Identification of the promising Ziziphus spina‐christi (L.) Willd. genotypes using pomological and chemical proprieties

Abstract Ziziphus spina‐christi (L.) Willd. is a multi‐purpose plant and is very popular found in local markets because of its high nutritional and medicinal values. The present work was carried out to study morphological and chemical properties of native accessions of this species. There were significant differences among the accessions investigated based on the morphological and chemical characters. Total phenolic content ranged from 4.84 to 49.58 mg/g fresh weight (FW). Total flavonoid content varied from 0.45 to 2.29 mg/g FW. Antioxidant activity measured with DPPH ranged from 0.32 to 16.99 mg/g FW, while it ranged from 6.64 to 84.15 µM FeSO4 FW with the FRAP method. The total phenol content showed significant and positive correlations with total flavonoid content (r = 0.33), antioxidant activity obtained with DPPH (r = 0.85), and antioxidant activity obtained with FRAP (r = 0.54). The ward dendrograms divided the accessions studied into two major clusters based on morphological and chemical characteristics. Based on the traits related to fruit quality such as fruit weight, fruit skin color, and fruit flavor, as well as in terms of chemical characteristics related to medicinal properties such as total flavonoids and antioxidant activity, 17 accessions were superior that could be used in breeding programs or cultivated directly. The present results can be used in defining conservation strategies, genetic improvement, and crop production.

the soil and are therefore highly tolerant of drought (Miehe, 1986). This plant is evergreen, but parts of its leaves fall off in very dry seasons (Maydell, 1986). Its height reaches between 5.00 and 10.00 m, and its trunk diameter is about 45.00 cm (El Amin, 1990;Saied et al., 2008).
Indigenous plants are part of the local agricultural and food source and are an important genetic source for maintaining biodiversity (Grivetti & Ogle, 2000). The potential of wild plant resources has not only been fully explored but has not been taken into account and has not been sufficiently involved in the supply of agricultural products (Saied et al., 2008;Schreckenberg et al., 2006). Local people have paid close attention to the importance of indigenous plants in terms of food supply, extra income, medicinal properties, and their use in preventing soil erosion (Gebauer et al., 2007). Due to the increasing population of the world and the need to feed them, the development of alternative crops is needed to improve the diet (El-Siddig et al., 1999). Identifying and introducing crops that tolerate harsh conditions such as drought, heat, and salinity, is especially important (Saied et al., 2008).
Recently, attention to medicinal plants has been increased because they are pharmaceutical agents and are important for the development of drugs. According to the World Health Organization (WHO), traditional and herbal medicine is used by 80.00% of the world's population in developing countries for their primary healthcare (Alhakmani et al., 2014). The Z. spina-christi is used to treat headaches, fractures, dandruff, chest pains, bruises, and blisters (Ghazanfar, 1994).
The Z. spina-christi, known locally as Konar in Iran, is highly distributed in the southern regions of the country, such as Khuzestan, Bushehr, Fars, Kerman, Sistan, and Hormozgan provinces. This plant is multipurpose and is very popular in local markets due to its high nutritional and medicinal values. Also, native accessions of this species have high tolerance to heat, drought, and salinity (Miehe, 1986). Thus, they can play an important role in breeding programs related to the mentioned stresses. Due to its nutritional value and medicinal uses in traditional medicine, considerable attention has been paid to this plant. Therefore, it should be prevented from genetic erosion.
Little is known about the morphological and chemical properties of this species in Iran. Thus, the current study was carried out to study morphological and chemical variation of some native accessions of this species selected from Shooshtar region in Khuzestan province from the south of Iran.

| Plant material
The present work was carried out to study morphological and chemical properties of 48 native accessions of Z. spina-christi selected from Shooshtar region in Khuzestan province from the south of Iran.
Shooshtar region is located at 31˚36'34"N latitude, 48˚35'22"E longitude, and 65 m height above sea level. To reduce the error and to prevent the collection of the clones belonging to a tree and ultimately increasing accuracy, a proper distance (200 m) between trees in each area was considered.

| Morphological analysis
The phenotypic evaluations were carried out using 44 morphological and pomological characters. Fifty replicates for the leaf and fruit were used for measurements, and the mean values were used for analysis. The fruits were harvested at full ripening time based on taste and color and then transported to a laboratory. A digital caliper was used to measure dimensions-related characters of leaf, thorn, fruit, and stone. Also, the weight of fruit and stone were measured using an electronic balance with 0.01 g precision. Furthermore, tree growth habit, tree vigor, tree height, branching, branch density, branch flexibility, trunk color, trunk type, trunk diameter, canopy symmetry, canopy density, tendency to suckering, thorn presence on current shoot, leaf density, leaf shape, leaf apex shape, leaf upper surface color, leaf lower surface color, leaf margin serration, leaf serration depth, fruit shape, fruit skin transparency, fruit skin color, fruit flesh color, fruit taste, fruit flesh texture, stone shape, stone surface, and stone terminal appendix were qualitatively estimated based on rating and coding.

| Total phenolic content
For extraction, samples (1.00 g) were homogenized with 10.00 ml of 80.00% methanol and the mixtures were centrifuged at 11200g G-force for 10 min. Supernatants were collected and analyzed for total phenolic content and antioxidant activity assays. Total phenolic contents of fruit extracts were measured using the Folin-Ciocalteu reagent method with spectrophotometry (Singleton & Rossi, 1965).
Briefly, 400 μl of the extract was combined with 2.00 ml of ten-folddiluted Folin-Ciocalteu reagent and 1.60 ml of sodium carbonate 7.50% and then placed at room temperature for 30 min. The absorbance was estimated at 756 nm. The concentration of total phenolic content was read in mg gallic equivalents per g fresh weight (FW) using a calibration curve prepared with gallic acid.

| Total flavonoid content
For determination of total flavonoid content, the method described by Grzegorczyk-Karolak et al. (2015) was adopted so that the 2.00 ml of fruit extracts were mixed with 2.00 ml of 2.00% AlCl 3 and the reaction mixture was allowed to stand for 15 min at room temperature. The absorbance was measured at 415 nm, and the findings were expressed as mg quercetin equivalents per g FW (mg QE/g FW) for total flavonoid content.

| Radical scavenging activity
The scavenging activity of the extracts prepared on 2,2-diphe nyl-1-picryl-hydrazyl-hydrate (DPPH) free radicals was determined. The 25 μl of the fruit extract was reacted with a 0.10 mM methanol solution of DPPH in a total volume of 3.00 ml, and the mixture was then placed in the dark at room temperature for 30 min. The absorbance was read at 517 nm. The DPPH scavenging activities were calculated based on the following formula: where A control and A sample represent the control absorbance and the sample absorbance, respectively (Zhu et al., 2009). The DPPH scavenging activity of fruits was expressed as mg ascorbic acid equivalents per g FW using the established ascorbic acid calibration curve.

| Ferric reducing antioxidant power (FRAP)
The method developed by Benzie and Strain (1996) was used for the FRAP assay. The FRAP reagent comprised 300 mM acetate buffer, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM ferric chloride (10:1:1, v/v/v). Three milliliters of FRAP reagent were added to 20 μl of fruit extract, and the reaction mixtures were placed in a 37℃ water bath for 10 min. The absorbance was read at 593 nm, and antioxidant activities were determined using the prepared FeSO 4 standard curve.

| Statistical analysis
Significant differences among the accessions in terms of the traits measured were determined using one-way analysis of variance (ANOVA) with SAS software (SAS institute, 1990). The parameters, including minimum, maximum, mean, standard deviation (SD), and coefficient of variation (CV), were calculated. Pearson correlation coefficient was used to determine the correlation between traits with SPSS ® software version 16 (SPSS Inc. Norusis, 1998). The relationship between the accessions was analyzed using principal component analysis (PCA) with SPSS software. Hierarchical cluster analysis (HCA) was performed using Euclidean distance coefficient and Ward method with PAST software .
Distance coefficients were standardized using the Z scale. Also, the two-dimensional plot was generated using the first and second principal components (PC1 and PC2) with PAST software.

| Morphological evaluations
There were significant differences among the accessions investigated based on the characters recorded (ANOVA, p < 0.01).
The highest CVs belonged to stone terminal appendix (720.00%), The range of fruit stalk length and diameter was from 2.45-6.82 mm and 0.45-0.87 mm, respectively. Stone length varied from 8.01 to 12.25 mm, stone width ranged from 6.73 to 10.08 mm, and the range of stone weight was from 0.17-0.84 g (Table 1). Differences in fruit size and weight under the same environmental and geographical conditions are probably the result of genetic effects (Karadeniz, 2002 Fruit skin color showed high variations and included maroon yellow (3 accessions), maroon (1), dark maroon (9), yellow-brown (1), light brown (28), and dark brown (6). Also, fruit flesh color was cream-yellow (19 accessions), cream (11) Tree growth habit -Spreading (24) Semi-erect (22) Erect (2)

TA B L E 3 (Continued)
The PCA reduces the number of correlated traits to a small number of components and interprets the relationship between variables (Khadivi, 2018). The PCA showed that the first 14 components accounted for 80.97% of the total variance (Table 3). The characters, including fruit length, fruit width, fruit fresh weight, fruit flesh thickness, stone length, stone width, and stone weight, were found to be correlated with PC1, accounting for 16.60% of the total variance, called fruit size-related characters and corresponded with findings of Norouzi et al. (2017). The PC2 included seven traits, including tree vigor, tree height, branching, branch density, trunk diameter, canopy density, and leaf density, with explaining 11.49% of the total variance, called vegetative-related traits. Three characters, including leaf length, leaf width, and petiole length, formed the PC3, accounting for 7.42% of total variance, called leaf-related traits. In the previous studies, PCA has shown the importance of fruit-related traits in describing the diversity between genotypes of Z. spina-christi (Baghazadeh-Daryaii et al., 2017;Norouzi et al., 2017).
The scatter plot created using PC1/PC2 showed phenotypic variations among the accessions (Figure 2). Starting from negative to positive values of PC1, the accessions showed gradual increases in fruit length, fruit width, fruit fresh weight, fruit flesh thickness, stone length, stone width, and stone weight. Also, starting from negative to positive values of PC2, the characters, including tree vigor, tree height, branching, branch density, trunk diameter, canopy density, and leaf density, showed gradual increases among the accessions studied.
Besides, HCA based on all the morphological data showed that the accessions studied were divided into two main clusters as revealed with the dendrogram created using Ward and Euclidean distance coefficients (Figure 3). The first cluster (I) was divided into two sub-clusters. Sub-cluster I-A included 12 accessions, and sub-cluster I-B consisted of seven accessions. Also, sub-cluster II-A included six accessions, and sub-cluster II-B consisted of 23 accessions. There was high phenotypic diversity among the accessions. Self and crossincompatibility is a common and unique phenomenon in the genus Ziziphus, which leads to an increase in its genetic and phenotypic diversity apart from clonal and bud mutations (Weekley et al., 2002).

| Chemical evaluations
The ANOVA revealed significant variations among the accessions studied in terms of the chemical properties measured (p < 0.01).  (Lin et al., 2005;Mallavadhani et al., 2006;Zheng & Wang, 2001). Phenolic compounds have also been reported to provide antimutagenic and anticarcinogenic properties in humans when approximately 1.00 g of them is consumed daily through a diet rich in vegetables and fruits (Tanaka et al., 1998).  (2015) reported an average of 21.30 mg/g for flavonoid content in Z. joazeiro. Flavonoids are a group of phenolic compounds presented in fruits and vegetables that are characterized by a benzoy-pyrone structure (Cheng et al., 2000). The antioxidant activity of flavonoids depends on the structure and substitution pattern of hydroxyl groups (Kaurinovic et al., 2011;Zheng & Wang, 2001).
Flavonoids exited in herbs are important in human health because of their pharmacological activity as antioxidants and radical scavengers, which are particularly scavengers of most oxidizing molecules such as singlet oxygen and various free radicals (Schubert et al., 2007;Turkmen et al., 2006). There were significant correlations between chemical properties (Table 5)

| CON CLUS ION
The Z. spina-christi is a multi-purpose plant species that has high medicinal uses and can also be used as a pasture and landscape. Besides, due to the high nutritional value of its fruit, it can be considered as a horticultural crop. The present study showed that the accessions studied of this species have a high diversity in terms of morphological traits and chemical properties that can be widely used to introduce cultivars in breeding programs and the pharmaceutical industry. The obtained results can be used in selecting and introducing cultivars with the desired traits. Based on the traits related to fruit quality such as fruit weight, fruit skin color, and fruit flavor, as well as in terms of chemical characteristics related to medicinal properties such as total flavonoids and antioxidant activity, 17 accessions, including Shooshtar-1, Shooshtar-7, Shooshtar-10, Shooshtar-17, and Shooshtar-20, were superior that could be used in breeding programs or cultivated directly. The present results can be used in defining conservation strategies, genetic improvement, and crop production.

ACK N OWLED G M ENT
None.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

R E S E A RCH I N VO LV I N G H U M A N PA RTI CI PA NTS A N D/ O R A N I M A L S
None.

I N FO R M E D CO N S E NT
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.