity (0.203) exist in Mazandaran, Chalos Neamat abad (population No.9), while the lowest amount of genetic polymorphism (22%) showed in population in Mazandaran, Noshahr (Table 3). AMOVA test showed that, 35% of total genetic diversity was within population and 65% was among population. Hedrick standardized fixation index makes the genetic distance among the studied populations. We have moderate level for AMOVA produced after 999 permutations (G'st = 0.632, P = 0.001) and Hedrick differentiation index (D-est = 0.271, P = 0.001). Our results showed that the populations of S. media are differentiated from each other.

Populations , genetic affinity
Neighbor-Net network and Nj tree revealed identitical results but here only Neighbor-Net network is discussed (Fig. 2). This network shows that the populations 3 and 7, as well as populations 4 and 11 are placed close to each other, which indicate that they have closer genetic affinity. The populations 2 and 6 and 8 are differentiated from the other populations. The studied specimen in PCoA plot revealed that they belong to different groups, which is in agreement with the AMOVA results (Fig. 3). The relationship between altitude distance and genetic distance indicated by Mantel test after 5000 permutations is significant in these populations (r = 0.22, P = 0.001). The isolation in S. media occurred because of low amount of gene flow due to geographically more distant of populations.

Populations genetic structure
The result carried out on STRUCTURE analyses by Evanno test makes a peak at k = 6 (Fig. 4). Furthermore, STRUCTURE analyses show genetic identity between populations 3 and 4 (similarly colored), populations 5 and 6, like populations 10 and 11. But it indicated genetic difference between populations 1 and 2 (differently colored).   The results of Reticulogram (Fig. 5), that is based on the least square method, indicates some of shared alleles among populations 3 and 5, 6 and between 8 and 2 and 11, also between 1, 4, 3 and 9 and 10. The mean Nm = 0.32 indicates very low level of genetic diversity and supports genetic stratification as showed by STRUCTURE analyses and K-Means. Nm result agreed with population assignment test and cannot showed gene flow among these populations. In total, ten ISSR primers produced 90 bands, fragment size ranged from 150 to 3000 bp.  Table 1).

Morphometric analyses
ANOVA tests for 110 plant specimens from 11 populations were performed. Our results indicated significant difference in comparison with the studied populations (P < 0.05). Ordination plot and other analyses produced similar results on these populations (Fig. 6). Our result revealed that among of the studied populations, morphological divergence exist and this divergence was due to quantitative traits. For example, length of stem leaves character separated population No. 2, but the populations 4 and 8 separated from the other populations due to difference in calyx length. We performed for both morphological and RAPD data-based a consensus tree (Fig. 7). It indicates that some populations are different from other populations due to both morphological and molecular characters.  According to Çalişkan (2012) genetic diversity provides information about adaptation to changing environments, understanding of positive influence in the conservation of endangered species, hybridization and gene flow among the populations. This study evaluates the use of RAPD markers for comparing the gene flow and relationships within the population of S. media in Iran. Verkleij et al. (1980) showed that Amylases isoenzymes could be successfully applied to assess inter-populational variation in S. media.
In this study, we have provided information on current taxonomic, molecular and geographical distance and data about gene flow and genetic structure of S. media in some parts of Iran. Chickweed can germinate and flower any time of the year. It is mainly self-pollinating, but sometimes cross-pollination occurs by flies and insects.
According to Chater and Heywood (1993) S,. media is a widespread weedy species. There are three subspecies viz., S. media subsp. media, S. media subsp. cupaniana and S. media subsp. postii but some authors showed that subsp. cupaniana (Scholte, 1978) and subsp. postii (Sinha, 1965) should be included in S. neglecta. According to Fedorov (1969) chromosome numbers that have been reported for S. media included 2n = 24, 28, 36, 38, 40, 42 and 44 from many parts of the world. However, chromosome numbers 2n =40, 42 and 44 are the most commonly reported and this species shows a high degree of genotypic variation that is highly correlated with its reproductive characters (Freeland et al., 2011).
S. media is annual, characterized by the presence of five sepals and petals which are usually bifid. Generally, within family Caryophyllaceae diversity of morphological features makes taxa complicated to be delineated and identified. S. media is occur on abandoned fields and commonly sensitive to disturbance of its habitat. S. pallida and S. media are self-pollinating and there are crossing barrier between them. This happened due to the presence of polyploidy in S. media (2n = 40-44), in contrast to the diploidy of S. pallida (2n = 22) (Scholte, 1978;Slatkin, 1993;Jolivet and Bernasconi, 2007). Therefore, breeding systems plays important role in low level of gene flow in S. media (Hutchison and Templeton, 1999;Medrano and Herrera, 2008). Our results shows that the seed morphologies of Stellaria media and S. pallida are similar. Seed coat cellsare rounded polygonal and V-shaped margin. Based on these characters, we decided that S. media could be differenced from S. pallida. Seed coat morphology was observed in 18 species of Stellaria by Chen (2010).