Morphological and chemical characterizations of jujube (Ziziphus jujuba Mill.) to select superior accessions

Abstract The fruits of jujube (Ziziphus jujuba Mill.) are consumed worldwide as food and herbal medicine because of their impact on human health and benefits. Here, phenotypic and chemical variation of this species was investigated to select superior accessions. The selected accessions showed significant differences based on the measured characteristics. Fresh fruit weight varied from 2.72 to 6.42 g with an average of 4.54, while dry fruit weight ranged from 0.89 to 2.57 g with an average of 1.55. Total phenolic content ranged from 1.69 to 14.05 mg gallic acid equivalent (GAE) g−1 fresh weight (FW) and total flavonoid content varied from 0.25 to 2.01 mg quercetin equivalents (QE) g−1 FW. Total anthocyanin content varied from 5.98 to 76.32 µg CyE g−1 FW. Radical scavenging activity (2,2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate [DPPH]) ranged from 1.32 to 5.82 mg ascorbic acid equivalents (AsAE) g−1 FW, while ferric reducing antioxidant power (FRAP) varied from 35.37 to 93.35 µM FeSO4. The present study showed high diversity in morphological and chemical properties of jujube accessions. 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 anthocyanin content and antioxidant activity, 13 accessions were superior and are recommended to use in breeding programs. The commercial orchard of those best accessions should be extensively constructed to take advantage of the high yield of this tree as a crop and its medicinal properties.

Jujube seeds are used because of their effect on reducing insomnia and anxiety. Jujube is used as a blood purifier, nerve sedative, stomach tonic, laxative, and antitussive (Gao et al., 2011).
Genetic diversity is the basis of agricultural programs and development. Genetic resources are the primary basis for creating promising improved cultivars, which today are considered to be the most valuable national resources and primary resources of any country (Behera et al., 2008). Gene banks are created to collect genetic material to examine genetic diversity and long-term protection. Materials collected in terms of population, species, and gene pool need to be diverse. In any country, it is essential to study the genetic diversity of plant species to use them in breeding, conservation, management, and establishment of plant species (Ghazaeian, 2015). Iran is one of the richest countries in the world in terms of plant genetic resources.
The country has an advantage in terms of plant diversity, which is part of the existing biological richness, due to exceptional ecological characteristics (Khadivi, 2018).
Morphological characterization is one of the first steps to identify plant genetic resources. Evaluation of morphological traits, genetic resources, and the collection of desirable traits in one cultivar is one of the important breeding goals in plants (Khadivi-Khub & Anjam, 2014). Also, knowledge about the chemical properties of a plant species can help the food and pharmaceutical industries.
Superior accessions can be used to produce different food, pharmaceutical, and antioxidant products (Halliwell & Gutteridge, 1990).
The present research aimed to investigate the phenotypic and biochemical characterizations of jujube (Z. jujuba) and then to select superior accessions for cultivation in the orchards as well as use in future breeding programs.

| Plant material
In the present study, 100 local accessions of jujube (Z. jujuba) in a collection site from Tootkan area in Lorestan province/Iran were selected.
The sampled accessions were chosen randomly based on their health and yield. Tootkan area is located at 32°54′57″N latitude, 49°39′31″E longitude, and 2025 m height above sea level (Figure 1).

| Morphological and pomological analysis
Phenotypic diversity of the accessions studied was investigated using 39 morphological traits according to the International Plant Genetic Resources Institute (IPGRI) guideline (Saha, 1997). The 50 mature leaves and 50 mature fruits in each accession were used to record the related characters. The length and width of leaf, fruit, and stone were measured using a digital caliper. Also, fresh fruit weight, dry fruit weight, and stone weight were measured using an electronic balance with 0.01 g precision. Furthermore, the qualitative characters were estimated based on rating and coding (Table 2) (IPGRI, Saha, 1997).

| Total phenolic content
For extraction, samples (1.00 g) were homogenized using 10.00 ml of 80.00% methanol, and the mixtures were centrifuged at 4472 g (revolutions per minute) for 10 min. Supernatants were collected and analyzed for total phenolic content and antioxidant activity assays. Total phenolic content of fruit extracts was 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 10-fold diluted 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 fruit 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 ml of fruit extracts was mixed with 2 ml of 2.00% aluminum chloride (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.

| Total anthocyanins
Total anthocyanins in fruits were extracted with mixing 0.50 g of fresh materials with 10 ml of acidified methanol containing 1% HCl (v/v). The extract was centrifuged at 4472 g for 10 min and the absorbance of supernatants was recorded at 530 nm (Nogues & Baker, 2000). The content of anthocyanins in fruit samples was calculated using the extinction coefficient of cyanidin-3-glucoside (cyd-3-glu) and expressed as mg Cyd-3-glu equivalents.

| Radical scavenging activity
The scavenging activity of the extracts prepared on 2,2-diphenyl-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 DPPH scavenging effect ( % ) = (A control − A sample ∕A control ) × 100.
scavenging activity of fruits was expressed as mg ascorbic acid equivalents (ASAE) 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 of 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). To 20 μl of fruit extract was added 3.00 ml of FRAP reagent, and the reaction mixtures were placed in a 37°C 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
The Spearman correlation coefficient with SPSS software (PSS Inc., Norusis, 1998) was applied to determine the simple correlations between the traits. Principal component analysis (PCA) was used to study the relationships among the accessions with SPSS software. Also, the scatter plot was created using the first three principal components (PC1/PC2/PC3) with SPSS software. The Ward's method and Euclidean distance were used to hierarchical cluster analysis (HCA) with PAST (PAleontological STatistics) software (Hammer et al., 2001).

| Morphological descriptions
Out of 39 morphological traits measured, nine characters, including branch color (gray in all the accessions), leaf density (high), leaf shape (ovate), leaf apex shape (obtuse), leaf upper surface color (dark green), leaf lower surface color (light green), leaf serration (present), fruit shape (oval), and fruit stone surface (coarse), had no differences among the studied accessions. Thus, they were excluded from analysis and Tables. The morphological traits without variation are more homogeneous and repeatable among the accessions, and therefore may be considered as stable traits. The remaining 30 morphological characters and also all five chemical properties had high variabilities. The highest CV (400.00%) belonged to thorn presence on annual shoot (Table 1).

Tootkan
The accessions showed three types of growth habit, including spreading (80 accessions), semi-erect (12), and erect (8). Tree growth vigor, tree height, canopy density, branching, and branch density were predominantly high (Table 2). In general, the higher tree growth vigor, the higher tree height, branching, and branch density, and as a result, the fruit-related traits will be more desirable, and thus the yield, fruit length, and dry fruit weight are increased (Hosseini et al., 2018).
The fruits in the majority of accessions were ripened in late-

September (70 accessions). Most of the accessions (98 out of 100)
showed a high yield. Fruit skin color was brown in 99 accessions, and also fruit flesh color was light green in 99 accessions (Table 2). In the study of a jujube collection from Ukraine, skin color showed strong diversity, ranging from brown-yellow to dark brown (Grygorieva et al., 2014).  (Ghazaeian, 2015). Also, the range of 0.14-6.33 g has been recorded for fruit weight in jujube from China (Liu et al., 2009).

TA B L E 1
Stone length ranged from 13.41 to 19.23 mm, stone width varied from 6.91 to 11.48 mm, and stone weighed from 0.31 to 0.47 g (Table 1). In the study of a jujube collection from the north of Iran, the ranges of stone length, stone width, and stone weight have been reported as 10.20-13.50 mm, 3.80-7.90 mm, and 0.26-1.93 g, respectively (Ghazaeian, 2015). Besides, in the study of a jujube collection from Ukraine, the ranges of stone length and stone width have been reported as 12.84-28.67 mm and 5.06-9.74 mm, respectively (Grygorieva et al., 2014). Also, the ranges of 0.28-0.65 g (Sivakov et al., 1988) and 0.06-1.90 g (Ghosh and Mathew, 2002)  Tree growth habit -Spreading (80) Semi-erect (12) Erect (8) Tree growth vigor --Intermediate (7) High (93) Tree height --Intermediate (4) High (96) Tree trunk type -Single-trunk (93) Multitrunk (7) (Table 1). Zhang et al. (2010) reported that the value of total phenolic content in Z. jujuba was 32.80 mg GAE g −1 DW. Phenolic compounds play an essential role in plants as primary antioxidants or free radical scavengers, and antioxidant activity is due to their redox activity, which plays a key role in the uptake and sterilization of free radicals, quenching singlet and triple oxygen, and decomposition of peroxides (Himesh et al., 2011). Phenolic compounds are a group of antioxidant agents that act as terminators of free radicals, and bioactivity may be due to their ability to chelate metals, inhibit lipoxygenases, and free radical scavenging (Lin et al., 2005;Mallavadhani et al., 2006). 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).
Total anthocyanin content was significantly and positively correlated with ripening date (r = .28), yield (r = .22), fresh fruit weight TA B L E 3 The most important fruit quality-related traits and chemical properties of superior accessions of Z. jujuba in this investigation and ferric reducing antioxidant power (r = .87) and corresponded with the previous findings in jujube (Gao et al., 2012;Wang et al., 2011;Zhang et al., 2010).
The scatter plot created using PC1/PC2/PC3 showed variations among the accessions (Figure 3). Tootkan-29 accession showed high differences with other, characterized by low fresh fruit weight, dry fruit weight, and fruit flesh thickness. Also, five accessions, including Tootkan-1, Tootkan-3, Tootkan-8, Tootkan-9, and Tootkan-10, formed another group, characterized by moderate fresh fruit weight, dry fruit weight, and fruit flesh thickness. The remaining accessions were placed into the same group.
Besides, HCA with Ward dendrogram showed that the accessions were clustered into two major clusters (Figure 4). The first cluster (I) con-

| CON CLUS ION
Genetic diversity of indigenous genotypes and their related wild accessions is the primary basis for many agricultural research programs, especially breeding programs. Therefore, it is necessary to know the characteristics and potential of these valuable resources collected to use in research programs, so that specialized experts can use them to improve the characters in their programs. The present study showed high diversity in morphological and chemical properties of some jujube accessions. Based on the traits related to fruit quality such as high fruit weight, soft fruit flesh texture, brown fruit skin color, and sweet fruit flavor, as well as in terms of chemical characteristics related to medicinal properties such as higher total anthocyanin content and higher antioxidant activity, 13 accessions, including Tootkan-50, Tootkan-97, Tootkan-99, Tootkan-61, Tootkan-55, Tootkan-59, Tootkan-67, and Tootkan-38, were superior. The commercial orchard of those best accessions should be extensively constructed to take advantage of the high yield of Z. jujuba as a crop and its medicinal properties.

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.