Morpho-phytochemical Screening and Molecular Diversity of Pomegranate Accessions Grown in Halabja Governorate, Kurdistan Region-Iraq

doi:10.21203/rs.3.rs-2851963/v1

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. In this study, phenotypical parameter, highly significant morphometric differences (P ≤ 0.05) among 24 genotypes, with the mean values of 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) 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 between 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. Alongside with the ISSR marker, which is a useful technique for allowing the identification of different selections and assessing the genetic similarity among studied pomegranate accessions, this would facilitate their use as identified genetic stock in future breeding programs.

Horticulture

genetic relationship

genetic structure

molecular marker DNA

physiochemical parameters

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 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 side, local cultivars could also provide a crucial 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 better orienting breeding programs (Ejjilani et al. 2022).

Genetically, the pomegranate genome is estimated to be 328 Mb and a diploid species with the number of the chromosome is (2n = 2x = 16 and 18) (Qin et al. 2017). Consequently, the molecular marker is a more reliable approach for assessing genetic diversity based on DNA sequence polymorphism to determine plant genotype within a population (Karapetsi et al. 2021). Hence, many molecular markers, such as ISSR, RAPD, SSR, SRAP, RFLP, AFLP, and ISTR, have been recently utilized efficiently to adjust genetic diversity among pomegranate cultivars, which differ in the generation of polymorphic bands and their complexity (Amar and El-Zayat 2017). Amongst them, ISSRs are preferred due to their advantages of being codominant, having redundancy in the genome, having high polymorphism, and having repeatability (Madadi et al. 2017). Because of this, the availability of information on the genetic relationships between accessions is very important. Therefore, inter simple sequence repeat (ISSR) molecular markers are recommended to study genetic diversity in pomegranates, which are becoming easier and quicker to illustrate germplasm and detect genotypes with desirable traits in breeding programs (Karapetsi et al. 2021). Kurdistan region, particularly Halabja governorate, is famous for growing pomegranate trees. Additionally, the application of genetic diversity for pomegranate accessions in Halabja is vital for the implementation of the degree addressed. Subsequently, implementing multivariate statistical methods for pomegranates, including cluster analysis, population structure, and principal component analysis (PCA) in Halabja will be developed for the first time using the ISSR marker.

The aim of the current study is to investigate the pomological and physico-chemical properties 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 cultivar-breeding approach and with high fruit quality attributes for potential commercial production.

Plant material, location, and physical trait determination

The study area was Halabja, located in the north-east of Iraq between 35°10'59.22"N latitude and 45°58'59.05"E longitude (Alwaely et al. 2015). Three locations with twenty-four accessions of pomegranate trees as experimental materials were randomly selected and distributed, including Khurmal (6 accessions), Tepe Toleke (7 accessions), and Bawakochak (11 accessions). The physical shape of pomegranate genotypes, arils color, and test were shown (Table 1). Sensory evaluation was performed according to the (Gadže, et al. 2011; Mayuoni-Kirshinbaum and Porat, 2014). Parameters are measured with the naked eye and classified according to the (Juss and Shaw, 2014; Lahouel and Belhadj, 2021).

Table 1

Genotypes, local names, and locations pomegranate shapes Aril’s color and taste.
Accessions	The local names of pomegranates	Locations	Pomegranate shapes	Aril’s color	Test
A1	Salakhani mekhosh	Tepe Toleke	Oval	Dark red	Sour-Sweet
A2	Wonderful	Tepe Toleke	Oval	Dark red	Sour-Sweet
A3	Salakhani Trsh	Khurmal	Oval	Medium pink	Sour
A4	Karaly	Tepe Toleke	Oval	Light red	Sweet
A5	Sazan	Tepe Toleke	Round	white	Sour-Sweet
A6	Hanary Trsh	Bawa kirchak	Round	Medium pink	Sour
A7	Salakhni Nawrsh	Tepe Toleke	Oval	Dark red	Sour-Sweet
A8	Salakhani Twekl spy	Bawakirchak	Oval	Dark red	Sweet
A9	Salakhni Tweklastur	Khurmal	Round	Dark red	Sweet
A10	Twekl Sury Sherin	Bawakirchak	Round	white pink	Sour
A11	Twekl Sury Trsh	Bawakirchak	Oval	Light red	Sour
A12	Trsh Drezh	Khurmal	Round	Medium pink	Sour
A13	Twekl Astury naw spy	Khurmal	Oval	White	Sweet
A14	Twekl Astury Mekhosh	Tepe Toleke	Round	Dark red	Sour-Sweet
A15	Sherna	Bawakirchak	Oval	Dark red	Sweet
A16	Malesa	Bawakirchak	Oval	Light red	Sour-Sweet
A17	Sura	Khurmal	Oval	Medium pink	Sour-Sweet
A18	Salakhni Darajayak	Tepe Toleke	Round	Dark red	Sour-Sweet
A19	Sawa Hanra	Bawakirchak	Oval	Dark red	Sour
A20	Malesay Twekl tank	Bawakirchak	Round	Light red	Sweet
A21	Malesay Twekl astuer	Bawakirchak	Oval	Dark red	Sour-Sweet
A22	Benawk Trsh	Khurmal	Oval	Light red	Sour
A23	Sury Trsh	Bawakirchak	Round	Dark red	Sour
A24	Kawa Trshy Draja yak	Bawakirchak	Oval	Dark red	Sour-Sweet

Morphological parameters studied

The six fully ripe fruits were randomly collected from each genotype 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 TFC, TPC, ABTS antioxidant, and TSS were estimated as following.

Total flavonoid content (TFC) estimation

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.

Measurement of total phenolic content (TPC).

The total phenolic content of pomegranate juice was determined using the Folin-Ciocalteu reagent, briefly, to setup the protocol 30 µl of each pomegranate juice sample was mixed with (2 mL) the Folin Ciocalteu reagent as deeply described by (Lateef et al. 2021; Tahir et al. 2022) with some modifications. The mean value was obtained after three samples were repeated.

ABTS antioxidant

Antioxidant activity of pomegranate juice was determined by the ABTS + decolorization assay. The protocol was started by adding 10 µl of juice samples to 3 mL of diluted ABTS radical cation solution. All tests have been repeated three times. The protocol was followed according to (Re et al. 1999) with some modifications.

Measurement of 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 stored at -20° C.

ISSR marker analysis

Table 2 shows the primers were used for this study. The designed primers were got in our lab (Rasul et al. 2022; 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 minutes. The stained gel was visualized by an ultraviolet transilluminator, and a digital imaging system was used to capture it.

Table 2

Synoptic of ISSR primer length, size of amplicon, and annealing temperature.
Primer numbers	Primer names	Primer sequences	Annealing temperature (°C)
1	ISSR-6	3-GCCTCCTCCTCCTCCTCC-5	50
2	ISSR-9	CACACACACACACACATG	50
3	ISSR-10	GAGAGAGAGAGAGAGAGG	50
4	ISSR-11	ACACACACACACACACGG	50
5	ISSR-12	AGAGAGAGAGAGAGAGCT	50
6	UBC-811	GAGAGAGAGAGAGAGAC	50
7	UBC-812	GAGAGAGAGAGAGAGAA	50
8	UBC-825	ACACAC ACACACACACT	50
9	UBC-841	GAGAGAGAGAGAGAGACTC	50
10	UBC-847	CACACACACACACACAGC	50
11	UBC-845	CTCTCTCTCTCTCTCTTG	50
12	UBC-849	GTGTGTGTGTGTGTGTGA	50

Statistical analysis

To analyze the morpho-phytochemical one-way ANOVA, 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. In addition, to calculate polymorphism information content (PIC), diversity of genes, and frequency of major alleles, Power Marker version 3.25 software was utilized (Liu and Muse 2005) and to evaluate molecular variance between and within populations, the software GenAlEx (version 6.5) was also used (Peakall and Smouse 2006). A model analysis of the software STRUCTURE (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

Agronomically interesting parameters, including 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 Table 3 shows that phenotypical parameters varied, ranging from 92.27 g (A6) to 538.18 g (A9), 1.45 mm (A20) to 6.52 mm (A4), and 18.26 g (A14) to 55.48 g (A19), with an average of about 299.21 g, 3.47 mm, and 38.59 g for the fruit weight, thickness of the pomegranate peel, and weight of 100 Arils, respectively. Morphometric characterization of different accessions, particularly fruit weight, could depend widely on genetic and ecological condition (Melgarejo-Sánchez et al., 2015). Therefore, our results are comparable with those of other researchers, including Ejjilani et al., (2022) confirmed that fruit weight varied for 14 pomegranate genotypes from 168.0 to 558.0 g, with an average of about 339.5 g, and the thickness of the pomegranate peel ranged from 1.2 to 3.4 mm. Lahouel and Belhadj (2022) reported that the average fruit weight varies between a maximum value of 317.39 g for genotype (Messaad) and 135.85 g for genotype (Amourah) as a minimum value. Fruit weight ranged from 103.38 to 505.00 g (Akbarpour et al. 2009). Arlotta et al. (2022) reported that minimum and maximum differences among pomegranate genotypes ranged from 255.87 g to 825.82 g and were observed 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 pomegranate genotypes possessing thick skin (5.1-6 mm). A previous researcher disputed that for 100 Arils (g) weighted among differing genotypes, the mean value for the weight of 100 arils was 34.7 g (Chater et al. 2018) and also Wetzstein et al. (2011) reported that the weight of 100 Arils for ‘Wonderful’ was reported to be 35.7 g, which is very close to and in agreement with our results. It is clearly noted that the pomological variations recorded on the 14 cultivars herein evaluated fall within the variation intervals for each single trait, with evidently a remarkable phenotypic effect with regards to the environment effect as well. 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 3

Shows a comparison of the genotypes for the morphological properties of the pomegranate accession.
Accessions	Fruit weight (g)	Thickness of pomegranate peel (mm)	weight of 100 Arils (g)
A1	339.14 c-f	4.18 de	42.98 cde
A2	352.74 b-e	4.45 cde	44.63 cd
A3	185.74 gh	2.19 jk	34.80 f-i
A4	382.05 bc	6.52 a	41.08 c-g
A5	367.97 bcd	3.46 e-i	41.91 c-f
A6	92.27 h	2.87 f-j	27.76 i
A7	450.82 ab	2.37 ijk	44.36 cd
A8	237.39 fg	4.12 def	28.51 i
A9	538.18 a	5.52 abc	47.90 bc
A10	368.32 bcd	2.83 g-j	39.72 d-h
A11	256.35 efg	2.54 h-k	33.88 ghi
A12	275.10 d-g	4.81 bcd	35.49 e-i
A13	119.58 h	3.63 d-i	30.09 i
A14	118.11 h	5.64 ab	18.26 j
A15	245.08 fg	3.84 d-g	33.54 ghi
A16	305.55 c-f	3.81 d-h	43.12 cde
A17	112.27 h	3.72 d-h	32.89 hi
A18	390.93 bc	2.74 g-j	53.78 ab
A19	338.73 c-f	2.88 f-j	55.48 a
A20	389.93 bc	1.45 k	40.59 c-h
A21	318.83 c-f	2.14 jk	40.86 c-g
A22	247.03 fg	2.58 g-k	39.04 d-h
A23	349.83 b-e	2.88 f-j	35.42 e-i
A24	399.00 bc	2.19 jk	40.10 c-h
Minimum	92.27	1.45	18.26
Maximum	538.18	6.52	55.48
Mean	299.21	3.47	38.59
Standard deviation	121.23	1.35	8.78

Influence of genotypes on biochemical traits

Results regarding biochemical characterization of the genotypes are summarized in Table 4, which shows that significant differences have been detected among 24 pomegranate accessions. The maximum TFC and TPC content were recorded in accession 3 with 60.07 µg/ml and accession 1 with 63.26 µg/ml, the minimum level was observed in accession 10 by 49.03 µg/ml and accession 19 with 38.91 µg/ml, respectively. The mean values of TFC and TPC were 54.50 and 47.97 µg/ml, respectively. In addition, antioxidant variability also was observed: accession 10 had the highest level of antioxidant, which was 31.16 µg/ml while accession 12 had lower content with 15.79 µg/ml and the mean value of 21.08 µg/ml was also observed. The total soluble solids content (TSS) showed significant variability with an interval of variation of 8.97–18.70 Brix with mean value of 14.59 Brix. The highest TSS content was recorded in accession 9, and the lowest levels were observed in accession 19. Our results are comparable to those found by Meena et al. (2021), who demonstrated that the flavonoid content of juice extracted from five different cultivars of pomegranates varied from 15.72 to 20.3 (CE mg 100 g-1) whereas the phenol content varied from 53.63 to 104.33 (GAE mg 100 g-1). In addition, total phenol content among genotypes was investigated that ‘Granato’ and ‘Roce’ showed a similar (1100 mg GAE L^− 1) and significantly higher than ‘San Pietro’, ‘Mondrone Dolce’ and ‘Dente di Cavallo’ (750 mg L^− 1); the highest concentration was found in cv. ‘Wonderful’ (Adiletta et al. 2018). Recent studies by El Moujahed et al. (2022) demonstrate that the total flavonoid content of juice extracted from different Moroccan local cultivars of pomegranates ranged from 62.47 to 140.44 mg GAE/100 ml, whereas the total phenol content for local varieties ranged from 885 mg to 1348 mg GAE/L. Nikdel et al. (2016) confirmed that ‘Rubab’ had the highest content of total flavonoids (4.93 mg GAE/100 ml) and 'Shahvar’ had the minimum of total flavonoids (2.24 mg GAE/100 ml), Rubab’ had the highest content of total phenolics (6.36 mg GAE/100 ml), and the lowest content of total phenolics was found in ‘Ghand’ as (1.78 mg GAE/100 ml). Reading antioxidant activity, the highest and the lowest antioxidant activity were detected in ‘Rubab’ (86.77%) and ‘Shahvar’ (79.54%), respectively. Additionally, levels of antioxidant activity in other research were 10.37–67.46% for seven cultivars of pomegranate juice in Turkey (Tezcan et al. 2009) and 18.6–42.8% for eight pomegranate juices from Iran (Mousavinejad et al. 2009).

Regarding total soluble solids (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 found by (Adiletta et al. 2018). In the investigated pomegranate genotypes, the TSS range was from 17.0 to 18.5°Brix; in particular, ‘Granato’ and ‘Roce’ had the lowest total soluble solid content, corresponding to the lowest reducing sugars. Ejjilani et al. (2022) confirmed that the total soluble solids content (TSS) showed a significant variability ranging from 13.00 to 17.00 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 17.57–19.99 ºBrix and from 13.13 to 16.55 ºBrix. Another researcher confirmed that TSS was reported 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 twenty-four accessions confirmed that varietal distinction impacts the biosynthesis of the proximate constitution and phytochemical components in pomegranate accessions. Therefore, these variabilities indicated the presence of an interesting variant among the pomegranate accessions for 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 agents, 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 in terms of 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 that 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 plant varieties. Furthermore, the ecosystem has a strong influence on morphological traits, and there are time and cost considerations. Therefore, morphological criteria alone are not sufficient to distinguish some different varieties that are morphologically similar. Therefore, the molecular fingerprinting of a plant variety is extremely important for protecting plant breeders’ rights (Pasquali et al. 2022).

Table 4

Phytochemical and antioxidant analysis of 24 pomegranate juices accessions.
Accessions	TFC (µg/ml)	TPC (µg/ml)	Antioxidant (µg/ml)	TSS (Brix)
1	58.01 abc	63.26 a	28.47 ab	16.90 cd
2	58.95 ab	60.64 ab	19.33 d-i	17.70 bc
3	60.07 a	58.20 bc	18.91 e-i	17.80 b
4	56.70 abc	59.51 abc	19.48 d-i	17.40 bcd
5	54.64 abc	43.78 fg	16.93 ghi	14.23 g
6	52.40 abc	56.14 cd	17.07 ghi	15.77 f
7	58.76 ab	45.09 fg	19.05 e-i	15.93 ef
8	53.52 abc	61.95 ab	20.96 d-g	15.60 f
9	57.64 abc	53.90 de	23.02 cde	18.70 a
10	49.03 c	40.79 gh	31.16 a	16.60 de
11	53.52 abc	41.72 gh	26.06 bc	16.77 d
12	52.02 abc	51.84 e	15.79 i	15.70 f
13	52.96 abc	43.03 fgh	20.96 d-g	15.43 f
14	49.59 bc	42.10 gh	21.95 c-f	17.30 bcd
15	52.21 abc	41.54 gh	19.05 e-i	9.53 l
16	52.58 abc	43.22 fgh	22.88 c-f	12.27 ij
17	52.96 abc	43.41 fgh	16.01 hi	13.40 h
18	53.71 abc	45.28 fg	22.10 c-f	12.17 j
19	55.21 abc	38.91 h	22.03 c-f	8.97 l
20	54.64 abc	42.28 fgh	20.54 d-h	11.23 k
21	56.33 abc	44.34 fg	19.97 d-i	13.00 hi
22	53.15 abc	41.72 gh	23.94 cd	13.17 h
23	55.77 abc	41.91 gh	18.20 f-i	13.80 gh
24	53.52 abc	46.78 f	22.17 c-f	10.83 k
Maximum	60.07	63.26	31.16	18.70
Minimum	49.03	38.91	15.79	8.97
Mean	54.50	47.97	21.08	14.59
Standard deviation	3.98	7.89	3.92	2.73

ISSR markers with polymorphism parameters

To analyze the genetic diversity of plants, a molecular marker study was used. In the current study, all ISSR primers gave scorable, good amplification products and presented high polymorphisms among the 24 pomegranate genotypes examined. The twelve random ISSR primers were utilized and generated 78 scorable polymorphic bands (Table 5 and Fig. 1). The number of amplified bands in our study ranged from 3 to 12 for ISSR markers (ISSR25, ISSR845) and 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 were recorded 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. (58%) were observed for the ISSR25 marker, which has major alleles with the highest frequency. Values of the diversity gene were detected in the cultivated pomegranates at 0.61–0.91, with an average value of 0.81 per ISSR marker. In the genetic diversity research, the number of polymorphism bands with each marker technique employed may be extra-straightly linked to the ability to detect genetic differences among different accessions.

Therefore, our consequences 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. Researchers demonstrated that nine primers were detected for the diversity and the genetic differences among different pomegranate varieties; total bands gave 88 bands, of which 72 bands 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 twenty 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 number of unique bands of 6, 6, and 9, respectively. Genotype C amplified the highest number of DNA fragments (115) and unique bands (13). (PIC) ranged from 0.449 to 0.768, while genetic diversity ranged from 0.56 to 0.80. These results indicated that the ISSR technique was sufficiently informative and powerful to assess genetic variability in pomegranate. It is clear that a good technique to identify the pomegranate genotype is ISSR, these results are conducted with the results of Ajal et al. (2014). In addition, researchers confirmed the analysis of genetic diversity among the different cultivated pomegranate genotypes from Azerbaijan using the ISSR marker (Hajiyeva et al. 2018).

Informative molecular markers are commonly exploited for genetic diversity measurement improvement, genotypes that identify genotypes, populations for breeding, and conservation applications. As the genomic regions are highly polymorphic, different approaches to markers will produce varied consequences. The accuracy of the information should improve as the number of markers and genome coverage expand. The ISSR markers can be utilized to detect samples of plants that are closely linked in different populations. The ISSR markers with the most polymorphisms demonstrate how each locus can be used to estimate genetic diversity. Because primers with more alleles cover more of the genome, they are better at detecting variations in genetic make-up. It's possible that the variation in primer polymorphism is due to the fact that different primers have different nucleotide sequences. The results of this study show that ISSR markers are active in analyzing pomegranate genetic diversity and that the accessions studied have important genetic variation. An increased PIC value designates a higher degree of ISSR marker polymorphism in the genetic divergence analysis.

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 frequencies, gene diversity, and PIC value that were obtained from 24 pomegranate accessions using twelve primers.

Marker

name

Number of amplifed bands

Number of polymorphic bands

Number of monomorphic bands

Major allele frequency

Gene diversity

PIC

ISSR6

10

0

0.38

0.72

0.67

ISSR9

9

0

0.33

0.83

0.81

ISSR10

7

0

0.29

0.85

0.84

ISSR11

8

7

1

0.25

0.86

0.85

ISSR12

4

0

0.29

0.80

0.77

ISSR25

3

0

0.58

0.61

0.58

ISSR811

7

6

1

0.25

0.85

0.83

ISSR812

6

5

1

0.29

0.83

0.81

ISSR841

10

9

1

0.21

0.90

ISSR845

3

0

0.29

0.81

0.79

ISSR847

12

11

1

0.21

0.91

0.90

ISSR849

4

0

0.33

0.78

0.74

Total

83

78

5

Mean

6.91

6.5

0.41

0.31

0.81

0.79

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 pomegranate accessions were grouped into 5 groups (A, B, C, D, and E) with a mean dissimilarity of 0.49 for twelve 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). Many researchers were recorded regarding pomegranate genetic diversity and their relationships, including Al-Mousa et al. (2019). They evaluated the genetic variation among five pomegranate genotypes using twenty 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 unweighted pair-group method with arithmetic average (UPGMA) dendrogram grouped the genotypes into two clusters. In addition, Ajal et al. (2014) demonstrated the genetic relationship and diversity of Moroccan pomegranate germplasm among cultivars, and two main groups were designed, 1 and 2, based on the UPGMA dendrogram. In addition, studies indicate that the characterization of genetic markers for cultivar is also very promising by ISSR, which indicates an appreciably higher level of polymorphism in pomegranate plants. Almiahy and Jum'a (2017) reported that they evaluated the genetic diversity and the relationship among ten 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, A and B.

Molecular variance (AMOVA)

The analysis of molecular variance (AMOVA) for the 24 pomegranate accessions in the ISSR investigation confirmed 87% of the total difference within the populations and 13% of the differences among populations (Table 6). Clustering and structure analyses were confirmed based on AMOVA results.

Table 6

Illustrated the molecular variance (AMOVA) of the five populations for 24 pomegranate accessions.
Source	df	SS	MS	Est. Var.	%	P-Value
Among Population	3	89.125	29.708	2.369	13%**	0.001
Within Population	20	315.125	15.756	15.756	87%**	0.001
Total	23	404.250		18.125	100%

Structure analysis among 24 accessions by ISSR Markers

Allele frequencies using STRUCTURE analysis was utilized to determine for 24 pomegranate accessions by Evanno et al. (2005). Results showed that four groups or sub-populations 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), and the rest shown is another background that may 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 that the peak was started at 3 and ISSR markers number 12 showed the real K value with the highest value of K = 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 STRUCTURE analysis. The clusters' accessions contain the majority of the diversity that may be imply the presence of admixed among accessions.

The current study is the first attempt to associate morpho-phytochemical analysis and genotypic level of pomegranate accessions in Halabja. Significant pomegranate accessions-dependent variability has been observed for the qualitative and chemical characters among the pomegranate accessions and the ISSR markers were suitable utilized for the assessment of variability in the 24-pomegranate germplasm for the preservation of this species. Many morphological desirable traits between pomegranate genotypes including fruit weight, thickness of pomegranate peel and weight of 100 arils have been significant role to identify the consumer preference (Khadivi-Khub et al. 2015). It will also be helpful for pomegranate producers, industrial point of view, for selection of superior cultivars and conduct of breeding programs to the breeders for improvement in desirable traits. The variations could be originated from genotype of cultivar or due to agro-climatic and environmental conditions.

Due to the relatively low number of cultivars studied and primers tested are seemed in this area therefore, recently the progress of a molecular method is appropriated for pomegranate genetic polymorphisms surveying. The consequences of this study confirmed the findings that the genetic differences within each genotype, in which these distinctions may be caused by natural or artificial breeding of pomegranate over centuries with the interaction effect between gene and environment to adapt to the special environment. In addition, the local genotypes were affected, by mutation with wrong naming by pomegranate growers and that cause the incorrect name.

Overall, the application of degrees addressed to their usages and preservations value of the genetic variety in Halabja pomegranate accessions is vital for breeding. Quantitative traits loci (QTL) analysis and genome wide associated with the development of quantity and quality of pomegranate genotypes are our strong recommendations for upcoming research. Eventually, to assist future conservation and breeding programmers can utilize more different places, genotypes and also numerous type markers SNIPs SRAPs, ALFPs, Scot and SSRs are best option to progress diversity of pomegranates.

Acknowledgment: The author would like to thank grateful farmers (Shakhawn Bawakochaki, Ahmad Ali Tape Toleke and Dahahir Sharif khurmali) and many thanks to Sarwar Saber and Aram Akram to help us during collecting samples.

Funding: This research received no external funding.

Conflicts of Interest: The author declares no conflict of interest.

Abdulrahman AB, Mhamad HJ, Talb SS, Aljabary AM (2021) November. Physicochemical Properties and Phenolic Contents of Fresh and Concentrated Juice of Four Pomegranate Cultivars in Iraq. In IOP Conference Series: Earth and Environmental Science (Vol. 910, No. 1, p. 012093). IOP Publishing
Adiletta G, Petriccione M, Liguori L, Pizzolongo F, Romano R, Di Matteo M, (2018) Study of pomological traits and physico-chemical quality of pomegranate (Punica granatum L.) genotypes grown in Italy. European Food Research and Technology, 244, pp.1427–1438.
Ahmed AA, Sirwa AQ, Nawroz AT (2022) CDDP and ISSR markers-assisted diversity and structure analysis in Iraqi Mazu (Quercus infectoria Oliv.) accessions. All Life, 15 (1): 247–261 https://doi.org/10.1080/26895293.2022.2042401
Ajal A, Jbir R, Melgarejo P, Hernández F, Haddioui A, Hannachi A (2014) Efficiency of Inter Simple Sequence Repeat (ISSR) markers for the assessment of genetic diversity of Moroccan pomegranate (Punica granatum L.) cultivars. Biochemical Systematics and Ecology, 56: pp. 24–31. https://doi.org/10.1016/j.bse.2014.04.003
Akbarpour V, Hemmati K Sharifani M (2009) Physical and chemical properties of pomegranate (Punica granatum L.) fruit in maturation stage. American-Eurasian Journal of Agricultural & Environmental Sciences, 6(4): pp.411–416
Almiahy FH, Jum’a FF (2017) Evaluation of the genetic diversity of pomegranate accessions some Iraqi Pomegranate (Punica granatum L.) genotypes using ISSR marker. IOSR Journal of Agriculture and Veterinary Science, 10: 44–49.
Al-Mousa R, Al-Biski F, Alshaal A (2019) Molecular Characterization of Some Syrian Pomegranate (Punica granatum L.) Genotypes. Jordan Journal of Agricultural Sciences, 15: (3) https://jjournals.ju.edu.jo/index.php/jjas/article/view/52
Alwaely AA, Al-qaralocy HN, Al-Asadi KA, Chaichan MT, Kazem HA (2015) The environmental aftermath resulted from chemical bombardment of Halabja Territory for the period 1988–2014. International Journal of Scientific & Engineering Research. 6 (9): 40–44.
Amar MH, El-Zayat MA (2017) Utilization of ISTR, ISSR and SRAP molecular markers to reveal and classify Egyptian pomegranates ('Punica granatum'L.). Plant omics, 10 (5): 237–245. https://search.informit.org/doi/abs/10.3316/informit.358807885554620
Arlotta C, Toscano V, Genovese C, Calderaro P, Puglia GD Raccuia SA (2022) Nutraceutical content and genetic diversity share a common pattern in new pomegranate genotypes. Molecules, 27(2), p.389.
Aziz RR Tahir NAR (2022) Growth and fruit morpho-physicochemical diversity assessment of local melon genotypes. Tikrit journal for agricultural sciences, 22(3), pp.191–204.
Chater JM, Merhaut DJ, Jia Z, Mauk PA Preece JE (2018) Fruit quality traits of ten California-grown pomegranate cultivars harvested over three months. Scientia Horticulturae, 237, pp.11–19.
Ejjilani A, Houmanat K, Hanine H, Hssaini L, Elfazazi K, Hernandez F, Hmid I Razouk R (2022) Pomegranate morpho-chemodiversity: computational investigations based on in-vivo and in-vitro screening. Heliyon, 8(5), p.e09345.
El Moujahed S, Dinica RM, Cudalbeanu M, Avramescu SM, Msegued Ayam I, Ouazzani Chahdi F, Kandri Rodi Y Errachidi F, (2022) Characterizations of Six Pomegranate (Punica granatum L.) Varieties of Global Commercial Interest in Morocco: Pomological, Organoleptic, Chemical and Biochemical Studies. Molecules, 27(12), p.3847.
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study Molecular Ecology 14(8): 2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.
Gadže, J., Prlić, M., Bulić, M., Leko, M., Barbarić, M., Vego, D., Raguž, M. (2011). Physical and chemical characteristics and sensory evaluation of pomegranate fruit of (Punica granatum L.) cv." Glavaš". Pomologia Croatica: Glasilo Hrvatskog agronomskog društva, 17(3–4), 87–98.
Hajiyeva SV, Akparov ZI, Hasanov NA, Mustafayeva ZP, Hajiyev ES, Mammadov AT, Izzatullayeva VI, Babayeva SM, Sharifova SS, Mammadov AM, Abbasov MA (2018) ISSR Analysis of Variability of Cultivated Form and Varieties of Pomegranate (Punica granatum L.) from Azerbaijan. Russian journal of genetics, 54(2): 188–197.https://doi.org/10.1134/S1022795418020072
Harel-Beja R, Sherman A, Rubinstein M, Eshed R, Bar-Ya’akov I, Trainin T, Ophir R, Holland D (2015) A novel genetic map of pomegranate based on transcript markers enriched with QTLs for fruit quality traits. Tree Genet Genomes 11:109
Hasnaoui N, Mars M, Ghaffari S, Trifi M, Melgarejo P, Hernandez F (2011) Seed and juice characterization of pomegranate fruits grown in Tunisia: comparison between sour and sweet cultivars revealed interesting properties for prospective industrial applications. Ind. Crop. Prod. 33, 374–381.
Hmid I, Hanine H, Elothmani D, Oukabli A (2018) The physico-chemical characteristics of Morrocan pomegranate and evaluation of the antioxidant activity for their juices. Journal of the Saudi Society of Agricultural Sciences, 17(3), pp.302–309.
Holland D, Bar-Ya’akov I (2018) Pomegranate (Punica granatum L.) breeding. In Advances in plant breeding strategies: Advance in plant breeding strategies: Fruits, Cham, Springer International Publishing: (601–647).https://doi.org/10.1007/978-3-319-91944-7-15
Holland D, Hatib K, Bar-Ya’akov I (2009) Pomegranate: Botany, Horticulture, Breeding. In Horticultural Reviews, 35(2):127–191.
Juss, P., Shaw, P. J. (2014). Protocol for Test on Distinctness, Uniformity and Stability. Community Plant Varienty Office (CPVO-TP), 1, 286–310.
Karapetsi L, Pantelidis G, Pratsinakis ED, Drogoudi P, Madesis P (2021) Fruit Quality Traits and Genotypic Characterization in a Pomegranate Ex Situ (Punica granatum L.) Collection in Greece. Agriculture, 11(6): 482. https://doi.org/10.3390/agriculture11060482
Khadivi-Khub A, (2015) Physiological and genetic factors influencing fruit cracking. Acta Physiologiae Plantarum, 37(1), p.1718.
Khadivi A, Mirheidari F, Moradi Y, Paryan S, (2020) Morphological variability of wild pomegranate (Punica granatum L.) accessions from natural habitats in the Northern parts of Iran. Sci. Hortic. 264, 109165.
Lahouel M, Belhadj S (2022) Morphological variability of the fruits of seven pomegranate (Punica granatum L.) cultivars grown in Messaad region in central Algeria. Biotechnology, Agronomy and Society and Environment 26(1), pp. 16–29.
Lateef, D., Mustafa, K. Tahir, N., 2021. Screening of Iraqi barley accessions under PEG-induced drought conditions. All Life, 14(1), pp.308–332
Li X, Wasila H, Liu L, Yuan T, Gao Z, Zhao B Ahmad I (2015) Physicochemical characteristics, polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese cultivars and the environmental factors analysis. Food chemistry, 175, pp.575–584.
Liu K, Muse S (2005) Power Marker: an integrated analysis environment for genetic marker analysis. Bioinformatics, 21(9): 2128–2129. DOI: 10.1093/bioinformatics/bti282
Madadi M, Zamani Z, Fatahi R (2017) Assessment of Genetic Variation within Commercial Iranian Pomegranate (Punica granatum L.) Cultivars, Using ISSR and SSR Markers. Turkish Journal of Agriculture - Food Science and Technology, 5: 622–628 https://doi.org/10.24925/turjaf.v5i6.622-628.1100
Martínez JJ, Hernández F, Abdelmajid H, Legua P, Martínez R, Amine AE, Melgarejo P (2012) Physico-chemical characterization of six pomegranate cultivars from Morocco: processing and fresh market aptitudes. Sci Hort 140:100–106
Mayuoni-Kirshinbaum, L., Porat, R. (2014). The flavor of pomegranate fruit: a review. Journal of the Science of Food and Agriculture, 94(1), 21–27.
Meena VS, Bibwe B, Bhushan B, Jalgaonkar K, Mahawar M (2021) Physicochemical Characterization of Selected Pomegranate (Punica granatum L.) Cultivars. Turkish Journal of Agricultural Engineering Research, 2(2), pp.425–433.
Mena P, García-Viguera C, Navarro-Rico J, Moreno DA, Bartual J, Saura D, Martí N (2011) Phytochemical characterisation for industrial use of pomegranate (Punica granatum L.) cultivars grown in Spain. J Sci Food Agric 91:1893–1906.
Melgarejo P, Calín-Sánchez A, Vázquez-Aráujo L, Hernández F, Martínez JJ, Legua P, Carbonell-Barrachina AA (2011) Volatile composition of pomegranates from 9 Spanish cultivars using headspace solid phase microextraction. J Food Sci 76:114–120
Melgarejo-Sánchez P, Martínez JJ, Legua P, Martínez R, Hernández F, Melgarejo P (2015) Quality, antioxidant activity and total phenols of six Spanish pomegranates clones. Scientia Horticulturae, 182, pp.65–72.
Melgarejo P, Núñez-Gómez D, Legua P, Martínez-Nicolás JJ, Almansa MS (2020) Pomegranate (Punica granatum L.) a dry pericarp fruit with fleshy seeds. Trends in Food Science & Technology, 102, pp.232–236.
Mousavinejad G, Emam-Djomeh Z, Rezaei K, Haddad Khodaparast MH (2009) Identification and and quantification of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight Iranian cultivars. Food chem, 115: 1274–1278. Doi: 10.1016/j.foodchem.2009.01.044
Nikdel K, SEIFI E, Babaie H, Sharifani M, Hemmati K (2016) Physicochemical properties and antioxidant activities of five Iranian pomegranate cultivars (Punica granatum L.) in maturation stage. Acta agriculturae Slovenica, 107(2), pp.277–286.
Pasquali E, Palumbo F, Barcaccia G (2022) Assessment of the Genetic Distinctiveness and Uniformity of Pre-Basic Seed Stocks of Italian Ryegrass Varieties. Genes, 13(11), p.2097.
Peakall R O, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology, 6 (1): 288–295. https://doi.org/10.1111/j.1471-8286.2005.01155.xCitations: 9,612
Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics, 155 (2): 945–959. https://doi.org/10.1093/genetics/155.2.945
Qin G, Xu C, Ming R, Tang H, Guyot R, Kramer E, Hu Y, Yi X, Qi Y, Xu X, Gao Z (2017) The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis. The Plant Journal, 91(6): 1108–1128. https://doi.org/10.1111/tpj.13625
Rasul KS, Grundler FM, Tahir NA (2022) Genetic diversity and population structure assessment of Iraqi tomato accessions using fruit characteristics and molecular markers. Horticulture, Environment, and Biotechnology, 63(4), pp.523–538. https://doi.org/10.1007/s13580-022-00429-3
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free radical biology and medicine, 26(9–10), pp.1231–1237.
Sarkhosh A, Zamani Z, Fatahi R, Ranjbar H (2009) Evaluation of Genetic Diversity among Iranian Soft-Seed Pomegranate Accessionsby Fruit Characteristics and RAPD Markers. Scientia Horticulturae, 121: 313–319. https://doi.org/10.1016/j.scienta.2009.02.024
Silva A, Rana S, Narzary D, Verma N, Meshram T, Ranade A (2013) Pomegranate biology and biotechnology: A review. Scientia Horticulturae 85–107. https://doi.org/10.1016/j.scienta.2013.05.017.
Tahir, N., Lateef, D., Rasul, K., Rahim, D., Mustafa, K., Sleman, S., Mirza, A. Aziz, R. (2023) Assessment of genetic variation and population structure in Iraqi barley accessions using ISSR, CDDP, and SCoT markers. Czech Journal of Genetics and Plant Breeding, 59 (3). doi: 10.17221/112/2022-CJGPB
Tahir N.A, Omer D.A, Lateef D.A, Ahmad S.H, Salih S.H, Hiwakhal L.K (2019) Diversity and population structure analysis of faba bean (Vicia Faba L.) accessions using SSR markers. Journal of Agriculture Sciences and Technology, 21(2): 463–474.
Tahir, N.A., Rasul, K.S., Lateef, D.D. Grundler, F.M., 2022. Effects of Oak Leaf Extract, Biofertilizer, and Soil Containing Oak Leaf Powder on Tomato Growth and Biochemical Characteristics under Water Stress Conditions. Agriculture, 12(12), p.2082. https://doi.org/10.3390/agriculture12122082
Talebi M, Bahar M, Sharifnabi B, Yamchi A (2011) Evaluation of genetic diversity among Iranian pomegranate (Punica granatum L.) cultivars, using ISSR and RAPD markers. journal of taxonomy and biosistematics, 3rd Year, 35–44.
Tehranifar A, Zarei M, Esfandiyari B, Nemati Z (2010) Physicochemical properties and antioxidant activities of pomegranate fruit (Punica granatum) of different cultivars grown in Iran. Horticulture Environment and Biotechnology, 51(6), pp.573–579.
Tezcan F, Gultekin-Ozguven M, Diken T, Ozcelik B, Erim FB (2009) Antioxidant activity and total phenolic, organic acid and sugar content in commercial pomegranate juices. Food Chem, 115: 873–877. Doi: 10.1016/j.foodchem.2008.12.103
Wetzstein HY, Zhang Z, Ravid N, Wetzstein ME (2011) Characterization of attributes related to fruit size in pomegranate. HortScience, 46(6), pp.908–912.

Morpho-phytochemical Screening and Molecular Diversity of Pomegranate Accessions Grown in Halabja Governorate, Kurdistan Region-Iraq

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Plant material, location, and physical trait determination

Morphological parameters studied

Biochemical juice analysis

Total flavonoid content (TFC) estimation

ABTS antioxidant

Extraction of genomic DNA from leaves of pomegranate

ISSR marker analysis

Statistical analysis

Results and Discussions

Morphometric characterization

Influence of genotypes on biochemical traits

ISSR markers with polymorphism parameters

Molecular variance (AMOVA)

Structure analysis among 24 accessions by ISSR Markers

Conclusions

Declarations

References

Status:

Version 1