Evaluation of Molecular and Morphological Diversity of Capparis Spinosa

The objective of this study was the evaluation of molecular and morphological diversity among 80 caper (Capparis spinosa L.) genotypes from the 12 regions of the central Zagros Mountains located in the west of Iran. The results showed a high level of morphological genetic variation among the caper samples. According to the morphological cluster analysis, 80 genotypes were clustered into ve main groups. The 15 factors justi ﬁ ed 78.7 % of the total variation based on factor analysis. ISSR primers produced a total of 108 polymorphic bands (85.04%) from 127 bands and the PIC for primers ranged from 0.01 to 0.52. SCoT primers produced a total of 165 polymorphic bands (86.84%) from 190 bands and the PIC for primers ranged from 0.06 to 0.55. Ordination and cluster analysis by ISSR markers showed that the genetic relationships among all accessions could be separated into three major groups and by SCoT markers separated into six groups. The results did not show a perfect match between the molecular diversity groupings and geographical regions, because many natural factors and human activities shape the amount and pattern of genetic diversity in a plant population. SCoT markers were more informative than ISSR markers for the assessment of genetic diversity of caper germplasm. The combined (ISSR+SCoT) markers haven't shown more information of genetic diversity than single analysis of ISSR and SCoT. The results indicated the existence of dispersion and different levels of morphological variation and molecular genetic diversity in the genotypes collected from west of Iran. genetic analysis, bulked segregant analysis, and quantitative trait loci mapping. Gristina et al. (2014) studied nineteen wild populations collected from different regions in Italy and belonging to two different subspecies, C. spinosa subsp. spinosa and subsp. rupestris, were evaluated by the ISSR marker. Ahmadi and Saeidi (2018) Studied 21 populations of caper Iran using markers, SCoT a novel method for generating plant DNA markers were designed based on the ATG start sequences and the regions between the start codons are amplied during the polymerase chain reaction and show the differences. The SCoT primers are typically 18–24 nucleotides and their G and C content is 50–72% (Bertrand et al. 2009). In a study, 16 Foeniculum vulgare populations in Iran were evaluated using SCoT markers, and the results showed that the studied populations were divided into 4 clusters (Nikkerdar et al. 2017). In the present study, genetic diversity of 80 C. spinosa samples collected from 12 regions in west of Iran were evaluated, and characterized by using morphological characteristics, ISSR, and SCOT markers.


Introduction
Recently, medicinal plants have been known for possess multiple health-promoting effects, and the high e cacy and low adverse effects for the treatment of different human diseases (Nabavi et al. 2016). Also, it is well known that synthetic drugs can cause a wide range of serious adverse events (Bertrand et al. 2014). Therefore, in recent years, medicinal plants have received much attention (Schulz 2006). C. spinosa belonging to the family Capparidaceae is a xerophytic plant. C. spinosa had been a part of the Mediterranean diet for over 5,000 years (Muharrem et al. 2009). Its commercial name is C. spinosa and its brand is caper. This plant is capable of growing in a broad range of climatic conditions, varying from dry deserts to cooler altitudes of mountains, this plant has developed special mechanisms in order to survive in the semiarid lands conditions and consequently its introduction may help to prevent the disruption of the equilibrium of those fragile ecosystems and the soil degradation. (Lansky et al. 2014;Pugnaire 1989;Manikandaselvi et al. 2016). C. spinosa is used as a traditional medicine for lowering blood sugar and blood fat, diuresis, as well as a rheumatism and arthritis treatment. Studies have shown that many of its chemical constituents have antimicrobial, antioxidative, anti-in ammatory, immunomodulatory and antiviral properties (Tlili et al. 2010;Patel et al. 2014; Zhang et al. 2012). Caper population structure has been low studied. Different taxonomists have recognized 250 morphologically different species in the genus caper.
All different parts of the caper like its young shoots, ower buds, fruits and seeds are used for human diet. Previous studies on the chemical composition of C. spinosa have reported the presence of a large number of bene cial compounds such as vitamins, minerals, alkaloids, and lipids (Matsuyama et al. 2009;Tlili et al. 2010;Patel et al. 2014). Capers ( ower buds), caper berries (fruits), leaves, roots, and seeds of this plant are used medicinally (Anonymous 1999). Fruit and the root of the plant were used in gout and also as diuretics, astringents, and tonic. It is known that its C. spinosa displays huge agro-based potentialities and a highly demand for exploitation due to a diversi ed international market. Today, it seems necessary to focus on the possibility of selection and improvement of this specie, especially in the east Mediterranean countries ( Aromatic and medicinal plants have less arable land compared to other crops. However, are contains a large number of plant species used, with the greatest variation in morphological traits and characteristics (Salamat et al. 2014). Patterns of morphological variation observed within and between plants populations indicate that morphology may vary in apparently random directions. Exactly unclear for the geographic origin of C. spinosa, however, it seems to be originated from somewhere in China, India or Central Asia (Liu et al. 2015). Zohary (1960) reported ve species with some varieties in Iran. Zokian (2015) reported the high diversity of morphological and anatomical characteristics of C. spinosa that grown wildly in Iraq. Musallam et al. (2012) collected twenty four populations of C. spinosa that covered different geographical regions of Jordan, and Reported that the phenotypic diversity of C. spinosa in Jordan was found to be high.
Increasingly, molecular marker technologies are playing an important role in assessing genetic diversity, identifying genetic relationships, and aiding germplasm ngerprinting in plant collections. Over the last few decades a variety of different genetic analytical techniques have emerged in the eld of molecular genetics along with several PCR-based genetic markers that have now been established and are used to provide information on genetic variations in plant species. Recently, studies were reported about a high genetic diversity in C. spinosa using DNA markers such as RAPD (O¨zbek and  ampli ed during the polymerase chain reaction and show the differences. The SCoT primers are typically 18-24 nucleotides and their G and C content is 50-72% (Bertrand et al. 2009). In a study, 16 Foeniculum vulgare populations in Iran were evaluated using SCoT markers, and the results showed that the studied populations were divided into 4 clusters (Nikkerdar et al. 2017).
In the present study, genetic diversity of 80 C. spinosa samples collected from 12 regions in west of Iran were evaluated, and characterized by using morphological characteristics, ISSR, and SCOT markers.

DNA extraction
Fresh young leaf samples were taken from each caper genotype to assess the molecular variation and genetic relationships of 80 genotypes. The samples were stored at -80°C temperature until DNA was extracted. Genomic DNA was extracted using a modi ed CTAB protocol by Doyle and Doyle (1987). Quantitative amount of DNA was determined with spectrophotometer at the wavelength of 260 nm and quality of DNA was determined with agarose gel electrophoresis.  (Table 3), and extension for 1 min 30 second at 72°C. This was followed by a nal extension of 6 min at 72°C. Generated products were separated on 2% agarose gel electrophoresis in 1×TBE buffer and stained with ethidium bromide (10 mg/ml). Fragment size was estimated by using a 1 kb DNA ladder and gels were visualized under UV light.
SCoT-PCR Ten primers were used for SCoT ampli cation (Table 3) (Table 3), and extension for 1 min 30 second at 72°C. This was followed by a nal extension of 6 min at 72°C. Generated products were separated on 2% agarose gel electrophoresis in 1×TBE buffer and stained with ethidium bromide (10 mg/ml). Fragment size was estimated by using a 1 kb DNA ladder and gels were visualized under UV light.

Statistical analysis
Frequency and percentage distribution of morphological traits were speci ed to qualitative descriptors. The Principal components analysis (PCA) based on the covariance matrix of the coe cients and factor analysis were performed by the SPSS version 22.0 software (SPSS Inc. 2004). Cluster analyses were conducted to specify the dissimilarity indices measure to be used in clustering with the Neighbour joining (NJ) method by the software DARwin5 (version: 5.0.145). PCR-ampli ed ISSR and SCoT fragments detected on gels were scored as absent (0) or present (1). The dissimilarity matrix was generated using Jaccard indices (Jaccard 1908). The DARwin program was used for cluster analysis based on a dissimilarity matrix. The Mantel test was performed using XLSTAT software. The matrix was analyzed by the Neighbour joining method (NJ) and relationships between the cultivars were illustrated as a dendrogram. Genetic diversity parameters such as the number of polymorphic loci (NPL), the percentage of polymorphic loci (PPL), effective number of alleles (Ne) (Kimura and Crow, 1964), Nei's genetic diversity (h), Shannon's information index (I) (Lewontin 1972), gene ow (Nm) and genetic differentiation coe cient (Gst) were calculated using POPGENE ver. 1.32 (Yeh et al. 1999). Analysis of molecular variance (AMOVA) and principal coordinate analysis (PCoA) were performed using GenALEx software ver. 6.5 (Peakall and Smouse 2006).

Results
Assessment of genetic variability and relationships among the caper genotypes using morphological traits Twelve C. spinosa populations were characterized according to general morphological characteristics. The genotypes collected from ''Gilangharb'' had medium to large size shrubs with green stem, medium owers with white petals and ag, ovate shape dark green leaves and glabrous, medium, nonsymmetrical and oblong fruits with the largest diameter in the middle, green fruit tail ( Fig. 2A). The genotypes belong to ''Gorese d'' had medium shrubs with violet stem, medium to small, non-symmetrical and oblong fruits with the largest diameter in the middle, green fruit tail, moderate or relatively large, glabrous dark green leaves, low inter node length, medium owers with white ags (Fig. 2B). ''Khosravi'' population had shrubs with medium growth power, wooden trunk, small and round leaves with abundant trichome, low internode length, small owers with white petals and purple ags, small and round fruits with a bright green color, and the largest diameter of the fruit in the middle (Fig. 2C). The genotypes collected from the ''Naftshahr'', ''Kerend'' and ''Somar'' had large shrubs with yellow stem, large owers with white ags, bold green, large and crusty leaves, ovate shape leaves and glabrous, sunk apex leaves, short middle node length, and medium fruits with the highest fruit diameter at the end of the fruit (Fig. 2D). ''Qasreshirin'' population had medium shrubs, hairless purple to green colored stem, round and small dark green leaves, Medium internode length, medium fruits and owers. The color of their fruit is dark green and their fruit tail color is green. They have ovate shape fruit with the largest diameter in the bottom, and red and white or cream colored ripe fruit. The genotypes collected from the ''Sarapulzahab'', ''Charmeleh'' and ''Kermanshah'' had large shrubs with large leaves, internode length, and petals. They had purple to green colored stem, white petals, pink and white colored ag, round hairy bright green leaves, bright ovate fruits, and green fruit tail (Fig. 2E, 2F).
The collected C. Spinosa fruits showed persistent variation (Fig. 3). Of the studied characteristics HSC, FTC, FPM and LSB showed higher coe cients of variation (CV), indicating a high level of variation, and the characteristics of HTS, FTL, P, SSH, LBRL, FL, and PS showed the least coe cients of variation (CV), representing the lowest level of changes ( Table 2).
The relationship between 80 C. Spinosa genotypes was drown in the dendrogram of a hierarchical cluster analysis using Euclidean dissimilarity with the the Neighbour joining (NJ) method as amalgamation rules (Fig. 4). According to the results of cluster analysis based on morphological characteristics,  Table 5). The loading factors greater than 0.5, regardless of the respective sign were considered as signi cant coe cients. The rst factor accounted for 6.70 % of the variation in total. This factor included type stem, fruit length, the maximum diameter, tail length, pedicel, aqueous meat, ower size, ag color, ag length and petal size. Therefore, the second factor explained 3.40 % of the total variation. Total growth power and branchesis were the main traits in second factor. The third factor explained 3.10 % of the total variation.  Assessment of genetic variability and relationships among the caper genotypes using molecular markers

Molecular analysis based on ISSR markers
Interpretation of obtained bands from gel electrophoresis showed a total of 127 ISSR bands ampli ed from 10 used ISSR primers. The mean number of band per assay was 12.7. The size of ISSR fragments generated by the different primers in this study ranged from 240 to 2500 bp, and the number of bands produced by the different primers ranged from 6 (UBC 864) to 18 (UBC 807). A total of 108 bands of 127 bands (85.04%) were polymorphic. The ISSR primers and their produced fragments in 80 caper genotypes are characterized in Table 6. The maximum number of polymorphic bands was ampli ed with the UBC 856, UBC 825, UBC 807 and UBC 808 primers, identifying 100% polymorphism and the minimum number of polymorphic bands were ampli ed with the ISSR864 primer, identifying 50% polymorphism.    % of the total genetic variance was attributed to between the populations, and 33% were explained by individual differences within populations (Table 8). The Jaccard dissimilarity indices ranged from 0.01 of 0.52. The hierarchical cluster analysis based on ISSR markers using the neighbour joining method generated a dendrogram with three main clusters (Fig. 5), which corresponded to the PCoA grouping (Fig. 6). . Two-dimensional PCoA plot of the C. spinosa also divided individuals into three groups same as grouping in the dendrogram (Fig. 6).
The Mantel test showed moderate correlation between morphological traits and ISSR-based genetic similarity (r = 0.289; P = 0.002) across all the genotypes (Table 9). SCoT primers generated 190 bands which 165 bands (86.84%) were polymorphic. The mean number of band per assay was 19. The size of SCoT fragments generated by the different primers, ranged from 150 to 3000 bp and the number of bands produced by the different primers ranged from 14 (SCoT29) to 24 (SCoT1). Table 10    populations. Two-dimensional PCoA plot divided 80 caper individuals into two groups (Fig. 8). The Mantel test showed the signi cant correlation between morphological characteristics and SCoT-based genetic distances (r = 0.462; P = 0.002) across all the genotypes (Table 9).

Molecular analysis of combined (ISSR + SCoT) markers
Genetic diversity parameters as NPL, PPL, Ne, h and I were calculated for the populations using the combined ISSR and SCoT markers. The obtained results based on the combined ISSR + SCoT data indicated the most variability in "Sarpolzahab" population and the least in "Ivan" and "Kerend" populations ( Table 12). The mean values of genetic differentiation (Gst), and gene ow (Nm) between populations were calculated as 0.470, and 0.563, respectively.
The dissimilarity coe cients ranged from 0.04 of 0.51. The dendrogram, constructed from combined ISSR + SCoT markers indicated that the caper cultivars grown in the western region of Iran could be clearly divided into three groups (Fig. 9) The Mantel test demonstrated the signi cant correlation between morphological traits and ISSR + SCoT genetic distances (r = 0.289; P = 0.001) across all the genotypes (Table 9).  (Fig. 4). The individuals collected from same locality were clearly included in one genetic cluster such as Kermanshah, Kerend, Naftshahr and Khosravi, but individuals of some populations such as Sarpolzahab, Somar, Gilanegharb and Charmelah were assigned to more than one cluster, in agreement with the result of Ahmadi and Saeidi study (2018). Having a hard seed shell makes the digestive tract of some animals and birds unable to digest the seeds of this plant, and seeds of these plants spread by the feces of animals and spread in different geographical locations. Also, being aware of the medicinal properties of this plant for thousands of years can play a role in the movement of seeds of different species and ecotypes of this plant. Overall locating some of the genotypes collected from the west of Iran habitats in separate groups and as well as the grouping of ecosystems in this region may be due to germplasm displacement and high plant diversity. The most of the traits examined had CV values greater than 30%, indicating high variation among the studied caper genotypes based on majority of the characteristics evaluated. Many natural factors and human activities shape the extent and pattern of genetic diversity in a plant species (Rao and Hodgkin 2002). In many plants, the effect of low temperature on the reduction of characteristics has been proven (Omidbaigi 2000). While with increasing altitude the most morphological characteristics could be increased signi cantly (Fakhri et al. 2008). In this study, the altitude difference of almost 1000 meters between the investigated locations could be one of the environmental causes of high variation among the genotypes studied.
In current study, most of the measured parameters showed high level of genetic diversity in Iranian germplasm of C. spinosa. This level of genetic diversity in Iran was previously reported by Ahmadi  The combined (ISSR + SCoT) markers compared to alone ISSR and SCoT markers were found same e cient with regards to polymorphism detection, and haven't shown more information of genetic diversity than single analysis of ISSRs and SCoTs. Molecular marker diversity studies did not show a perfect match between the molecular diversity groupings and geographical regions, but the combined (ISSR + SCoT) markers shown a perfect match between the molecular diversity groupings and geographical regions, Except Somar and Qasreshirin individuals were assigned to more than one cluster.
Mantel test demonstrated moderate correlation between the genetic relationships estimated using ISSR and SCoT data and morphological data. In both dendrograms for ISSR and SCoT markers, samples belonging to each population were almost clustered into one group. The samples clustered with morphological traits were classi ed in different groups from grouping based on molecular data and even from geographical groups. Grouping based on morphological traits could be in uenced by environmental factors (such as sea level elevation, amount of light exposure, air humidity, soil moisture content, soil texture, etc).
This study showed considerable gene ow for ISSRs (Nm = 0.518) and SCoTs (Nm = 0.613), and low level of differentiation of ISSRs (Gst = 0.491) and SCoTs (Gst = 0.449) among populations of C. spinosa. But, the results of Ahmadi and Saeidi (2018) study showed low level of gene ow (Nm = 0.455) and considerable differentiation (Gst = 0.523) among populations of C. spinosa. Caper is an andromonoecious species, bearing both male and perfect owers on the same plant (Zhang and Tan 2008), which causes high level of gene ow and consequently low level of genetic differentiation among populations.
The existence of dispersion and different levels of morphological variation and molecular genetic diversity in the studied genotypes indicates that C. spinosa germplasm in west of Iran is applicable and useful for breeding programs. Caper is long lived and it is perennials, possible that actual rate of out-crossing and gene ow are enough to maintain observed level of genetic variation. Hence, individuals belonging to populations with su cient genetic distance could be introduced as potentially appropriate parents in different caper breeding programs. Of course, further populations are needed to introduce the best populations in the natural habitats of this species and studying other species of this genus.