Morpho-phytochemical screening and molecular diversity of pomegranate accessions grown in Halabja Governorate, Kurdistan Region-Iraq

Halabja governorate in the Kurdistan region-Iraq is famous for having high-quality pomegranate accessions. The current study was conducted to evaluate the morpho-phytochemical amount of pomegranate functional food and the genetic diversity as an important tool for the characterization of the genetic resources for germplasm management and the identification of the best genotypes for genetic improvement. There were highly significant morphometric differences (P ≤ 0.05) among 24 genotypes, whose mean values for fruit weight, peel thickness, and weight of 100 Arils, total flavonoid content (TFC) and total phenolic content (TPC), antioxidant activity, total soluble solid content (TSS) were 299.21 g, 3.47 mm and 38.59 g, 54.50,47.97 µg/ml, 21.08 µg/ml and 14.59 Brix, respectively. In addition, inter-simple sequence repeat (ISSR) was utilized to assess the genetic diversity of the collected pomegranate genotypes. Twelve random primers of produced products plus a number of the amplified primer bands ranging from 3 to 12 and total number of 83 amplified bands were produced, among which 78 bands were polymorphic and 5 bands were monomorphic. The highest, lowest, and mean values of polymorphic bands were (11, 3, and 6.5), respectively. The PIC values ranged from 0.58 to 0.90. The dendrogram clusters for all the selections showed dissimilarity coefficients ranging from 0.22 to 0.23 (G4 vs. G5) to 0.63 (G13 vs. G14), and five groups (A, B, C, D, and F) with a mean dissimilarity (0.49). According to the results, morphometric and biochemical properties are significant aspects of development, discernment, and classification. ISSR markers allow the identification of different selections and assessing the genetic similarity among pomegranate accessions, which would facilitate their use as genetic stock in breeding programs.


Introduction
Pomegranate (Punica granatum L.), a vital horticultural plant belonging to the Punicaceae family, is one of the most important and oldest edible fruits (Holland and Bar-Ya'akov 2018).It is native from Iran to the Himalayan Mountains in northern India, and domestication is reported to have begun around 3000-4000 BC (Silva et al. 2013).Many different geographical regions, a wide range of weather and soil conditions are extremely adaptive to the growth of pomegranate fruit (Holland et al. 2009).Recently, increasing interest in pomegranate fruits as functional foods has been documented from both the scientific community and economically, which has significantly Vol:.( 1234567890) increased the number of publications focused mainly on the fruit's characteristics, benefits, and nutritional composition (Melgarejo et al. 2020).In addition, nutritional and healthful pharmaceutical sources with a symbol of beauty and fertility are major reasons for requesting this fruit.Therefore, the demand for pomegranate consumption has been significantly increased by consumers in the last two decades (Melgarejo et al. 2020).
In addition, pomegranate accessions for economic and industrial purposes were greatly necessary and selected depending on the development of agronomic and biochemical potential, databases, and genetic programs (Ejjilani et al. 2022).Thus, many researchers have focused mostly on local cultivars for morphological fruit properties such as fruit fresh weight, skin thickness, and physio-chemical descriptors (Hmid et al. 2018).Morphometric and biochemical characters can enhance the significance of the discernment and classification of local germplasm such that it could be the basis for further use and also contribute to the improvement of industrial processes using pomegranate as a raw material (Ejjilani et al. 2022).On the other hand, local cultivars could also provide a source of genes for breeding, especially in pedoclimatic conditions (Karapetsi et al. 2021).All of the above aspects are guiding pillars for the enhancement and development of the species and contribute to its competitiveness globally.Therefore, to obtain novel pomegranate cultivars with higher yield and better quality, it is essential to screen available pomegranate genetic resources and conserve them for breeding programs (Ejjilani et al. 2022).
The pomegranate genome is estimated to be 328 Mb.This diploid species has 16 or 18 chromosomes (Qin et al. 2017).DNA marker provide a reliable approach for assessing genetic diversity based on DNA sequence polymorphism within a population (Karapetsi et al. 2021).Hence, molecular markers have been recently utilized efficiently to assess genetic diversity among pomegranate cultivars, which differ in the generation of polymorphic bands and their complexity (Amar and El-Zayat 2017).Amongst them, inter simple sequence repeats (ISSRs) are preferred due to their advantages of being codominant, having redundancy in the genome, showing high polymorphism, and exhibiting repeatability (Madadi et al. 2017).Because of this, the availability of information on the genetic relationships between accessions is very important.Therefore, ISSRs are recommended to study genetic diversity in pomegranates.They are detecting genotypes with desirable traits in breeding programs (Karapetsi et al. 2021).The Kurdistan region, particularly Halabja governorate, is famous for growing pomegranate trees.
The aim of this study was to investigate the pomological and physico-chemical characteristics for 24 pomegranate accessions and also determine the overall degree of genetic polymorphism using ISSR markers.The generated knowledge will be useful for its hybridization and germplasm improvement through developing cultivars with high fruit quality for potential commercial production.

Morphological characteristics
The six fully ripe fruits were randomly collected from each tree, The ripeness degree was detected visually when fruits lost all traces of green color and became tinged with a yellowish or reddish color, depending on the genotypes.After that, morphological traits, such as the weight (gm) per fruit, with a weight of 100 Arils (gm) were recorded using an accuracy balance, while the thickness of the pomegranate peel (mm) was determined using a digital caliper (Ejjilani et al. 2022).

Biochemical juice analysis
A juice sample was extracted from each fruit by manually squeezing the arils.After that, the chemical analyses of total flavonoid content (TFC), total phenolic content (TPC), ABTS antioxidant, and total soluble solids (TSS) were estimated as indicate below.

Total flavonoid content (TFC)
The colorimetric assay was used for the determination of the total amount of flavonoid in pomegranate juice using three replicates.To initiate the assay, 100 μl of the sample extract was used according to the Lateef et al. (2021) protocol with some modifications.

Total phenolic content (TPC)
The total phenolic content of pomegranate juice was determined using the Folin-Ciocalteu reagent.To setup the protocol, 30 μl of each pomegranate juice sample were mixed with 2 ml of the Folin Ciocalteu reagent as described by Lateef et al. (2021) and Tahir et al. (2022) with some modifications.The mean value was obtained from three samples.

ABTS antioxidant
Antioxidant activity of pomegranate juice was determined by the ABTS + decolorization assay.The protocol started by adding 10 μl of juice samples to 3 ml of diluted ABTS radical cation solution.All tests were done three times.The protocol was according to Re et al. (1999) with some modifications.Total soluble solids (TSS) A digital refractometer was used to determine TSS from juice, and three replications were used to calculate the average value of each genotype.The TSS obtained was expressed as a Brix unit measured at 20 °C (Aziz and Tahir 2022;Rasul et al. 2022).

Extraction of genomic DNA from leaves of pomegranate
Total DNA from fresh leaves was extracted according to Ahmed et al. (2022) protocol with some modifications.Fresh leaves were ground by liquid nitrogen, genomic DNA was collected, and the quantity and quality of isolated genomic DNA were determined using a nanodrop spectrophotometer, and thereafter stored at − 20 °C.

ISSR marker analysis
Table 2 shows the primers used for this study.The designed primers were from Rasul et al. (2022) and Tahir et al. (2023).Amplification of ISSR was performed in a 25 μl reaction volume containing 50-100 ng genomic DNA, × 10 master mix buffer, 10 pMOL of single primer.The thermocycler was programmed as follows: an initial cycle of 5 min at 94 °C, followed by 36 cycles of 1 min at 94 °C, an annealing temperature of 50 °C for 1 min, an extension step of 2 min at 72 °C, and a final extension step of 10 min at 72 °C.Polymerase chain reaction products were separated by gel electrophoresis on 2% agarose gels with 1X TBE (tris-base, boric acid, and EDTA) buffer and added 0.20 μg/mL ethidium bromide.Gels were run at a constant voltage of 85 V/ cm for 90 min.The stained gel was visualized by an ultraviolet transilluminator, and a digital imaging system was used to capture it.

Statistical analysis
The analysis of variance and the Duncan's new multiple range tests were performed to analyze the differences between the means (p ≤ 0.05) among pomegranate accessions using XLSTAT software version 2017.In addition, the scorable bands were coded manually as either present (1) or absent (0).These data were used to create a dendrogram using the Jaccard method by using XLSTAT 2017 software (Tahir et al. 2019).Principal component analysis (PCA) was performed using XLSTAT 2017 software to indicate the association among different genotypes.Power Marker version 3.25 software (Liu and Muse 2005) was utilized (Liu ad Muse 2005).In addition, to calculate polymorphism information content (PIC), diversity of genes, and frequency of major alleles.The software GenAlEx (version 6.5) was also used (Peakall and Smouse 2006) to evaluate molecular variance between and within population.A model analysis of the software STRU CTU RE (version 2.3.4) was performed for the population structure to infer the genetic structure and clarify the number of subpopulations (Pritchard et al. 2000).

Morphometric characterization
Fruit weight, thickness of pomegranate peel, and weight of 100 Arils are key traits for the fresh market and breeding programs (Arlotta et al. 2022).Data in  Regarding TSS, which estimate the level of dissolved sugars but also the presence of other soluble compounds such as acids, salts, water-soluble vitamins, and other chemical compounds, our results are similar to those of Adiletta et al. (2018).In the investigated pomegranate genotypes, the TSS ranged from 17.0 to 18.5°Brix.'Granato'and 'Roce' had the lowest total soluble solid content, corresponding to the lowest reducing sugars.Ejjilani et al. (2022) confirmed that TSS had a significant variability ranging from 13 to 17 Brix.The TSS values ranged from 16.167 ('Salakhani') to 17.467 ºBrix ('Wonderful') in fresh juice (Abdulrahman et al. 2021).Hasnaoui et al. (2011) also reported similar TSS values in wild and cultivated pomegranate fruits, with respective ranges of 17.57-19.99ºBrix and from 13.13 to 16.55 ºBrix.Other researchers confirmed that TSS was between 14.31 and 15. ºBrix (Melgarejo et al. 2011), 13.73-17.60 ºBrix (Mena et al. 2011) and 12.36-16.32 ºBrix (Martínez et al. 2012). 11.4-16.2°Brix in Iran (Sarkhosh et al. 2009).
It is clear that the differences in chemical composition among the 24 accessions confirmed that varietal distinction impacts the biosynthesis of the proximate constitution and phytochemical components in pomegranate accessions.Therefore, this significant indicated the presence of an interesting variant among the pomegranate accessions with the ability to accumulate these compounds and suggested differences in genetic potential between them.Many researchers confirmed that phytochemical parameters may be influenced by many factors, including the growth year, growing region, cultivar, and maturity degree of the fruit (Abdulrahman et al. 2021).In addition, it is important to emphasize that several factors related to genotype, climate, and agricultural practices may explain differences for the juice (Ejjilani et al. 2022).Furthermore, researchers verified that the polyphenol and antioxidant potential levels of pomegranate juice could be significantly affected by the genotype (anthocyanin composition, and phenolic content) of some pomegranate cultivars with temperature during the maturity period and latitude of the growing region (Li et al. 2015).Moreover, the desirable trait in pomegranate juice is TSS, which is definitely important for fresh consumption, the food industry, and processing as it combines sweetness and flavor (Khadivi et al. 2020).These results showed the levels of sugar and other physicochemical properties were different among various cultivars of pomegranate, which could be due to the existence of high genetic heterogeneity within the cultivars (Tehranifar et al. 2010).Eventually, morphological characteristics were used for descriptive purposes and are now commonly utilized to differentiate cultivars.Furthermore, the ecosystem has a strong influence on morphological traits, and there are time and cost considerations.Morphology alone does not distinguish among different cultivars or accesions that are morphologically similar.Therefore, the molecular fingerprinting of a plant variety is extremely important for protecting plant breeders' rights (Pasquali et al. 2022).

ISSR markers showing polymorphism
In the current study, all ISSR primers gave scorable, good amplification products and presented high polymorphisms among the 24 pomegranate genotypes examined.The 12 random ISSR primers were utilized and generated 78 scorable polymorphic bands (Table 5 and Figs. 1, 2).The number of amplified bands in our study ranged from 3 to 12 for ISSR25 and ISSR845, as well as for ISSR847, respectively.In total 83 bands were scored among which 78 bands were polymorphic and five bands were monomorphic.The mean values, highest and lowest of polymorphic bands were 6.5, 11 and 3, respectively.Our results showed that the polymorphism information content (PIC) values ranged from 0.58 to 0.90 with an average value of 0.79 per ISSR marker.The major allele frequency ranged from 0.21 to 0.58, with 0.31 as the average allele per marker; and 58% were observed for the ISSR25 marker, which has major alleles with the highest frequency.Diversity detected in the cultivated pomegranates ranged from 0.61 to 0.91, with an average of 0.81 per ISSR marker.
Our results are comparable to those of Talib et al. ( 2011), who found that the total number of polymorphic markers and percentage of polymorphism were 64% and 36.99%,respectively.The mean PIC value was 0.163, and the lowest and highest PIC values were 0.099 (ISSR5 and ISSR6) and 0.257 (ISSR11), respectively.Nine primers were detected for the diversity and the genetic differences among different pomegranate cultivars; There were 88 bands, of which 72 had a polymorphism of 80.66% (Almiahy and Jum'a 2017).Al-Mousa et al. ( 2019) evaluated the genetic variation among five pomegranate genotypes using 20 ISSR markers.Twelve ISSR primers were successfully used as fingerprinting tools and amplified 137 DNA fragments, of which 78 were polymorphic (56.93%).Primers 17, NLSSR3, and 16, which showed the highest polymorphism percentages of 90.41, 80.00, and 76.47%, respectively, with the highest a number of unique bands (6, 6, and 9, respectively).Genotype C amplified the highest number of DNA fragments (115) and unique bands (13).The PIC ranged from  2014) and the analysis of genetic diversity among the different cultivated pomegranate genotypes from Azerbaijan using the ISSR markers (Hajiyeva et al. 2018).
Informative molecular markers are commonly exploited for genetic diversity measurement improvement, genotypes that identify genotypes, Because primers with more alleles cover more of the genome, they are better at detecting variation in genetic make-up.It is possible that the variation in primer polymorphism was due to the fact that different primers have different nucleotide sequences.The results of this study show that ISSR markers were useful for analyzing pomegranate genetic diversity and that the accessions included in our research have important genetic variation.
Clustering and population structure analysis of pomegranate genotypes Multivariate statistical methods are important in studying genetic diversity.One of them, cluster analysis, divides individuals into graphs based on intervals.The unweighted pair-group method (UPGMA) was used based on the Jaccard similarity coefficients to analyze the data, assessing and clustering for connections among pomegranate accessions (Rasul et al. 2022).The dissimilarity coefficients ranged from 0.23 (G4 vs. G5) to 0.63 (G13 vs. G14), and all 24 Fig. 3 The UPGMA method created a cluster tree based on 12 ISSR markers among 24 pomegranate accessions pomegranate accessions were grouped into 5 groups (A, B, C, D, and E) with a mean dissimilarity of 0.49 for 12 ISSR markers (Fig. 3).Cluster A includes only (G13 and G14), cluster B (G8 and G10), cluster C (G7, G9, G11, G19, G23, and G24), cluster D (G16, G6, G4, G5, G15, G2, G12, G3, G1, G18, and G22), and cluster E (G17, G20, and G21).In addition, other research indicated that the characterization of genetic markers for cultivar is also very promising by ISSR, which indicates a high level of polymorphism in pomegranate plants.Almiahy and Jum'a (2017) reported that they evaluated the genetic diversity and the relationship among 10 genotypes of pomegranates from different geographical regions of Iraq.According to the results of ISSR genetic distance using the UPGMA method, they showed that the 10 genotypes were distributed into two main groups.

Analysis of molecular variance
The analysis of molecular variance (AMOVA) for the 24 pomegranate accessions based on the ISSR markers indicated that 87% of the total variation was within the populations and 13% of the differences among populations (Table 6).Clustering and structure analyses were confirmed based on AMOVA results.

Structure analysis among 24 accessions by ISSR markers
Allele frequency using STRU CTU RE analysis was utilized for 24 pomegranate accessions following Evanno et al. (2005).Results showed that four groups or subpopulations were represented by colors, including group 1 (red line), group 2 (green line), group 3 (blue line), and group 4 (yellow line) according to Delta K genotypes (Fig. 4B).In addition, many genotype combinations were observed with more than one background, including 2, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 23, and 24, wheeras the rest had other background that could have had a complex history from the gene flow among taxa that caused connecting intercrossing or practicable consequences.In addition, altering weather conditions within the locations may be contributing to the high variability between accessions.
The right number of clusters (K) was determined and detected in a sample of individuals.The highest value of K was 4 (Fig. 4A).Unlike the dendrogram plot, our analysis shows that the genotypes can be divided into four distinct populations.The accessions distribution patterns in dendrogram plots are also the most similar to the distribution patterns in STRU CTU RE analysis.The clusters' accessions contain the majority of the diversity, and could imply the presence of admixtures among accessions.

Conclusion
The current study is the first attempt to associate morpho-phytochemical analysis and genetic diversity for pomegranate accessions from Halabja, noreast Iraq.Significant pomegranate accessions-dependent variability has been observed for the qualitative and chemical characters among the pomegranate accessions and the ISSR markers were suitable for the assessment of variability in the 24 pomegranate germplasm included in our study.Many morphological desirable traits between pomegranate genotypes including fruit weight, thickness of pomegranate peel and weight of 100 arils have a significant role to identify the consumer preference (Khadivi-Khub et al. 2015).

Fig. 1
Fig. 1 ISSR profile of the 24 pomegranate accessions amplified with the UBC-841 primer, M: DNA ladder Previous research by Al-Moussa et al. (2019) evaluated pomegranate genetic diversity and accessions or cultivar relationships (Al-Mousa et al. (2019).They evaluated the genetic variation among five pomegranate genotypes using 20 ISSR markers and found that the genetic distance ranged from 0.26 to 0.37.Some genotypes showed wide divergence (C and A; C and D), while genotypes A and B were closely related.The UPGMA dendrogram grouped the genotypes into two

Fig. 4
Fig. 4 Number of subpopulations considering the value of K by the ΔK procedure

Table 3
(Harel-Beja et al. 2015)5.87gto825.82gandwereobserved with an average fruit weight of 413.96 g.Adiletta et al. (2018)observed that Granato', 'Roce' and 'Wonderful' showed a thin thickness of skin (3-3.9 mm), while 'Dente di Cavallo', 'San Pietro' and Mondrone Dolce' were ranked among pomegranates possessing thick skin (5.1-6 mm).A previous researcher weighted 100 Arils (g) among differing genotypes, and noted that the mean value for the weight of 100 arils was 34.7 g(Chater et al. 2018).Wetzstein et al. (2011)also noticed that the weight of 100 Arils for 'Wonderful' was 35.7 g, which is very close to and in agreement with our results.It is clearly noted that the pomological variation recorded on the 14 cultivars herein evaluated fall within the variation intervals for each single trait.Most of the fruit traits are greatly affected by the orchard management and environmental conditions, although in pomegranate these traits are also controlled by multiple genes(Harel-Beja et al. 2015).

Table 5
Summary of PCR-ISSR amplified products including marker names, number of amplified bands, number of polymorphic bands, number of monomorphic bands, major allele These results indicated that the ISSR technique was sufficiently informative and powerful to assess genetic variability in pomegranate.Our research agrees with the results ofAjal et al. (