Analysis of Morphological and Molecular Genetic Diversity of Salicornia Iranica Akhani Wildlife Populations around Urmia Lake, Iran

Salicornia is a halophyte plant capable of being irrigated with seawater, which can be used as an alternative food. Given this, it is necessary to study the potentials of this plant's morphological diversity in the natural environment. In this study, 33 wild populations of Salicornia were collected from different geographical areas around Urmia Lake during the owering stage, and some morphological traits and 25 ISSR loci of the plant were measured. Based on morphological traits and the cluster analysis, Salicornia populations were divided into four groups. Overall, the high percentage of polymorphic gene loci (65.69%), the average number of effective alleles per gene locus (1.63), and the Shannon data index (0.540) indicate that ISSR markers can be used in Identify genetic diversity to be used. Molecular data cluster analysis divided the studied populations into two main groups, which included 12.12% and 87.88% of the populations, respectively. Based on the effective analysis of the population's genetic structure and the precise classication of individuals into suitable sub-populations, the value of K = 2 was calculated. The research ndings indicated that markers UBC823, B, A7, and K, and with the Shannon index, effective allele, and large heterozygosity values are markers with the highest effectiveness compared to other markers utilized, and they are used better than other compounds in genetic distance. The ndings of this study will aid in parental selection studies for breeding programs of salicornia in future. to estimate the morphological and molecular variation among 33 wild salicornia populations, 2: to search for genetic structure of salicornia populations and identify the most effective ISSR markers, and 3: to identify the relationships between morphological characteristics and ISSR markers that could aid breeding programs. In this study, 33 populations of Salicornia grown around Urmia Lake were collected, and to evaluate the morphological and genetical variation between different populations, 55 different morphological traits were studied.


Introduction
Genetic diversity in crops and orchards is an issue long considered by plant breeders searching for new sources of germplasm to perform gene transfer, phylogenetic testing, chromosome control genes, and marker selection, among other things (Liebhard et al. 2002).
Given the role of genetic diversity in advancing breeding programs and the importance of the local population, it is necessary to study the local population's genetic diversity (Solouki et al. 2008). A variety of natural genetic resources in an area can provide bene cial genes for plant breeding. These genes have been formed and stored mainly in native plants for centuries (Naghdiabadi H.A. 2002). Many of these native species have been being introduced as new plants due to their medicinal and industrial properties (Heywood 1999). It is necessary to study genetic diversity among different species using morphological features to nd desirable traits for further production (Noroozloo 2015). Morphological markers obtained from visible mutations in morphology include a wide range of genes that control morphological characteristics based on the appearance or phenotypes and serve as the rst markers. They used time immemorial, that is, the location of a gene chromosome determined (Naghavi 2007). Salicornia is one of the well-known genera of the Chenopodiaceae family that grows and evolves as a herbaceous (an annual plant with juicy and eshy stems) on the seashores and in the margins of brackish water (Isca et al. 2014). Salicornia genus consists of approximately 15 genus and 68 species (Shepherd et al. 2005). It is challenging to classify this plant species mainly due to self-pollination and diversity in local populations.
The most treasured resources in any country include genetic resources. Plant stocks are used by breeders as a resource for genetic material for generating new varieties. For utilizing genetic resources at the highest e ciency, the stored genetic material should be known. Samples can be evaluated in accordance with the purpose of germplasm usage, including pathological, agronomic, morphological, biochemical, molecular, and histological dimensions. With the evaluation of germplasm, information about the weaknesses and strengths of the genotypes and populations and their potentials can be obtained, and genetic basis of each trait can be determined by these evaluations. Investigating genetic diversity in plants is signi cant from various dimensions. Generally, when genetic diversity is determined, it is bene cial for researchers for managing collections, conservation, maintenance, and speci cation of plants, as well as usage of plant collections (Quilot et al. 2005).
Besides the loss of leaves and morphological identi cation indices and the small amount of dry matter compared to wet tissue, the accurate identi cation of species is di cult (Ball 1964). Salicornia Iranica Akhani, an endemic species of Salicornia in Iran, grows in central Iran and is a diploid genus of Salicornia (Akhani 2008). The habitats of this plant in Iran are Fars, Semnan, Gorgan, Bushehr, Hormozgan, Yazd, Khorasan, Khuzestan, Markazi, West, and East Azerbaijan, Isfahan, Qom, and Tehran provinces (Milić et al. 2011). According to studies, species collected from seven regions surrounding Urmia lake have been identi ed as Salicornia Iranica (Milić et al. 2011). Salicornia species are of great importance due to their application in human therapy. In addition to the pharmaceutical industry, Salicornia is used as an additive for glass and soap (Davy et al. 2001). Salicornia, an important salinity-resistant crop, can be found in human and livestock food products (Lu et al. 2010).
The Salicornia is important as a medicinal plant, and given the fact that there are not adequate and comprehensive studies in different elds of production, The current survey was conducted in order to 1: to estimate the morphological and molecular variation among 33 wild salicornia populations, 2: to search for genetic structure of salicornia populations and identify the most effective ISSR markers, and 3: to identify the relationships between morphological characteristics and ISSR markers that could aid breeding programs. In this study, 33 populations of Salicornia grown around Urmia Lake were collected, and to evaluate the morphological and genetical variation between different populations, 55 different morphological traits were studied.

Materials And Methods
In this study, 33 wild populations of Salicornia in full bloom and plant seeds were collected from different geographical areas in the lake's vicinity ( Table 1, Fig. 1). At the time of data collection, features such as the geographic area's location and characteristics (altitude and latitude) were recorded. Some populations were geographically less than a few hundred meters apart, which were considered separately based on eld observations. 55 morphological traits were evaluated. 15 specimens were sampled per population, and for each plant, all 55 traits were calculated ( Table 2). The morphological traits were measured in the Horticulture Department's plant physiology laboratory a liated to the faculty of agriculture at Urmia University and the herbarium of the faculty of pharmacy at Tabriz University. The properties were measured using a ruler, digital caliper, scrubber, and optical microscope (

Statistical Analysis Of Data
The ANOVA and variation within-group expressed as coe cient of variation for quantitative descriptors calculated for each group and the whole collection. Mantel test, and principal components analysis (PCA) performed using XLSTAT 2018.1 statistical software. The rst and second principal component axes scores were plotted to aid visualization of origin group differences and detect morphological variation in the collection.
Analysis of Data: population structure was studied using bands from all marker matrices. Using different algorithms, such as UPGMA, Single linkage, and Complete linkage, cluster analysis was performed. These algorithms were employed as zero (absence) and one (presence) scoring. The clusters were drawn in the present work using Mega software. Also other data were analyzed using the following software: NTSys version 2.0.1.5, SAS 9.2, SPSS, GenAlex, Mega, Excel, and PopGene.

Results
The variation and the mean traits were examined for different populations. Among the studied populations of Salicornia, the non-fertile parts on the longest secondary branch (V29) (84.75%), the fertile parts on the longest secondary branch (V28) (81.49%0 and the owering plants in the rst lateral branch (V34) (66.13%) had the highest diversity (Table 3). According to the results, the highest and lowest number of primary lateral branches (V9) were observed in (P27, 43) and (P22, 13.4), respectively.
Complete information about other variables is given in Table 3.
According to the results of cluster analyses by the Ward method, Salicornia populations were assigned to four groups ( Figure 4). The rst group contained 8.18% of populations (P16, P18, P24, P31, P20, and P22). In this group, populations with a short height, long spike, greater weight of 1000 seed, low number of Stomata, and the width across the apex on the third fertile segment were more abundant than other populations. The morphotype and in orescences of this group was distinct from other groups. The second group covered 15.15% of the whole population (P3, P11, P23, P2, and P33), comprising populations that were within the average range of trait sizes for diverse traits. The third group hosted 15.15% of the population (P4, P6, P1, P8, and P10), and the fourth group included 51.51% of the population (P6, P30, P25, P27, P21, P26, P15, P12, P28, P28, P7, P14, P17, P5, P9, P19, P29, P13, and p32). These populations had a great height, more internodes, more lateral branches, more stomata, a great weight of 1000 seeds and the width of the third fertile segment on the terminal spike. The accurate number of groups was identi ed using the detection function.

Genetic diversity of Salicornia populations
We evaluated genetic diversity in 33 Salicornia populations using 42 ISSR primers. 23 primers out of 42 primers under study generated a polymorphic band design at the suitable resolution, which were employed for the subsequent analysis phases (Table 5). Totally, 204 locations (averagely 8.87 locations per primer) were produced by these primers, 134 of them were polymorphic (65.69%). The ratio of markers to primer was 1 to 14, averagely 5.82 ( gure 5).
The number of effective (Ne) alleles in UBC849 was 1.25 and in PB it was 1.92, averagely 1.63 in each gene locus. Maximum value of this statistic shows that alleles have identical frequency in this location, and this statistic's minimum shows the rarity of other alleles and one allele's high frequency in samples.
In investigating allelic diversity, the highest observed heterozygosity was found in the B marker as 0.477, and the lowest observed heterozygosity was noticed in the UBC849 marker as 0.199. Besides, the highest expected heterozygosity was observed at approximately 0.484 in B marker, and the lowest expected heterozygosity was observed at approximately 0.203 in the UBC849 marker. Examining Shannon index (I) values showed that the highest value for this index was in marker B with a 0.670 and the lowest value was in UBC849 marker as 0.351 (Table 6).
The Jaccard similarity coe cient and UPGMA algorithm were used for dividing different populations into two separate groups. The rst group contained 12.12% and the second group included 87.88% of the masses. Two subgroups were made in the rst group, which the rst one included P24, P22, P26 and P1. The second group contains the residual 29 populations (P13, P20, P18, P30, P32, P29, P19, P8, P15, P17, P5, P27, P12, P33, P28, P31, P16, P21, P23, P14, P4, P3, P2, P7, P25, P6, P10, P11, P9), which was classi ed into two subgroups. The rst one is composed of just the P13 population. Also, this population was approximately different from other ones (Fig. 6). Structure 2.3.1 software was used for analyzing genetic population structure and precise classifying individuals into proper subpopulations. As shown by a two-way diagram of optimal determination of K with ISSR indicator, the ISSR primer shows the best K as 2, i.e., two subpopulations (K = 2) in the cultivars under study. The group was speci ed ( Table 7, Table 8).
The stabilization index (Fst) is a common and appropriate measure for genetic differentiation among groups and populations. When the Fst is higher, a better allele differentiation is obtained, with a higher allele stabilization rate. Potential subgroups in K = 2 show the difference among the populations under study in two potential groups. Besides, the individuals' matrix of the share in these groups (Table 4-5) indicated belonging populations with high coe cients to one group. Barplot results demonstrated inclusion of 26 Salicornia populations in the rst group (Red), and 5 populations in the second group (Green), with 2 populations had a complex structure (Fig. 7).

Discussion
The results showed that there was a signi cant difference between the studied populations in terms of traits in the question. Based on the mean of traits measured in the population, traits with a high percentage of variance had a wide range of trait quantities and offered a more extensive choice for traits. This difference is due to the impact of both genetic and environmental factors. Studies have shown that uctuations in soil and water salinity lead to physiological and phenotypic changes in the plant. Also, high plant density in a population restricts the number of branches and glaciers formed in the plant In S. Biglawi species, raising the salinity of irrigation water to 45 ds/m reduces the height and dry weight of the plant. In Persica species, increasing the irrigation water salinity had no effect on plant height but signi cantly decreased the dry weight (Rezapour 2018).
The cluster analysis results showed (Fig. 4) the clustering of populations is incompatible with geographical distribution. It may be due to sources of seed diversity caused by migration to different areas. Therefore, it may not be limited to different geographical regions in selecting parents for breeding projects, but it should be consistent with each population's speci c capacities. By studying Salicornia Pusilla, researchers have found that the plant seeds remain attached to the in orescence after ripening, and the spikes are trapped by a separating layer of the plant isolated in the water that may keep moving with the ow of water up to three months. They may even germinate but do not grow until the seeds are deposited in sediments (Dalby 1962). This feature may explain the common seed origin in the studied populations. Using 22 growth parameters, the researchers evaluated 11 Salicornia Bigelovii populations in the eld and divided the cluster analysis of studied populations into four groups (Lyra et al. 2016). Contrary to our study, the results of research on the genetic diversity of six Salicornia Ramosissima populations in central Germany showed that it is consistent with geographical distribution (Krüger et al. 2002). A review of the genetic diversity of the two species of saline Salsola manifested a signi cant difference in this plant and the environmental conditions of the plant, suggesting that disparity in salinity, nutrition, pH, and soil moisture changes the vegetative type of plants (Shuyskaya et al. 2017). The results of analyzing the main components con rmed the clustering obtained from the cluster decomposition. Though the association between regional diversity was not that evident, a close look at the scatter plot revealed some regional adaptation level was observed. Such regional variability could be due to geographic isolation and microclimatic differences between regions. Factors such as plant population isolation, adaptation to the environment due to declining lake water levels, and strong self-pollination within the plant population may contribute to Salicornia's population diversity. The degree of morphological differentials is signi cantly noticeable in different populations from four groups.
The research ndings indicated that markers UBC823, B, A7, and K, and with the Shannon index, effective allele, and large heterozygosity values are markers with the highest effectiveness compared to other markers utilized, and they are used better than other compounds in genetic distance.
As stated by Dirlewanger et al. (2002), there is a relationship between the alleles number in each gene locus and the number of used markers and the samples' number. According to the ndings of research on the genetic diversity of six populations of S. herbacea in South Korea, where 6 ISSR markers were used, 39 polymorphic bands were obtained out of 49 bands, with an average of the effective allele for each gene locus as 1.22. The mean genetic index was 0.249 and the mean Shannon index was 0.382. These researchers mentioned that for achieving high diversity in populations Salicornia, a wider research scope is required to be chosen (Kim et al. 2017).
These populations were separately gathered because of varying morphological types compared to other populations. Also, this difference is shown in the results. The second subgroup included P26 and P1 populations, with different appearances compared to other populations. They had a height higher than average, particularly the highest height was observed in P26 population among all populations. Moreover, long glazes were observed in these two populations. Additionally, it shows all botanical properties of S. Iranica (Akhani 2008).
In earlier Iranian research works on Salicornia, 36 samples of Salicornia were collected by (Heydarian 2001)from different saline areas. He speci ed this plant's genetic diversity by the use of 17 RAPD molecular markers, Jaccard similarity coe cient, and UPGMA approach. The subjects were categorized into 7 classes. Moreover, 18 Salicornia populations were evaluated by (Mohammadi 2012)that were collected from different regions in Iran. He used AFLP markers and categorized the individuals into 4 groups by the use of UPGMA method and Jaccard similarity coe cient. As shown by the research in this work, the researcher collected species from 7 regions near Lake Urmia and S. Iranica are presented, all in a group. In this research, S. Iranica species were separated from S. persica species using the AFLP marker, and they were placed in a subgroup. Additionally, the populations gathered from each area were put in a different subgroup. The genetic diversity in 11 S. brachiata populations were evaluated in India by (Badlani 2011) by 15 ISSR and 15 RAPD primers. The investigated populations showed high diversity. It was also observed in both markers of the populations under study. They were grouped into 3 groups.
The resulting Barplot showed that when the membership percentage to a cluster for a genotype is higher than or equal to 0.7, the genotype is allocated to that cluster, while if the percentage is below it, it is considered as a mixed genotype (hybrid) (Spataro et al. 2011). Generally, when the average effective allele numbers per gene locus (1.63), the polymorphic gene loci percentage (65.69%), and the Shannon data index (0.540) are high, it is indicated that we can use ISSR markers for identifying genetic diversity.

Conclusion
This study showed that Salicornia populations growing around Urmia Lake had considerable diversity in morphological and ISSR characteristics. The incompatibility of population clustering with their geographical distribution may be due to different populations' exact seed origins. The populations under the genetic study were divided into two major groups by cluster analysis of molecular data, including 12.12% and 87.88%. The K value was obtained as two according to the practical analysis of the population's genetic structure and the accurate individuals' classi cation to suitable sub-populations. The populations under study were classi ed into two groups that are because of Salicornia's self-pollination.
Differences in morphological and genetic grouping may be due to the environment's effect on morphological traits. While in genetic traits, the difference between the populations was may be due to the populations' isolation due to the lowering of the lake water, and the plant was directed towards selfbreeding. Combining morphological and ISSR data may be more effective for de ning genetic variation and genetic diversity within the salicornia population.

Declarations Acknowledgment
The Urmia lake studies research institute sincerely thanks for providing part of this research's necessary funds (Grant No. 608/53).

Disclosure statement
We declare no con ict of interest.    Cluster analysis of Salicornia populations based on algorithm UPGM