Physicochemical characterization and trait stability in a genetically diverse ex situ collection of pomegranate (Punica granatum L.) germplasm from Cyprus

Scientia Horticulturae 263 (2020) 109116

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Physicochemical characterization and trait stability in a genetically diverse ex situ collection of pomegranate (Punica granatum L.) germplasm from Cyprus

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Marios C. Kyriacoua,*, Sotiroula Ioannidoua, Nikolaos Nikoloudakisb, Nicos Seraphidesa, Lambros C. Papayiannisa, Angelos C. Kyratzisa a b

Agricultural Research Institute - Ministry of Agriculture, Rural Development and Environment, 1516 Nicosia, Cyprus Department of Agricultural Science, Biotechnology and Food Science, Cyprus University of Technology, Lemesos, Cyprus

ARTICLE INFO

ABSTRACT

Keywords: Antioxidant activity Anthocyanins Genetic polymorphism Organic acids Phenolic compounds Postharvest quality Soluble carbohydrates Texture analysis

Proximity to the center of origin and geographical isolation shaped a unique genetic diversity of pomegranate in Cyprus that constitutes a valuable resource for the crop. Physicochemical characters and trait stability were studied for three years in an ex situ collection of 29 pomegranate accessions from Cyprus. Accession signature traits with exceptional yearly stability were titratable acidity and the citrate/malate ratio. Overall, the Cypriot germplasm was characterized by juice of low anthocyanin content (x¯ = 20.6 mg/L) and moderate phenolic content (x¯ = 613.0 mg/L) with glucose (x¯ = 69.1 g/L) and fructose (x¯ = 74.2 g/L) as predominant sugars. Juice antioxidant capacity was associated primarily with total phenolics and less so with juice color and anthocyanin content. Total sugar content was higher in large-fruited accessions with darker juice. In most accessions the titratable acidity was low (< 0.50% w/v), with citric (x¯ = 44.7%), malic (x¯ = 39.1%) and succinic (x¯ = 15.8%) being the main organic acid fractions detected. Based on the maturity index (SSC/TA), six accessions were sweet-sour or borderline sweet-sour and 23 accessions were sweet. Cluster analysis of phenotypic characteristics and genetic data revealed a core group of thirteen genetically and phenotypically close accessions constituting the Cypriot pomegranate landrace, characterized by moderate fruit weight, high juiciness, thin rind, moderately hard seeds and light-colored juice low in acidity, anthocyanins, phenolics and antioxidant capacity. The present work advances the understanding of genetic and environmental contribution to the configuration of pomegranate physicochemical fruit composition.

1. Introduction Formerly an underutilized crop, the pomegranate (Punica granatum L.) has gained economic significance and expanding acreage in the past two decades compelled by consumer interest in foods supporting health and longevity (Kyriacou and Rouphael, 2018; Teixeira da Silva et al., 2013). Highly esteemed since antiquity for its health-promoting properties, the pomegranate has been termed a superfood owing to its rich bioactive composition which confers antioxidant and anti-inflammatory capacities contributing to the prevention of cancer, degenerative ailments, cardiovascular and metabolic diseases (Mayuoni‐Kirshinbaum and Porat, 2014). These properties are attributed largely to the high concentration of polyphenols in the peel and juice, particularly hydrolysable tannins (punicalagin, gallic and ellagic acid) and flavonoids (anthocyanins and catechins; Pareek et al., 2015). Nevertheless, the

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scientific knowledge on the biochemical or physiological properties of this plant and their modulation by genotype and agro-environment is still moderate (Ophir et al., 2014). Pomegranate draws its botanical origin from the area of Iran to Afghanistan, wherefrom it spread to India and the Mediterranean forming two independent sub-centers of genetic variability (Stover and Mercure, 2007; Teixeira da Silva et al., 2013; Ophir et al., 2014; Holland and Bar- Ya’akov, 2018). In the Mediterranean basin it was cultivated as early as the third millennium B.C.E., cherished for its therapeutic properties by the Egyptians, Greeks and Romans. Archaeobotanical evidence dates the presence of the species in Cyprus from the 3rd millennium B.C.E. wherefrom it reached north-western Africa and the western Mediterranean (Jacomet et al., 2002). The extensive variability of the species has prompted the establishment of numerous ex situ collections for the characterization of local germplasms in the

Corresponding author. E-mail address: [email protected] (M.C. Kyriacou).

https://doi.org/10.1016/j.scienta.2019.109116 Received 8 October 2019; Received in revised form 4 December 2019; Accepted 6 December 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Scientia Horticulturae 263 (2020) 109116

M.C. Kyriacou, et al.

Mediterranean and elsewhere (Holland and Bar- Ya’akov, 2018). Ex situ collections constitute a valuable tool for conserving, characterizing and comparing local and introduced genetic material under uniform agro-environmental conditions and, as such, a valuable resource for breeding programs. The characterization and assessment of pomegranate germplasm relies on morpho-physiological, physicochemical and technological traits many of which have agronomic and commercial significance. Since many of these traits however can be affected by agro-environmental conditions, assessing their stability against seasonal variation is critical. Moreover, given that phenotypic characteristics such as epidermal, aril and juice color may vary across different agro-environments synonymy of genetic material is not uncommon (Stover and Mercure, 2007). Complementing physicochemical with genetic characterization using molecular markers, such as Simple Sequence Repeats (SSRs), carries the advantage that the latter are not affected by the environment, they can distinguish closely related genotypes and detect duplicates or redundancies within collections (Alamuti et al., 2012; Hasnaoui et al., 2012; Jbir et al., 2012; Luo et al., 2018). Therefore, studies combining pomegranate physicochemical and genetic data are extremely useful for optimizing conservation of genetic resources and enhancing their valorization in plant breeding programs (Alamuti et al., 2012; Ferrara et al., 2014) as well as for dissecting genomic regions associated with agronomically important traits (Singh et al., 2015). The current work entails the characterization of physicochemical variability in pomegranate germplasm from Cyprus conducted on an ex situ collection of 29 select accessions along with commercial reference variety Wonderful. Characterization was repeated for three years in order to assess trait stability against yearly agro-environmental variation. Physicochemical characterization entailed fruit, aril and seed mass and size measurements, colorimetric assessment of rind and juice, seed and aril mechanical texture analysis, aril and fruit juice content, juice refractometry, titratable acidity, soluble carbohydrates, organic acids, total phenols, anthocyanins and ascorbate contents as well as juice in vitro radical scavenging capacity. Suitability of the genetic material for table or industrial use was designated. Phenotypic polymorphism based on physicochemical characterization was further combined and juxtaposed with genetic polymorphism employing microsatellite markers (SSRs). The present work advances the understanding of genetic and environmental contribution to the configuration of pomegranate physicochemical traits of agro-industrial significance. It also constitutes the first comprehensive characterization of pomegranate germplasm from Cyprus, one of the oldest centers of the species’ variability and dispersal in the Mediterranean.

estimates. Fertilization was provided in a combination of winter base dressing, covering 1/3 N, 1/1 P and 1/2 K requirements, with the rest of NPK requirements provided through fertigation and corrected according to soil and tissue analyses. NPK rates at the fourth year after planting reached 180, 100 and 160 kg/ha, respectively. Standard pest, disease and weed control practices were applied. Evaluation of maturity profiles was performed to identify the optimal harvest maturity period for each accession. Twenty-six early accessions were harvested in the period September 10–20 while four late accessions, including ‘Wonderful’, were harvested in the period October 20-28. Characterization was conducted in 2014, 2015 and 2016 using six replicated trees, from which eight representative healthy fruits free of visible defects, were collected from all external sides of the canopy at the harvester’s height. 2.2. Fruit morphometric characteristics Fruit gross weight, aril yield, aril gross weight and aril net weight were determined to ± 0.01 g on a Precisa XT 4200C electronic balance (Precisa Gravimetrics, Dietikon, Switzerland). Aril yield represented the total weight of arils as the percentage fraction of fruit gross weight. Aril gross weight was the mean unit aril weight obtained from 50 arils from each fruit. Aril net weight represented the aril gross weight less the seed weight expressed as the percentage fraction of aril gross weight [(aril gross weight – seed weight) × 100/ aril gross weight]. Seed weight was obtained as the mean weight of 50 seeds from each fruit. Aril and seed weights were determined to ± 0.001 g on a Precisa XT120A analytical balance. Shape Index (SI) was the ratio of fruit equatorial diameter to fruit height (stem end to calyx base) measured using an electronic caliper, also used for determining rind thickness as a mean of two representative points in cross-sectioned fruit and seed length. Juice was extracted mechanically by squeezing the arils through nylon organza. The juiciness of each fruit was expressed in the form of an index calculated as: aril yield (%) × aril net weight (%). Juice samples for phytochemical analyses were extracted from arils maintained at −80 °C. 2.3. Colorimetric and textural characterization Fruit external color and juice color were determined using Minolta CR-400 and CR-410 Chroma Meters (Minolta, Osaka, Japan), respectively, as described by McGuire (1992). Seed hardness was determined on a TA.XT plus Texture Analyser (Stable Micro Systems, Surrey, UK) equipped with a 50 kg load cell. Seed hardness was the maximum kgforce of resistance to 80% strain compression test, applied individually on each seed using a 75-mm flat probe at a test speed of 2 mm s−1. Ten seeds per fruit were tested. Aril texture was expressed as an index relating positively to the overall kinesthetic value of the arils, represented by the ratio of aril net weight to seed hardness.

2. Materials and methods 2.1. Plant material and experimental conditions Commercial groves and private collections were surveyed across traditional pomegranate producing districts of Cyprus at 0−800 m elevation and phenotypes of distinct characters and potential agronomic value were identified. Two-year old rooted cuttings of 29 select phenotypes, hereafter termed accessions, were used for establishing an ex situ collection in 2010. Commercial variety Wonderful was also included for comparison purposes, being the main introduced variety cultivated in Cyprus. The collection was planted at the Zygi Experimental Station (34° 44′ 00″ N; 33° 20′ 15″ E) of the Agricultural Research Institute of Cyprus where the prevailing climate is typical Mediterranean, average precipitation is 370 mm and the soil is alkaline (pH 8.2) clay-loam. The experimental design was completely random with six replicate trees per accession spaced 5 m × 6 m and supported by a Y-shaped trellis. Supplemental irrigation, provided through drippers at a frequency of 3–7 days between May and November, reached a total volume of 3500m3/ha at the fourth year after planting, distributed seasonally according to plant developmental stage and pan evaporation

2.4. Soluble solids content, titratable acidity, soluble carbohydrates and organic acids composition The titratable acidity and soluble solids content (% w/v SSC) of the juice were determined as previously described (Kyriacou et al., 2016), using respectively an automatic titrator (794 Basic Trinitro; Metrohm Ltd, Herisau, Switzerland) and a digital refractometer (RFM870; Bellingham-Stanley Ltd, Kent, UK). Analysis of non-structural carbohydrates (glucose, fructose and sucrose) in the juice was performed by liquid chromatography on an Agilent HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a 1200 Series quaternary pump and a 1260 Series Refractive Index detector operated by ChemStation software. Separation was performed on a Waters 4.6 × 250 mm carbohydrate column (Waters, Milford, MA, USA) at 35 °C using an ACN:H2O (75:25) mobile phase at a flow rate of 1.0 ml min−1. Quantification was performed against fructose, glucose and sucrose external 2

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standard calibrating curves with a coefficient of determination (R2) > 0.999. Organic acids were separated and quantified on the same HPLC system equipped with an Agilent 1260 Series Photo Diode Array detector. Injection volume was 15 ml and separation was performed using a Supelcogel C-610H (30 cm × 7.8 mm ID) ion exchange chromatography column, guarded by a Supelguard® (5 cm × 4.6 mm ID) column (Supelco Park, Bellefonte, USA) operated at 35 °C. Phosphoric acid 0.1% aqueous solution was used as the mobile phase at an isocratic flow rate of 0.5 ml/min. Peaks were detected at 210 nm and quantified against linear calibration curves (R2 > 0.999) constructed using 0.1–1.0 % (w/v) concentrations of citric, malic and succinic acid standards.

2.7. Statistical analysis Data were subjected to analysis of variance (ANOVA) and mean comparisons were performed by LSD test. Partial Eta Squared (η2) values [η2=SS effect / (SS effect + SS error)] were calculated to describe the proportion of the total variance attributed to main effects (accession and year) and their interaction. Pearson correlations were computed to estimate correlations between traits based on mean values across years. Hierarchical cluster analysis was conducted to explore relations between accessions. Squared Euclidean distances were calculated on standardized Z values, with a mean of 0 and a standard deviation of 1, from mean phenotypic data across years. Clustering was performed using the “between groups linkage” method. All analyses were carried out using SPSS (IBM, SPSS ver. 25). Genetic similarities were calculated based on Jaccard coefficient and a dendrogram was constructed with FreeTree software (Pavlicek et al., 1999) using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). The dendrogram was visualized with the implementation of TreeView (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html).

2.5. Juice total anthocyanin content, total phenolic content, antioxidant capacity and ascorbate content The total monomeric anthocyanin pigment content in the fruit juice was determined according to the AOAC Official Method 2005.02, based on the reversible color change from the colorless hemiketal form at pH 4.5 to the colored oxonium form at pH 1.0 (Lee et al., 2005). Quantification was performed at 520 nm and haze correction at 700 nm on a Jasco V-550 UV–vis spectrophotometer (Jasco Corp., Tokyo, Japan). Monomeric anthocyanin pigment content was expressed as cyanidin-3glucoside (cyd-3-glu) equivalents in mg/L. The total phenols content (TPC) of the juice was determined according to the method of Singleton et al. (1999). Quantification was performed on a Jasco V-550 UV–vis spectrophotometer against linear calibration with external gallic acid standards over the range of 50−500 mg L−1, yielding a regression coefficient R2 > 0.99. The TPC of the juice was expressed in gallic acid equivalents mg L−1. The ascorbate equivalent antioxidant capacity (AEAC) of the juice extracted in methanol was determined against a methanolic solution of 1,1-diphenyl-2-picrylhydrazyl free-radical (DPPH) based on the decrease in absorbance at 517 nm, and quantitated against ascorbate external standards (100–1000 μM) and expressed as mM AEAC (Drogoudi et al., 2005). The total ascorbic acid concentration of the juice was determined according to the method of Klein and Perry (1992) based on the reduction of 2,6-dichloro-indolphenol and expressed in mg L−1. Quantification was performed at 515 nm against linear calibration with external L-ascorbate standards (10−100 mg L−1) yielding regression coefficients R2 > 0.99.

3. Results and discussion 3.1. Fruit morphometric and colorimetric traits As further discussed below (paragraph 3.6), accessions 'Wonderful' and 'Mazotos1 shared the same genetic and phenotypic profiles and were therefore considered synonyms; henceforth, reference to Cypriot accessions excludes 'Mazotos1'. The fruit gross weight of Cypriot accessions was significantly lower than that of ‘Wonderful’ and its synonym 'Mazotos1' (Table 1; Supplementary Figs. 1 and 2). Fruit gross weight was low (< 350 g) in five accessions, intermediate (350−500 g) in 21 accessions and high (> 500 g) only in the late-maturing 'Dymes', 'Mazotos3', 'Mazotos1' and 'Wonderful'. The range and overall mean fruit weight of Cypriot accessions (273−527 g) were similar to those reported for Croatian (284−596 g; Radunić et al., 2015), Israeli (186−551 g; Dafny-Yalin et al., 2010) and Tunisian (196−674 g; Hasnaoui et al., 2011) germplasms, but higher than that of select USDAARS National Clonal Germplasm Repository (255−443 g; Chater et al., 2018), Iranian (197−315 g; Tehranifar et al., 2010), Spanish (325−414 g; Martinez-Nicolas et al., 2016) and Greek collections (246−445 g; Drogoudi et al., 2005). Aril yield for Cypriot accessions ranged 48.9% ('Marinouda1') to 69.0% ('Sotira') as opposed to 49.0% in 'Wonderful' (Table 1). Aril yield correlated negatively with rind thickness (-0.713; p < 0.01) but not with gross weight (Table 2), corroborating previous workers indicating it is an inappropriate criterion for screening genotypes for juice processing (Drogoudi et al., 2005). Rind thickness in Cypriot accessions ranged 2.4–4.7 mm, being significantly thinner than 'Wonderful' (5.3 mm; Table 1). Remarkably, several widely cultivated Cypriot accessions (e.g. 'Sotirkatiko', 'Chocolata', 'Sotira', 'Finikaria' and 'Kato Mylos1-3') yielded much higher aril yield (65.0–69.0 %) than 'Wonderful' (49.0%), which highlights their suitability for juice making or blending (Mena et al., 2014). The aril yields of these accessions exceeded the range reported for Spanish varieties (56.0–62.0 %; MartinezNicolas et al., 2016), Croatian varieties (35.7–62.1 %; Radunić et al., 2015) and select USDA germplasm (41.5–64.4 %; Chater et al., 2018). Yearly variation in juiciness index related to the pronounced year effect on rind thickness (Tables 1 and 3), which is partly a transient juvenility factor, with thicker fruit rind observed on juvenile trees (Schwartz et al., 2009a). This is corroborated by the fact that rind thickness declined progressively from 2014 to 2016 (Table 1). Information on pomegranate fruit dimensions is pertinent to the logistics of packing, handling and storage (Pareek et al., 2015). Fruit diameter in Cypriot accessions ranged 80.5–101.4 mm and fruit height ranged 70.2–85.0 mm (Table 1). 'Geroskipou' (SI = 1.088) had the most

2.6. DNA extraction and PCR amplification DNA was extracted from young leaves, using the DNeasy Plant Mini Kit (Qiagen, Venlo, Netherlands). DNA concentration and quality was assessed by Nanodrop 1000 (Thermo Fisher Scientific, Waltham, United States) and verified with agarose electrophoresis. Ten microsatellite markers (SSRs) were selected (Supplementary Table 1) based on their polymorphism in previous studies (Ebrahimi et al., 2010; Hasnaoui et al., 2012; Jbir et al., 2012; Parvaresh et al., 2012; Ferrara et al., 2014; Zarei and Sahraroo, 2018). The forward primers were 5´-end labeled with FAM (5-carboxy-fluorescent). Amplification reactions and PCR conditions were set up according to the kit protocol (Type-it® Microsatellite PCR kit, Qiagen). Annealing temperature varied depending of the primer pair (Supplementary Table 1). PCR amplification was performed in a PTC-200 thermocycler (Bio-Rad). Amplified PCR products were run on an ABI3130 genetic analyzer (Applied Biosystems, Foster City, United States). Size standard GeneScanTM 500LIZ® (Applied Biosystems) was added to each sample to delineate allele sizes. Data were analyzed using GeneMapper Software version 4.1 (Applied Biosystems). 3

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Table 1 Analysis of variance, Partial Eta Squared, means ± std and Least Significant Difference (LSD) for fruit gross weight, aril yield, fruit diameter, fruit shape index, rind thickness and fruit external hue angle of 30 pomegranate accessions from the Cypriot germplasm ex situ collection evaluated for three years. Source of variance Accession Year Accession × Year Accession Achelia 1 Achelia 2 Achelia 3 Achelia 4 Akrotiri Chocolata Dymes Emba 1 Emba 2 Emba 3 Emba 4 Finikaria Ftanofyllo Geri Geroskipou Kato Mylos 1 Kato Mylos 2 Kato Mylos 3 Kouklia 1 Kouklia 2 Marinouda 1 Marinouda 2 Marinouda 3 Mazotos 1 Mazotos 2 Mazotos 3 Paphos Sotikartiko Sotira Wonderful LSD Year 2014 2015 2016 LSD

Fruit gross weight Pr > F *** *** ***

2

η 0.453 0.048 0.154

Aril yield Pr > F *** *** ***

Fruit diameter 2

η 0.526 0.176 0.180

Pr > F *** *** ***

Fruit height

2

η 0.469 0.109 0.189

Pr > F *** *** ***

Shape 2

η 0.392 0.136 0.143

Pr > F *** *** ***

Rind thickness 2

η 0.258 0.014 0.087

Pr > F *** *** ***

2

η 0.397 0.547 0.301

Rind hue Pr > F *** *** ***

(g) 336 ± 65 428 ± 106 273 ± 62 463 ± 105 408 ± 78 430 ± 95 521 ± 120 424 ± 74 385 ± 89 474 ± 125 413 ± 76 486 ± 98 416 ± 99 284 ± 74 395 ± 100 463 ± 95 480 ± 94 477 ± 81 348 ± 95 401 ± 89 311 ± 76 500 ± 110 443 ± 84 680 ± 162 458 ± 142 527 ± 119 375 ± 63 445 ± 103 460 ± 100 575 ± 93 29

(%) 54.1 ± 5.2 59.2 ± 5.8 56.8 ± 3.8 59.7 ± 5.6 62.7 ± 3.4 67.6 ± 4.4 58.8 ± 6.3 56.3 ± 6.5 60.0 ± 4.7 57.2 ± 6.1 62.0 ± 7.9 65.0 ± 5.1 60.6 ± 5.4 54.7 ± 5.1 54.9 ± 6.7 65.3 ± 3.2 65.0 ± 5.2 65.4 ± 5.2 61.5 ± 4.4 59.2 ± 5.0 48.9 ± 4.9 58.5 ± 5.7 55.4 ± 4.0 51.8 ± 7.3 61.6 ± 8.9 62.2 ± 6.4 61.8 ± 3.9 68.0 ± 4.1 69.0 ± 4.5 49.0 ± 7.1 1.521

(mm) 86.8 ± 5.9 93.5 ± 7.5 80.5 ± 6.1 97.1 ± 7.8 93.0 ± 6.0 93.5 ± 6.6 100.3 ± 7.8 93.3 ± 6.1 90.6 ± 7.1 97.9 ± 8.6 91.9 ± 5.5 98.5 ± 7.0 93.0 ± 7.4 82.2 ± 7.8 90.2 ± 8.3 97.2 ± 7.7 98.2 ± 7.2 98.2 ± 6.2 89.6 ± 8.1 91.8 ± 7.3 82.7 ± 6.8 99.3 ± 7.6 94.0 ± 6.1 108.2 ± 8.6 98.1 ± 10.6 101.4 ± 8.4 89.5 ± 5.7 96.4 ± 7.9 96.6 ± 7.2 103.7 ± 6.6 2.065

(mm) 74.2 ± 4.4 81.5 ± 7.3 70.2 ± 4.4 82.4 ± 6.5 81.4 ± 5.0 79.8 ± 5.8 85.0 ± 7.1 81.8 ± 4.8 78.2 ± 5.8 82.7 ± 7.8 78.0 ± 4.8 82.2 ± 6.2 81.4 ± 6.7 73.2 ± 7.5 83.0 ± 7.3 81.6 ± 6.8 83.4 ± 6.1 82.5 ± 5.5 74.8 ± 6.5 80.4 ± 6.6 75.3 ± 6.5 84.0 ± 7.0 82.3 ± 5.0 94.7 ± 7.7 83.0 ± 8.2 84.2 ± 7.5 78.6 ± 4.9 81.1 ± 6.8 82.9 ± 7.1 89.4 ± 5.8 1.823

(index) 1.170 ± 0.05 1.148 ± 0.04 1.148 ± 0.06 1.179 ± 0.04 1.144 ± 0.04 1.172 ± 0.05 1.181 ± 0.05 1.142 ± 0.04 1.159 ± 0.05 1.186 ± 0.05 1.179 ± 0.05 1.200 ± 0.05 1.144 ± 0.04 1.125 ± 0.04 1.088 ± 0.05 1.193 ± 0.05 1.178 ± 0.05 1.191 ± 0.04 1.199 ± 0.05 1.143 ± 0.04 1.100 ± 0.05 1.184 ± 0.05 1.143 ± 0.05 1.144 ± 0.05 1.182 ± 0.05 1.207 ± 0.06 1.141 ± 0.05 1.190 ± 0.06 1.168 ± 0.05 1.160 ± 0.04 0.015

(mm) 3.3 ± 0.7 2.9 ± 1.0 2.7 ± 0.9 4.2 ± 1.8 2.8 ± 1.1 2.4 ± 1.2 3.9 ± 1.7 3.4 ± 1.5 3.0 ± 0.9 3.4 ± 1.2 3.3 ± 1.8 3.1 ± 1.1 3.0 ± 1.2 3.3 ± 1.1 4.0 ± 1.2 2.8 ± 0.9 3.2 ± 1.4 3.3 ± 1.4 3.7 ± 1.3 3.0 ± 1.0 4.7 ± 1.0 3.5 ± 1.2 3.2 ± 0.9 4.6 ± 1.8 3.6 ± 1.8 3.4 ± 1.7 3.4 ± 1.1 2.6 ± 0.7 2.5 ± 1.1 5.3 ± 1.6 0.257

(0–360 °) 77.58 ± 14.0 69.02 ± 9.4 74.67 ± 12.4 63.38 ± 14.2 74.80 ± 9.7 70.55 ± 12.1 45.21 ± 13.2 65.03 ± 10.6 60.33 ± 11.9 38.40 ± 10.1 73.69 ± 9.3 69.45 ± 9.9 65.37 ± 10.7 48.41 ± 12.0 70.05 ± 17.1 62.61 ± 10.8 62.58 ± 11.9 60.83 ± 10.0 35.39 ± 10.8 64.59 ± 10.1 93.82 ± 12.4 59.80 ± 10.0 69.50 ± 7.3 25.92 ± 4.1 28.93 ± 5.8 53.63 ± 14.1 70.12 ± 12.5 68.38 ± 10.1 70.91 ± 10.7 27.02 ± 4.6 3.029

482 ± 14 449 ± 13 408 ± 10 9

55.6 ± 6.9 59.8 ± 7.1 62.1 ± 7.4 0.481

98.0 ± 9.5 96.1 ± 10.3 91.3 ± 7.4 0.653

85.0 ± 7.8 82.6 ± 7.9 78.3 ± 6.6 0.577

1.154 ± 0.05 1.163 ± 0.06 1.169 ± 0.05 0.005

4.5 ± 1.3 3.8 ± 1.2 2.3 ± 0.9 0.081

51.28 ± 17.3 64.33 ± 19.8 61.46 ± 18.9 0.958

η2 0.734 0.157 0.176

***ignificant effect at the 0.001 level.

spherical fruit and 'Mazotos3' (SI = 1.207) the most flattened. Fruit gross weight, height and diameter varied across years, however the SI remained relatively sTable (1 .164-1.159) as indicated by the low η2 value (0.014) for the year effect, suggesting that fruit shape is primarily a genotypic trait. The weak association of fruit shape with gross weight is underlined by their low correlation coefficient (r = 0.424, p < 0.05; Table 2). Fruit blush coloration (hue angle, h°) demonstrated wide variability (Table 1; Fig. 1). The reddest blush (h° < 30°) was that of 'Mazotos1', 'Wonderful' and 'Mazotos2', which developed full external red coloration and significantly narrower h° at than the rest accessions. Earlymaturing 'Mazotos2' is particularly noteworthy for its intense external red coloration developed as early as the first week of August. Four more accessions ('Kouklia1', 'Emba3', 'Dymes' and 'Geri') demonstrated extensive blush of reddish hue (30° < h° > 50°), while the rest had extensive yellow ground color with rudimentary red blush (Fig. 1).

for Spanish (Martinez-Nicolas et al., 2016) and Tunisian (Hasnaoui et al., 2011) germplasm, higher than that of USDA (Chater et al., 2018) and Greek (Drogoudi et al., 2005) germplasm and similar to that of Iranian (Tehranifar et al., 2010) germplasm and UMH Germplasm Bank and commercial cultivars from Spain (Alcaraz-Mármol et al., 2017). Aril net weight ranged 88.7–96.5 % and correlated significantly with aril gross weight (r = 0.722; p < 0.01; Table 2) but not with seed weight, suggesting that genotypes having large arils tend to be more appropriate for juice making. It is noteworthy that juiciness index was lowest in 'Wonderful' (46.1%) while in Cypriot accessions it averaged 57.1% (Table 3). Accessions 'Chocolata', 'Sotikartiko' and 'Sotira' had remarkably high juiciness index (65.1–66.3 %), which renders them prime candidates for the juice industry. The highest positive correlation with juiciness was obtained for aril yield (r = 0.988; p < 0.01) and aril gross weight (r = 0.674; p < 0.01), the highest negative correlation with rind thickness (r=-0.665; p < 0.01) whereas correlation with fruit gross weight or seed weight was non-significant (Table 2), as previously observed in other germplasm collections (Tehranifar et al., 2010). The correlation matrix indicates that juiciness is a trait associated primarily with thin rind and high aril yield, and secondarily with high aril gross weight. 'Wonderful' had darker juice (L* = 26.96) than all Cypriot accessions, which ranged 34.82–50.61 (Table 3). Juice red color was also

3.2. Aril and seed morphometric, colorimetric and textural traits Aril gross weight in Cypriot accessions ranged 0.234-0.589 g (Table 3). Values ≥0.400 g approximating the aril gross weight of 'Wonderful' (0.408 g) were obtained in 14 accessions (Table 3; Fig. 2). Overall, aril weight in Cypriot accessions was lower than that reported 4

5

ns ns 0.424* 0.585** −0.374* 0.412* ns ns 0.583** ns ns ns ns ns −0.390* −0.456* ns ns

0.951** ns ns

AEAC

ns −0.713** −0.384* ns ns ns −0.665** −0.633** 0.572** 0.413* 0.543** ns 0.446* 0.567** ns ns ns ns ns −0.369* 0.463** ns 0.633** ns −0.403* −0.378* 0.377*

ns ns 0.424* 0.585** −0.374* 0.412* ns ns 0.583** ns ns ns ns ns −0.390* −0.456* ns ns ns ns ns ns ns −0.561** ns 0.423* ns ns ns

Fruit shape

Rind thickness

Fruit shape

ns ns

Phenolics

ns −0.557** ns ns ns ns −0.633** 0.441* 0.803** 0.627** −0.417* ns ns 0.677** 0.498** −0.398* 0.607** 0.571** −0.425* 0.570** −0.613** 0.586** ns ns ns ns

Fruit gross weight

0.890**

Glucose

ns 0.596** ns 0.387* 0.988** 0.368* −0.437* ns −0.378* ns −0.641** −0.747** ns ns ns ns ns ns ns ns −0.487** ns 0.368* 0.387* ns

Fruit net weight

Fructose

ns ns ns ns 0.738** −0.648** −0.557** −0.471** ns ns ns −0.364* ns ns ns ns ns −0.410* ns −0.726** ns ns ns ns

Fruit hue

Fructose/ Glucose

Citrate

−0.540** ns ns ns ns 0.397* −0.468** ns ns ns ns ns ns ns ns ns ns ns ns 0.855** ns −0.878**

Seed weight

Total sugars

ns 0.722** 0.674** ns −0.468** ns ns ns −0.538** −0.596** ns ns −0.629** ns 0.535** −0.418* 0.412* −0.526** ns ns ns 0.721** ns

Aril gross weight

Malate

0.523** ns ns ns ns ns −0.543** −0.517** ns ns ns ns 0.433* ns ns −0.523** ns ns ns 0.460* 0.396*

Aril net weight

Citrate/ Malate

ns −0.453* ns ns ns −0.679** −0.773** ns ns ns ns ns ns ns ns −0.457* ns ns 0.436* ns

Juiciness

Succinate

−0.909** −0.736** −0.523** ns −0.382* −0.460* −0.561** −0.508** ns −0.550** ns ns ns ns −0.873** ns ns 0.563** ns

Juice L*

Anthocyanins

0.551** 0.438* ns 0.385* 0.462* 0.370* 0.431* ns 0.411* ns ns ns ns 0.708** ns ns −0.707** ns

Juice a*

Ascorbate

0.514** ns ns ns 0.905** 0.780** ns 0.869** 0.389* ns 0.381* −0.524** 0.693** ns ns −0.403* ns

SSC

Seed length

ns ns ns ns 0.513** ns −0.851** 0.910** −0.860** 0.508** ns ns ns ns ns

SSC/TA

(continued on next page)

Seed hardness

−0.882** 0.362* ns 0.381* ns −0.559** ns 0.805** −0.840** 0.931** −0.523** 0.543** 0.400* ns ns ns

TA

Table 2 Pearson correlation coefficients for fruit, aril and seed physicochemical characteristics of 30 pomegranate accessions from the Cypriot germplasm ex situ collection evaluated for three years ns = non-significant; *p < 0.05, **p < 0.01.

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Scientia Horticulturae 263 (2020) 109116

Scientia Horticulturae 263 (2020) 109116

ns −0.642** 0.616** −0.652** 0.488** ns −0.393* −0.386* −0.396* ns

ns ns ns ns 0.501** ns ns −0.447* ns

−0.906** 0.926** −0.817** ns 0.443* ns ns ns

−0.930** 0.500** ns −0.404* ns ns ns

−0.644** 0.404* 0.528** ns ns ns

ns ns ns ns ns

ns ns −0.427* ns

0.383* ns ns

ns −0.986**

ns

most intense in 'Wonderful' (a* = 22.42), its synonym 'Mazotos1' (23.09) and in 'Kouklia1' (20.23), whereas 'Achelia3' (3.16), 'Sotirkatiko' (4.24) and 'Chocolata' (5.25) had the least colored juice. Overall, Cypriot accessions demonstrated less dark (L* = 42.12) but more intensely red (a* = 12.46) juice than Israeli germplasm (L* = 21.29; a* = 2.87) that was rather achromatic (Dafny-Yalin et al., 2010). The latter colorimetric traits however are likely to have resulted from increased transfer of peel phenolics to the juice during extraction and their subsequent oxidation or from differences in instrumentation and calibration procedures (McGuire, 1992). Seed weight in Cypriot accessions ranged 0.015-0.028 g and seed length 6.8–8.1 mm (Table 3). Mean seed weight in Cypriot accessions (0.022 g) was lower than that reported for Spanish germplasm (AlcarazMármol et al., 2017), although mean aril weights were very similar (0.414 and 0.410 g), which explains the high aril net weight (94.4%) of Cypriot cultivars. High aril net weight improves the palatability of pomegranates for table use although it is of limited consequence to aril yield and therefore to the juice industry (Tehranifar et al., 2010). Seed hardness is another critical organoleptic trait of pomegranates for table consumption. It is often absent from germplasm evaluations or it is usually assessed by ad hoc sensory panels as opposed to the instrumental texture analysis performed in the current study (Zarei et al., 2013). Seed hardness across Cypriot accessions ranged 31.0–49.4 kgforce with an intermediate hardness for 'Wonderful' (40.4 kg-force; Table 3). Seed hardness correlated positively with seed weight (r = 0.855, p < 0.01), indicating that large-seeded genotypes tend to bear the hardest seeds (Tables 2 and 3). Performing mechanical seed texture tests on pomegranate genotypes Zarei et al. (2013) determined a similar correlation (r = 0.823; p < 0.01) between seed hardness and weight. The overall kinesthetic value of pomegranate arils is influenced both by seed hardness and juice content (Zarei et al., 2013). Using the ratio aril-net-weight/seed-hardness as an index of overall aril texture, we identified the best texture in 'Marinouda3' (3.11) and 'Marinouda1' (2.94), both of which had been collected as soft-seeded germplasm (Table 3). 'Wonderful' had a moderate aril texture index (2.35) owing to its semi-hard seeds and moderate aril net weight. Genotype effect was predominant on most fruit, aril and seed morphometric, colorimetric and textural traits examined, as reflected in the higher η2 values of accession vs. year (Tables 1 and 3). Fruit physical traits most resilient to year effect included aril weight, seed hardness, fruit gross weight (confirming Chater et al., 2018), fruit shape and rind hue. Moderate yearly variation was found in aril yield, seed weight, juiciness index and juice color whereas the most prominent year effect was found in rind thickness.

ns 0.971** ns ns ns ns 0.405* ns −0.424* −0.523** 0.361*

3.3. Juice soluble solids content (SSC), titratable acidity (TA), soluble carbohydrates and organic acids

ns 0.973** ns ns ns −0.374* 0.561** ns ns ns ns

The SSC of Cypriot accessions ranged 14.8–17.6 % as opposed to 18.4% in ‘Wonderful’ (Table 4). Overall, Cypriot germplasm demonstrated higher SSC than Spanish germplasm (12.6–15.3 %; MartinezNicolas et al., 2016) but similar to USDA (14.9–17.3 %; Chater et al., 2018), Greek (14.4–17.0 %; Drogoudi et al., 2005), Turkish (15.5–16.9 %, Cam et al., 2009) and Israeli (13.7–17.8 %, Dafny-Yalin et al., 2010) germplasms. However, comparing the present SSC of 'Wonderful' (18.4%) to that of the USDA collection (16.5%; Chater et al., 2018) suggests that, aside from genotypic differences, variation also reflects different agro-environmental conditions (Schwartz et al., 2009b) and different harvest maturity standards (Fawole and Opara, 2013). The SSC correlated (p < 0.01) positively with fruit gross weight (r = 0.803) and juice red color (a*; r = 0.551), and negatively with juice color-lightness (L*; r=-0.736) and rind hue (r=-0.557). These correlations indicate a tendency for higher SSC in accessions bearing large fruit with reddish external hue and intense dark red juice color, exemplified by 'Wonderful'. The proximate η2 values for accession (0.577) and year (0.547) effects suggest that yearly fluctuation in biotic

ns ns ns ns ns ns 0.456* ns ns ns ns ns ns ns ns ns −0.561** ns 0.423* ns ns ns

ns ns ns ns ns ns 0.554** ns ns −0.398* ns

AEAC Fruit shape

Table 2 (continued)

Phenolics

Glucose

Fructose

Fructose/ Glucose

Total sugars

Citrate

Malate

Citrate/ Malate

Succinate

Anthocyanins

Ascorbate

Seed hardness

Seed length

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6

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and abiotic pressures and shifting in the harvest maturity period contribute to variation in SSC (Schwartz et al., 2009a). Glucose and fructose were the predominant soluble carbohydrates detected in pomegranate juice with only sporadic trace concentrations of sucrose (mean = 0.2 g/L), corroborating previous findings from Tunisian (Hasnaoui et al., 2011), Israeli (Dafny-Yalin et al., 2010) and Turkish (Cam et al., 2009) germplasms. In Cypriot accessions glucose concentration ranged 60.8–74.0 g/L and fructose 65.4–81.3 g/L as opposed to 81.00 g/L and 79.8 g/L in 'Wonderful', respectively (Table 4). Total sugars in Cypriot accessions ranged 129.08–155.2 g/L, as opposed to 161.3 g/L found in 'Wonderful', and were comparable to concentrations reported for Israeli (Dafny-Yalin et al., 2010) and Turkish (Caliskan and Bayazit, 2012; Cam et al., 2009) germplasms. Glucose (r = 0.905), fructose (r = 0.780) and total sugar concentrations (r = 0.869) correlated significantly (p < 0.01) with SSC (Table 2). Moreover, total sugar concentration correlated positively with fruit gross weight (0.607) and negatively with juice L* (r=-0.550) favoring large-fruited accessions with darker juice. Year effect was higher for glucose (η2 = 0.198) than fructose (η2 = 0.092), however annual variation in the mean total sugar concentration across accessions was limited (143.1–147.7 g/L; year-η2 = 0.027). Furthermore, the fructose/ glucose ratio was assessed as it carries both sensory and health implications since fructose contributes to sweetness sensation twice as glucose and high levels of fructose consumption were linked to the metabolic syndrome (Mayuoni‐Kirshinbaum and Porat, 2014). The fructose/glucose ratio was 0.99 in 'Wonderful' and averaged 1.08 across Cypriot accessions, ranging from 1.01 in 'Chocolata' to 1.14 in 'Akrotiri' (Table 4). In 2014 and 2016 this ratio was 1.00 and 1.02, respectively; but in 2015 it spiked to 1.20, suggesting this is not entirely under genetic effect but it is subject to yearly variation deriving possibly from the shifting of harvest maturity (Fawole and Opara, 2013). The sensory profile of pomegranate varieties favored by consumers includes high sweetness and moderate to low acidity (Mayuoni‐Kirshinbaum and Porat, 2014). Varieties with acidity < 1% are considered sweet, 1.2% sweet–sour and > 2% sour (Pareek et al., 2015). In this respect, all Cypriot accessions evaluated can be termed sweet (TA < 1%), except from the two late-maturing accessions 'Dymes' and 'Mazotos3' which are marginally sweet-sour (Table 4). Most Cypriot accessions were in fact of insipid acidity (TA < 0.50%) and demonstrated overall mean acidity (TA = 0.50%) similar to Tunisian pomegranates (0.60%; Hasnaoui et al., 2011), lower than USDA germplasm (1.33%; Chater et al., 2018) but higher than Spanish germplasm (0.32%; Martinez-Nicolas et al., 2016). The acidity of 'Wonderful' in the current collection (1.04%) was notably lower than that obtained by Chater et al. (2018; 1.71%); which is indicative of the different harvest maturity standards applied in different markets but may also reflect variation in agro-environmental conditions (Shwartz et al., 2009b). As opposed to the US market, where pomegranate TA < 1.85% is acceptable (Stover and Mercure, 2007), the Mediterranean market perceives pomegranate fruit with TA > 1.2% as displeasingly sour. Low acidity generally characterizes germplasm adapted to temperate Mediterranean climate where phenotype selection has favored sweet over sour pomegranates (Schwartz et al., 2009b). Mean TA across accessions was 0.575% in 2014 and 0.534% in 2015 and 2016 with TA variance derived mainly from accession (η2 = 0.815) with very limited year (η2 = 0.016) and accession × year (η2 = 0.086) contributions. The main organic acid fractions detected across accessions were citric (x¯ = 44.7%), malic (x¯ = 39.1%) and succinic (x¯ = 15.8%; Table 4), underscored by a wide range of citrate/malate (0.20–2.67). Remarkably, the TA correlated positively with the citrate fraction (r = 0.805, p < 0.01) and negatively with malate (r=-0.840, p < 0.01), whereas the highest positive correlation was obtained with the citrate/malate ratio (r = 0.931, p < 0.01; Table 2). Thus highly acidic late-maturing genotypes tend to have higher citrate/malate ratio than early-maturing genotypes of low acidity. Similar conclusions on the contribution of organic acids to pomegranate acidity were drawn

from previous germplasm evaluations (Melgarejo et al., 2000; Hasnaoui et al., 2011; Dafny-Yalin et al., 2010) showing citric as the dominant acid in sour varieties (Mayuoni‐Kirshinbaum and Porat, 2014). Furthermore, yearly variation in the citrate/malate ratio was limited (1.16–1.34) and the η2 values for year effect were very low for all acidity traits (Table 4). The present study clearly indicates that signature traits for pomegranate accessions are those related to juice acidity, including TA and the citrate/malate ratio. These traits demonstrated exceptional resilience to year effect and accession × year interaction (Supplementary Fig. 1A and B). The maturity index (MI = SSC/TA) is commonly applied for categorizing pomegranate varieties into sweet, sweet-sour and sour (Martínez et al., 2012). However, the MI can be influenced by environmental conditions (Pareek et al., 2015), moreover it is varietydependent and, optimally, must be defined for specific varieties (Mayuoni‐Kirshinbaum and Porat, 2014). Out results indicate that MI yearly variation (η2 = 0.111) was moderate compared to accession–based variation (η2 = 0.799; Table 4); however, it was less resilient to yearly variation than TA (η2 = 0.016). The MI of Cypriot pomegranates (x¯ = 36.1) was on average lower than Spanish germplasm (54.3; Martinez-Nicolas et al., 2016) but higher than select USDA germplasm (22.3; Chater et al., 2018). However, the MI range for Cypriot germplasm (15.8–57.3) was wide, with 'Wonderful' MI at 18.5 (Table 4). According to the classification proposed by Martínez et al. (2012), latematuring 'Dymes' (15.8) and 'Mazotos3' (17.9) can be safely classified as sweet-sour while four more accessions with MI 25–30 (Emba1, Emba4, Kouklia2, Marinouda2) can be considered borderline sweet-sour, but the majority of accessions were clearly sweet (MI > 30). Correlations between phenomenally related variables provide useful information for pomegranate breeding programs and may also elucidate the origins of seasonal variation in certain traits. For instance, the SSC correlated significantly (p < 0.001) with total soluble sugars (r = 0.869), anthocyanin (r = 0.693) and TA (r = 0.514) concentrations (Table 2); however, total sugar concentration did not correlate significantly with TA. This highlights that sour genotypes do not necessarily lack in sugar content compared to sweet ones (Mayuoni‐Kirshinbaum and Porat, 2014). Late-maturing sweet-sour accessions (e.g. 'Mazotos1', 'Mazotos3', 'Dymes' and 'Wonderful') reached high sugar content early, well before acidity decreased to levels warranting optimal harvest maturity (data not shown). Thus critical harvest maturity indices for early genotypes are juiciness and sugar content whereas for late genotypes it is acidity. This also highlights the importance of assessing sweetness in terms of actual sugar concentrations in pomegranate juice rather than by SSC alone, the refractometric determination of which is influenced by the presence of organic acids in the juice (Magwaza and Opara, 2015). 3.4. Juice total anthocyanin content, total phenolic content, ascorbate equivalent antioxidant capacity (AEAC) and ascorbate content Anthocyanins contribute to color and antioxidant capacity of pomegranate juice, hence they are considered desirable in pomegranates destined for either processing or table use (Pareek et al., 2015). The anthocyanin content of Cypriot pomegranate germplasm ranged 1.2–100.0 mg/L, in cyanidin-3-glucoside equivalents (Table 5). Notably high anthocyanins were found in 'Mazotos2'′ (100.0 mg/L) but much lower than found in 'Wonderful' (188.8 mg/L). Fourteen accessions had very low (0−15 mg/L) and thirteen others moderately low (15−50 mg/L) anthocyanin concentration, which expectedly correlated significantly (p < 0.01) with juice lightness L* (r=-0.873) and redness a* (r = 0.708). Given the overall insipid juice coloration of Cypriot germplasm, it is not surprising that anthocyanin concentrations were low. Yearly variation in anthocyanin concentration ranged 22.6–44.0 mg/L, reflecting yearly differences in day-night temperature, irradiance levels and consequent shifts in maturity (Schwartz et al., 2009b). Anthocyanin content was predominantly influenced by 7

8

(%) 92.5 ± 1.6 93.2 ± 1.0 94.6 ± 0.9 95.0 ± 0.7 95.3 ± 0.7 96.3 ± 0.6 94.2 ± 0.8 92.8 ± 1.2 94.4 ± 1.2 94.1 ± 1.0 92.8 ± 1.0 94.6 ± 0.6 95.0 ± 0.7 88.7 ± 2.0 94.4 ± 1.3 94.6 ± 0.7 94.7 ± 0.7 94.8 ± 0.8 96.5 ± 0.6 93.1 ± 1.0 95.3 ± 0.6 93.5 ± 0.8 95.7 ± 0.6 94.3 ± 0.8 94.8 ± 0.7 95.1 ± 0.7 95.0 ± 1.0 96.4 ± 0.5 96.1 ± 0.6 94.7 ± 0.7 0.222 94.3 ± 1.7 93.7 ± 2.0 94.9 ± 1.4 0.070

0.414 ± 0.087 0.396 ± 0.106 0.421 ± 0.103 0.006

Pr > F *** *** ***

(g) 0.304 ± 0.047 0.358 ± 0.054 0.431 ± 0.060 0.344 ± 0.059 0.382 ± 0.043 0.573 ± 0.072 0.418 ± 0.073 0.370 ± 0.043 0.446 ± 0.099 0.388 ± 0.062 0.348 ± 0.037 0.400 ± 0.053 0.395 ± 0.047 0.234 ± 0.046 0.376 ± 0.061 0.439 ± 0.062 0.414 ± 0.050 0.425 ± 0.048 0.482 ± 0.083 0.354 ± 0.046 0.358 ± 0.052 0.386 ± 0.045 0.346 ± 0.041 0.421 ± 0.046 0.390 ± 0.067 0.571 ± 0.113 0.498 ± 0.076 0.583 ± 0.077 0.589 ± 0.070 0.408 ± 0.047 0.019

η2 0.639 0.043 0.105

η2 0.829 0.372 0.253

(%)

(mg)

Pr > F *** *** ***

Aril net weight

Aril gross weight

***significant effect at the 0.001 level.

Accession Achelia 1 Achelia 2 Achelia 3 Achelia 4 Akrotiri Chocolata Dymes Emba 1 Emba 2 Emba 3 Emba 4 Finikaria Ftanofyllo Geri Geroskipou Kato Mylos 1 Kato Mylos 2 Kato Mylos 3 Kouklia 1 Kouklia 2 Marinouda 1 Marinouda 2 Marinouda 3 Mazotos 1 Mazotos 2 Mazotos 3 Paphos Sotikartiko Sotira Wonderful LSD Year 2014 2015 2016 LSD

Accession Year Accession × Year

Source of variance

52.4 ± 6.8 56.1 ± 7.1 59.0 ± 7.2 0.455

(index) 50.2 ± 5.2 55.2 ± 5.4 53.7 ± 3.8 56.7 ± 5.3 59.7 ± 3.3 65.1 ± 4.4 55.4 ± 5.8 52.0 ± 5.8 56.7 ± 4.5 53.8 ± 6.0 57.6 ± 7.6 61.5 ± 4.9 57.6 ± 5.3 48.5 ± 5.1 51.8 ± 6.4 61.8 ± 3.1 61.5 ± 5.0 62.0 ± 5.0 59.3 ± 4.3 55.1 ± 5.0 46.5 ± 4.7 54.9 ± 5.3 53.0 ± 3.8 49.3 ± 7.4 58.3 ± 8.4 59.1 ± 6.0 58.8 ± 4.0 65.5 ± 4.0 66.3 ± 4.3 46.1 ± 6.1 1.438

Pr > F *** *** ***

Juiciness

η2 0.567 0.197 0.184

39.81 ± 7.2 44.00 ± 5.0 38.73 ± 6.4 0.307

(0–100) 42.86 ± 3.6 43.97 ± 3.2 50.61 ± 2.5 37.09 ± 4.5 41.40 ± 2.8 44.98 ± 2.3 39.21 ± 5.2 35.97 ± 5.9 42.95 ± 3.0 39.87 ± 3.9 37.76 ± 5.5 42.96 ± 4.8 40.67 ± 4.5 42.64 ± 3.5 43.32 ± 3.5 41.67 ± 5.7 42.27 ± 5.0 42.17 ± 6.5 36.92 ± 4.5 41.45 ± 5.5 46.18 ± 3.6 40.40 ± 3.8 43.22 ± 3.9 27.67 ± 4.5 34.82 ± 5.2 45.25 ± 5.0 47.41 ± 2.5 46.61 ± 2.8 44.60 ± 2.7 26.96 ± 3.9 0.969

Pr > F *** *** ***

(0–100)

Juice L*

η2 0.744 0.397 0.333

14.16 ± 6.2 10.08 ± 6.0 15.93 ± 7.6 0.364

(-60/+60) 14.93 ± 4.6 11.57 ± 6.0 3.16 ± 2.1 16.61 ± 4.9 9.14 ± 3.6 5.25 ± 1.7 17.92 ± 7.0 19.31 ± 5.5 10.48 ± 4.7 15.76 ± 4.7 17.84 ± 5.0 12.42 ± 6.2 14.94 ± 4.5 14.29 ± 5.7 10.30 ± 5.2 14.39 ± 5.8 12.09 ± 6.3 11.23 ± 6.1 20.23 ± 5.2 15.61 ± 6.9 9.37 ± 4.1 16.16 ± 5.4 12.75 ± 5.7 23.09 ± 3.6 19.76 ± 4.4 6.09 ± 7.2 6.35 ± 3.6 4.84 ± 1.6 5.97 ± 2.0 22.42 ± 3.4 1.148

Pr > F *** *** ***

(-60/+60)

Juice a*

η2 0.658 0.361 0.353

η2 0.667 0.300 0.138

0.023 ± 0.004 0.023 ± 0.004 0.020 ± 0.004 0.000

(g) 0.022 ± 0.002 0.024 ± 0.002 0.023 ± 0.002 0.017 ± 0.003 0.018 ± 0.002 0.021 ± 0.003 0.024 ± 0.003 0.026 ± 0.003 0.024 ± 0.003 0.023 ± 0.003 0.025 ± 0.003 0.022 ± 0.002 0.019 ± 0.002 0.026 ± 0.003 0.021 ± 0.003 0.024 ± 0.003 0.022 ± 0.002 0.022 ± 0.003 0.017 ± 0.002 0.024 ± 0.002 0.017 ± 0.002 0.025 ± 0.003 0.015 ± 0.002 0.024 ± 0.002 0.020 ± 0.003 0.028 ± 0.004 0.025 ± 0.002 0.021 ± 0.003 0.023 ± 0.003 0.022 ± 0.002 0.001

Pr > F *** *** ***

(mg)

Seed weight

7.3 ± 0.6 7.6 ± 0.6 7.3 ± 0.5 0.041

(mm) 7.0 ± 0.4 7.5 ± 0.3 7.9 ± 0.4 7.2 ± 0.4 7.2 ± 0.4 8.1 ± 0.5 7.1 ± 0.4 7.6 ± 0.4 7.7 ± 0.6 7.3 ± 0.4 7.3 ± 0.4 7.2 ± 0.4 7.0 ± 0.4 6.8 ± 0.3 7.7 ± 0.4 7.3 ± 0.4 7.3 ± 0.4 7.2 ± 0.4 7.5 ± 0.4 7.4 ± 0.4 7.4 ± 0.3 7.5 ± 0.4 7.6 ± 0.5 7.2 ± 0.3 7.0 ± 0.4 8.0 ± 0.7 8.1 ± 0.4 7.9 ± 0.6 8.1 ± 0.5 6.9 ± 0.4 0.128

Pr > F *** *** ***

(mm)

Seed length

η2 0.445 0.128 0.080

42.8 ± 6.3 43.6 ± 5.0 41.9 ± 5.2 0.280

(kg-force) 42.7 ± 2.7 43.8 ± 3.1 45.5 ± 2.6 33.4 ± 4.9 36.0 ± 2.0 46.1 ± 2.7 45.6 ± 3.9 46.4 ± 2.3 45.9 ± 4.2 41.6 ± 3.5 43.2 ± 3.6 43.8 ± 2.7 44.6 ± 2.3 45.9 ± 3.9 41.2 ± 4.3 44.3 ± 2.7 43.6 ± 2.3 42.8 ± 2.8 35.2 ± 2.1 44.9 ± 3.6 32.6 ± 2.8 45.3 ± 3.8 31.0 ± 2.5 41.0 ± 3.0 44.0 ± 2.7 48.5 ± 3.8 49.4 ± 3.2 45.9 ± 2.8 46.3 ± 2.3 40.5 ± 3.2 0.884

Pr > F *** *** ***

(kg-force) η2 0.723 0.071 0.263

Seed hardness

2.3 ± 0.4 2.2 ± 0.3 2.3 ± 0.3 0.016

(index) 2.18 ± 0.1 2.14 ± 0.2 2.09 ± 0.1 2.90 ± 0.4 2.66 ± 0.1 2.10 ± 0.1 2.08 ± 0.2 2.01 ± 0.1 2.07 ± 0.2 2.28 ± 0.2 2.17 ± 0.2 2.17 ± 0.1 2.14 ± 0.1 1.95 ± 0.2 2.32 ± 0.3 2.14 ± 0.1 2.18 ± 0.1 2.23 ± 0.2 2.75 ± 0.2 2.09 ± 0.2 2.94 ± 0.3 2.08 ± 0.2 3.11 ± 0.3 2.32 ± 0.2 2.16 ± 0.1 1.97 ± 0.2 1.93 ± 0.1 2.11 ± 0.1 2.08 ± 0.1 2.35 ± 0.2 0.051

Pr > F *** *** ***

Aril texture

η2 0.776 0.111 0.267

Table 3 Analysis of variance, Partial Eta Squared (η2), means ± std and Least Significant Difference (LSD) for aril gross weight, aril net weight, juiciness index, juice lightness (L*) and redness (a*), seed weight, seed length, seed hardness and aril texture index of 30 pomegranate accessions from the Cypriot germplasm ex situ collection evaluated for three years.ss.

M.C. Kyriacou, et al.

Scientia Horticulturae 263 (2020) 109116

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M.C. Kyriacou, et al.

Fig. 1. Representative intact fruit from the 30 accessions of the Cypriot pomegranate germplasm ex situ collection.

Fig. 2. Representative equatorially sectioned fruit from the 30 accessions of the Cypriot pomegranate germplasm ex situ collection.

9

10

0.575 ± 0.28 0.534 ± 0.24 0.534 ± 0.24 1.680

η 0.815 0.016 0.086

15.8 ± 1.1 16.1 ± 1.1 17.4 ± 1.1 0.071

Pr > F *** *** ***

(%w/v) 0.545 ± 0.06 0.508 ± 0.05 0.442 ± 0.04 0.324 ± 0.12 0.401 ± 0.03 0.412 ± 0.05 1.146 ± 0.32 0.667 ± 0.07 0.515 ± 0.06 0.496 ± 0.06 0.634 ± 0.10 0.454 ± 0.06 0.453 ± 0.05 0.290 ± 0.04 0.367 ± 0.05 0.445 ± 0.06 0.431 ± 0.05 0.432 ± 0.04 0.429 ± 0.06 0.570 ± 0.07 0.472 ± 0.04 0.574 ± 0.07 0.440 ± 0.05 1.086 ± 0.22 0.351 ± 0.05 0.957 ± 0.26 0.519 ± 0.06 0.386 ± 0.04 0.407 ± 0.04 1.042 ± 0.24 0.036

η 0.577 0.547 0.190

2

(%w/v) 16.7 ± 1.1 15.5 ± 1.2 14.8 ± 1.1 16.5 ± 1.0 17.6 ± 1.1 15.9 ± 1.0 17.0 ± 1.0 16.6 ± 1.1 16.4 ± 1.5 16.4 ± 1.3 15.9 ± 1.2 17.0 ± 1.0 16.7 ± 1.0 15.6 ± 1.1 16.5 ± 0.6 16.7 ± 1.1 16.8 ± 0.8 16.8 ± 0.8 16.3 ± 0.9 16.3 ± 1.0 15.2 ± 0.8 16.5 ± 1.1 16.8 ± 1.0 18.6 ± 0.8 16.6 ± 1.4 16.5 ± 0.8 16.1 ± 1.3 16.9 ± 1.3 16.5 ± 1.0 18.4 ± 0.8 0.224

Pr > F *** *** ***

(% w/v)

(% w/v)

2

TA

SSC

32.5 ± 13.0 34.2 ± 11.2 36.9 ± 11.2 0.533

2

η 0.799 0.111 0.136

(ratio) 31.2 ± 4.7 30.9 ± 3.6 33.6 ± 2.9 57.3 ± 15.2 43.9 ± 3.3 39.5 ± 7.0 15.8 ± 4.6 24.9 ± 3.3 32.2 ± 5.0 33.7 ± 5.3 25.9 ± 5.5 38.0 ± 5.8 37.2 ± 4.7 54.3 ± 6.1 45.8 ± 7.1 38.4 ± 6.3 39.4 ± 4.7 39.3 ± 4.2 38.6 ± 5.2 28.7 ± 3.8 32.5 ± 3.3 29.2 ± 4.2 38.5 ± 4.8 17.6 ± 3.9 47.7 ± 7.0 17.9 ± 4.6 31.6 ± 4.8 44.3 ± 6.6 41.0 ± 5.2 18.5 ± 4.4 1.680

Pr > F *** *** ***

SSC/TA

72.2 ± 1.4 64.9 ± 0.9 73.1 ± 0.6 0.767

2

η 0.298 0.198 0.221

(g/L) 70.7 ± 9.5 64.6 ± 7.9 63.7 ± 5.4 74.0 ± 9.1 71.5 ± 10.8 69.0 ± 8.4 72.0 ± 10.0 67.6 ± 7.0 67.0 ± 10.7 65.4 ± 9.9 64.9 ± 9.8 73.4 ± 7.7 70.0 ± 6.7 66.7 ± 8.6 73.8 ± 8.5 68.0 ± 7.2 73.6 ± 10.4 73.2 ± 9.7 69.2 ± 7.5 67.9 ± 7.0 60.8 ± 7.3 71.2 ± 8.1 73.6 ± 7.2 81.9 ± 13.7 67.3 ± 9.8 69.5 ± 7.3 66.9 ± 9.1 69.5 ± 9.0 68.9 ± 8.7 81.0 ± 11.4 0.242

Pr > F *** *** ***

(mg/L)

Glucose

72.2 ± 1.3 77.9 ± 1.0 74.3 ± 0.5 0.782

2

η 0.274 0.092 0.177

(g/L) 74.9 ± 8.5 71.2 ± 7.6 65.3 ± 4.9 80.9 ± 7.6 80.5 ± 8.5 70.0 ± 9.3 75.1 ± 8.5 73.9 ± 7.5 69.8 ± 10.0 73.5 ± 9.7 69.1 ± 10.3 79.9 ± 6.5 75.3 ± 6.8 73.4 ± 9.9 81.3 ± 8.4 75.6 ± 7.8 78.9 ± 8.7 77.7 ± 8.7 75.3 ± 8.0 74.0 ± 7.2 68.0 ± 7.2 75.8 ± 8.6 79.3 ± 9.3 83.0 ± 9.1 73.8 ± 9.8 71.5 ± 9.5 70.3 ± 8.5 73.0 ± 8.8 70.6 ± 7.1 79.8 ± 9.3 0.247

Pr > F *** *** ***

(mg/L)

Fructose

1.00 ± 0.0 1.20 ± 0.0 1.02 ± 0.0 0.003

(ratio) 1.06 ± 0.1 1.11 ± 0.1 1.03 ± 0.1 1.10 ± 0.1 1.14 ± 0.1 1.01 ± 0.1 1.05 ± 0.1 1.10 ± 0.1 1.05 ± 0.1 1.13 ± 0.1 1.07 ± 0.1 1.10 ± 0.1 1.08 ± 0.1 1.10 ± 0.1 1.11 ± 0.1 1.11 ± 0.1 1.08 ± 0.1 1.07 ± 0.1 1.09 ± 0.1 1.09 ± 0.1 1.12 ± 0.1 1.07 ± 0.1 1.08 ± 0.1 1.03 ± 0.1 1.10 ± 0.1 1.03 ± 0.1 1.05 ± 0.1 1.06 ± 0.1 1.03 ± 0.1 0.99 ± 0.1 0.009

Pr > F *** *** ***

Fru/Glc

η 0.601 0.916 0.357

2

η 0.277 0.027 0.195

2

144.6 ± 2.6 143.1 ± 1.8 147.7 ± 1.0 0.154

(g/L) 146.0 ± 17.4 135.9 ± 14.5 129.7 ± 9.5 155.0 ± 15.8 152.1 ± 18.5 139.2 ± 16.7 147.3 ± 16.7 141.6 ± 13.7 137.3 ± 20.1 139.8 ± 17.8 134.4 ± 19.5 153.7 ± 12.3 145.4 ± 12.3 140.1 ± 17.7 155.2 ± 16.4 143.7 ± 13.9 152.7 ± 18.0 151.1 ± 17.3 144.4 ± 15.6 141.9 ± 13.1 129.1 ± 13.1 147.1 ± 15.9 153.4 ± 15.1 165.0 ± 21.2 141.2 ± 18.9 141.2 ± 14.7 137.4 ± 16.9 143.2 ± 15.8 139.5 ± 14.1 161.3 ± 19.5 0.486

Pr > F *** *** ***

(mg/L)

Total sugars

– 47.1 ± 11.8 42.7 ± 11.2 0.451

2

η 0.842 0.170 0.114

(%) 45.4 ± 6.2 41.2 ± 3.5 41.8 ± 5.8 25.2 ± 12.9 39.5 ± 4.2 46.7 ± 4.2 64.0 ± 5.2 51.0 ± 5.1 42.3 ± 4.6 45.4 ± 5.4 43.8 ± 5.5 44.5 ± 3.8 45.6 ± 3.3 11.6 ± 2.9 32.8 ± 7.5 45.9 ± 4.9 44.2 ± 3.6 44.8 ± 3.9 48.4 ± 7.7 42.8 ± 3.6 44.7 ± 4.0 41.8 ± 4.3 38.9 ± 3.8 61.3 ± 4.8 38.9 ± 4.2 67.3 ± 4.2 45.3 ± 4.7 45.2 ± 5.0 48.0 ± 4.1 62.6 ± 5.4 1.743

Pr > F *** *** ***

(%)

Citrate

– 38.6 ± 9.2 40.6 ± 8.7 0.556

2

η 0.652 0.051 0.107

(%) 41.1 ± 9.6 40.0 ± 4.4 32.8 ± 6.3 52.0 ± 16.1 45.8 ± 3.5 40.7 ± 4.5 25.0 ± 5.9 37.7 ± 4.1 37.3 ± 3.7 41.4 ± 6.8 34.9 ± 5.3 42.2 ± 3.9 40.0 ± 3.2 56.9 ± 5.1 39.9 ± 14.2 41.8 ± 4.9 42.1 ± 4.3 42.4 ± 3.8 40.5 ± 5.1 38.8 ± 4.2 35.5 ± 2.8 39.1 ± 4.4 45.2 ± 4.2 27.7 ± 3.8 42.1 ± 8.8 25.9 ± 3.6 35.3 ± 3.7 40.0 ± 3.7 40.2 ± 3.5 28.3 ± 5.0 2.150

Pr > F *** *** ***

(%)

Malate

– 1.34 ± 0.6 1.16 ± 0.6 0.026

(ratio) 1.08 ± 0.3 1.04 ± 0.2 1.26 ± 0.2 0.63 ± 0.6 0.87 ± 0.1 1.16 ± 0.2 2.58 ± 0.7 1.38 ± 0.3 1.15 ± 0.2 1.11 ± 0.3 1.30 ± 0.3 1.07 ± 0.2 1.15 ± 0.2 0.20 ± 0.1 0.78 ± 0.2 1.12 ± 0.2 1.07 ± 0.2 1.07 ± 0.2 1.23 ± 0.4 1.12 ± 0.2 1.28 ± 0.2 1.09 ± 0.2 0.87 ± 0.2 2.23 ± 0.3 0.94 ± 0.3 2.67 ± 0.5 1.30 ± 0.2 1.14 ± 0.2 1.20 ± 0.2 2.26 ± 0.5 0.101

Pr > F *** *** ***

η 0.793 0.094 0.096

2

Citrate/Malate

– 14.5 ± 7.1 16.7 ± 6.8 0.415

(%) 11.4 ± 5.0 18.8 ± 3.5 25.0 ± 8.2 19.5 ± 4.8 14.7 ± 4.8 12.7 ± 5.8 10.7 ± 3.9 11.3 ± 3.6 20.4 ± 4.9 12.9 ± 3.8 21.3 ± 4.1 13.3 ± 3.8 14.4 ± 3.7 31.5 ± 4.7 24.2 ± 5.7 11.9 ± 4.0 13.6 ± 3.9 12.8 ± 3.5 11.0 ± 5.1 18.4 ± 3.1 19.8 ± 3.6 19.1 ± 3.0 15.9 ± 3.6 10.8 ± 4.8 18.1 ± 5.8 7.0 ± 3.9 19.4 ± 5.5 14.8 ± 5.5 11.8 ± 5.5 8.7 ± 6.0 1.602

Pr > F *** *** ***

(%)

Succinate

η2 0.606 0.053 0.148

SSC, TA, SSC/TA, glucose, fructose, total sugar contents and fructose/glucose ratio were evaluated for three years (2014–2016); citric, malic, succinic acid contents and citrate/malate ratio were evaluated for two years (2015 and 2016). ***significant effect at the 0.001 level.

§

Accession Achelia 1 Achelia 2 Achelia 3 Achelia 4 Akrotiri Chocolata Dymes Emba 1 Emba 2 Emba 3 Emba 4 Finikaria Ftanofyllo Geri Geroskipou Kato Mylos 1 Kato Mylos 2 Kato Mylos 3 Kouklia 1 Kouklia 2 Marinouda 1 Marinouda 2 Marinouda 3 Mazotos 1 Mazotos 2 Mazotos 3 Paphos Sotikartiko Sotira Wonderful LSD Year 2014 2015 2016 LSD

Accession Year Accession × Year

Source of variance

Table 4 Analysis of variance, Partial Eta Squared, means ± std and Least Significant Difference (LSD) for juice soluble solids (SSC), titratable acidity (TA), SSC/TA ratio, glucose, fructose and total sugar contents, fructose/ glucose ratio, relative content of citric, malic and succinic acids, and the citrate/malate ratio of 30 pomegranate accessions from the Cypriot germplasm ex situ collection§.

M.C. Kyriacou, et al.

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accession, as reflected in the respective η2 values for accession (0.893) and year (0.177) effects. With the exception of 'Mazotos2', which has a conspicuously red-colored juice, anthocyanin levels in Cypriot pomegranates were generally in the low end of the range reported for Spanish (Hernández et al., 1999), Israeli (Tzulker et al., 2007), Tunisian (Hasnaoui et al., 2011), Turkish (Caliskan and Bayazit, 2012) and Italian (Adiletta et al., 2018) accessions. The anthocyanin concentration found in 'Wonderful' (188.8 mg/L) corroborates the concentration (≈210 mg/L) found by Holcroft et al. (1998) in Fresno, California but it is much lower than that found by Adiletta et al. (2018) in Italy (555 mg/L). Given that the present germplasm collection was established on the southern coastal zone of Cyprus, where autumnal temperatures are mild, it is possible that the same germplasm would attain higher anthocyanin concentrations where it cultivated at higher elevations (Schwartz et al., 2009b). Total phenolic content (TPC) of the juice of Cypriot accessions averaged 613.0 mg/L and ranged from 384.3 mg/L in 'Ftanofyllo' to 858.1 mg/L in 'Emba4' (Table 5). Overall, the TPC was below the 2.5 g/ L maximum concentration tolerable in terms of astringency (Pareek et al., 2015). The TPC obtained in ‘Wonderful’ (896.6 mg/L) was nearly half of that (2117 mg/L) obtained for the same variety in California (Holcroft et al., 1998) which may reflect different agro-environmental conditions and different harvest maturities, given that phenolic concentrations decline during ripening (Pareek et al., 2015). Cypriot accessions had TPC comparable to Greek germplasm (225−697 mg/L; Drogoudi et al., 2005) and low end Italian germplasm (797−1190 mg/ L; Adiletta et al., 2018) but significantly lower than Turkish germplasm (1245−2076 mg/L; Özgen et al., 2008). These differences may reflect variation in genetic background and agro-environmental conditions but also juice extraction methods, e.g. squeezing arils vs. pressing fruits, which may carryover the high TPC present in the fruit rind and membranes to the juice, as commonly observed with industrial pomegranate juice (Gil et al., 2000). Phenolic concentration correlated positively (r = 0.951, p < 0.01) with the antioxidant capacity of the juice (AEAC), and less so with juice redness (a*; r = 0.462, p < 0.05; Table 2). Moreover, the low correlation coefficients of AEAC with juice lightness-L* (r=-0.382; p < 0.05), redness-a* (r = 0.385; p < 0.05) and anthocyanin content (r = 0.456; p < 0.05) suggest that juice color and anthocyanin content do not constitute reliable indices of antioxidant capacity. Our findings corroborate Gil et al. (2000) who concluded that only a limited fraction of pomegranate juice’s radical scavenging potential seems linked to flavonoid content as the major antioxidant phenolic components are ellagitannins and ellagic acid. The TPC of juice had significant (p < 0.01) negative correlation to juiciness index (r=-0.773), aril yield (r=-0.747) and aril net weight (r=-0.517) but no significant correlation to fruit gross weight which suggests that phenolic concentration is higher in pomegranate accessions bearing thick fruit rind and low juice content. Yearly, the TPC ranged from 529.3 mg/L in 2015 to 685.3 mg/L in 2014. This attests to the strong genotypic effect underlying TPC, reflected also in the lower η2 value for the year effect (0.248) compared to accession effect (0.694; Table 5). The AEAC of Cypriot pomegranate juice, expressed in ascorbate equivalents, ranged 3.624–8.030 mM (Table 5). These values are on the low end of the range reported by previous workers (Gil et al., 2000; Tzulker et al., 2007) and reflect the low TPC of Cypriot accessions. However, aside from genotypic and agro-environmental factors, different juice extraction methods may influence carryover of phenolic components from the rind and membranes to the juice causing a spike in antioxidant capacity and astringency (Gil et al., 2000). The AEAC correlated more with TPC (r = 0.951, p < 0.01) than anthocyanins (r = 0.456, p < 0.05) or juice redness-a* (r = 385, p < 0.05; Table 2). The AEAC demonstrated significant (p < 0.01) negative correlation with juiciness index (r=-0.679), aril yield (r=-0.641) and aril net weight (r=-0.543) but not with fruit gross weight, which suggests that AEAC is higher in accessions characterized by thick rind and low juice content. Yearly mean AEAC ranged 4.996–6.287 mM while η2

values for year (0.279) and accession effects (0.596) indicate it is predominantly under genetic control. Ascorbate concentration in the juice of Cypriot accessions ranged 23.2–318.9 mg/L with an overall mean of 175.4 mg/L (Table 5). Similar concentrations (170−300 mg/L) were reported in Israeli germplasm (Schwartz et al., 2009a, 2009b) and lower in Iranian pomegranates (97.8 mg/100 ml; Zarei et al., 2011). Ascorbate concentration correlated significantly with the citrate/malate ratio (r = 0.528, p < 0.01) but not with most other physicochemical variables, including the AEAC. This confirms that the contribution of ascorbate to the antioxidant capacity of pomegranate juice is low compared to that of phenolic components (Gil et al., 2000). 3.5. Cluster analysis based on phenotypic traits Hierarchical cluster analysis based on phenotypic characters grouped the accessions into nine major sub-clusters (Fig. 3A; Supplementary Table 2). Half of the accessions (15/30) formed cluster VIIc, which represents accessions of average fruit weight, fair juice index, thin rind, rather hard seeds, poor juice color and low anthocyanin, phenolic and AEAC values. 'Wonderful' and 'Mazotos1' formed group Ia that was phenotypically the most disparate. This cluster of late-maturing accessions demonstrated the highest fruit size and gross weight, the reddest external coloration, the darkest and reddest juice coloration, the highest SSC, TA, phenolic content and AEAC, together with an average seed hardness and aril texture; however, it also had the thickest rind and consequently a very low juiciness index. Group IIIa of latematuring accessions 'Dymes' and 'Mazotos3' demonstrated large fruit combined with thin rind, high aril weight and high juiciness index, and faintly colored juice of average sugar content and high acidity. Local accessions of distinct character were also 'Marinouda1' (IVa) and 'Geri' (IIa), both bearing very low fruit weight and low aril weight, SSC and juiciness index. Finally, accessions 'Chocolata', 'Sotirkatiko' and 'Sotira' formed cluster VIb, distinguished by very thin rind, very high juiciness index and low acidity. 3.6. Genetic diversity and cluster analysis The microsatellites selected for this study were polymorphic (Supplementary Table 1). Thirty-three alleles were detected with an average of 3.3 alleles per locus. Nine alleles appeared with frequency < 5% and four were unique to a single accession. PIC values ranged 0.122 to 0.563, while Expected Heterozygosity (He) ranged 0.210 to 0.633. Average Shannon Information Index (I), PIC and He were 0.779, 0.393 and 0.453, respectively. The level of polymorphism detected in the present study was moderate, slightly lower or comparable to previous works employing SSRs (Curro et al., 2010; Ebrahimi et al., 2010; Pirseyedi et al., 2010; Hasnaoui et al., 2012; Jbir et al., 2012; Jian et al., 2012; Ferrara et al., 2014; Zarei and Sahraroo, 2018). For example, Alamuti et al. (2012) assessed 738 Iranian accessions with 12 microsatellites and reported mean PIC and He values of 0.458 and 0.521, respectively. Parvaresh et al., 2012 revealed 38 alleles with an average of 3.17 alleles per locus and mean PIC of 0.358 in a collection of 75 accessions of diverse origin. In a collection of 136 varieties, most of them originating from China, Luo et al., 2018 reported PIC values ranging 0.14 to 0.29, with an average of 0.22. Average He was 0.473 in a collection containing 88 wild and cultivated accessions from India (Singh et al., 2015). For some primers, allele range differed from that reported in other studies employing common SSRs (Ebrahimi et al., 2010; Hasnaoui et al., 2012; Ferrara et al., 2014; Zarei and Sahraroo, 2018) suggesting the presence of unique genetic diversity within the Cypriot pomegranate germplasm. It can be inferred that the long history of the species on the island (Jacomet et al., 2002), proximity to the center of origin (Teixeira da Silva et al., 2013; Holland and Bar- Ya’akov, 2018) and geographical isolation shaped pomegranate genetic diversity in Cyprus. Further to 11

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Table 5 Analysis of variance, Partial Eta Squared, means ± std and Least Significant Difference (LSD) for juice anthocyanin, total phenolic and ascorbate contents, and ascorbate equivalent antioxidant capacity (AEAC) of 30 pomegranate accessions from the Cypriot germplasm ex situ collection evaluated for three years § ascorbate content was evaluated for two years (2014 and 2016). Source of variance

Anthocyanins

Accession Year Accession × Year

Pr > F Pr > F *** *** ***

Accession Achelia 1 Achelia 2 Achelia 3 Achelia 4 Akrotiri Chocolata Dymes Emba 1 Emba 2 Emba 3 Emba 4 Finikaria Ftanofyllo Geri Geroskipou Kato Mylos 1 Kato Mylos 2 Kato Mylos 3 Kouklia 1 Kouklia 2 Marinouda 1 Marinouda 2 Marinouda 3 Mazotos 1 Mazotos 2 Mazotos 3 Paphos Sotikartiko Sotira Wonderful LSD Year 2014 2015 2016 LSD

Phenolics Eta η2 0.893 0.177 0.347

Ascorbate§

AEAC

Pr > F Pr > F *** *** ***

Eta η2 0.694 0.248 0.280

Pr > F Pr > F *** *** ***

Eta η2 0.596 0.279 0.185

Pr > F Pr > F *** *** ***

(mg/L) 26.9 ± 13.9 18.4 ± 13.8 1.2 ± 1.5 31.4 ± 17.9 9.8 ± 6.5 3.4 ± 1.5 15.1 ± 9.4 46.1 ± 28.7 12.4 ± 11.1 27.7 ± 12.8 25.6 ± 19.1 14.5 ± 9.9 21.6 ± 10.6 30.4 ± 17.3 12.7 ± 13.5 14.5 ± 9.4 13.7 ± 9.9 12.9 ± 10.3 39.9 ± 15.6 25.2 ± 17.5 8.2 ± 8.2 27.7 ± 17.4 16.9 ± 10.0 191.2 ± 51.0 100.0 ± 58.7 7.0 ± 10.0 5.4 ± 4.4 3.8 ± 2.1 3.8 ± 3.4 188.8 ± 54.1 5.592

(mg/L GAE) 743.5 ± 139.3 567.6 ± 85.6 711.3 ± 126.3 695.4 ± 135.2 705.8 ± 89.2 413.5 ± 75.4 568.9 ± 113.5 748.2 ± 209.4 616.0 ± 154.7 628.3 ± 111.8 858.1 ± 166.2 466.2 ± 68.7 384.3 ± 65.1 817.7 ± 107.7 678.2 ± 145.7 507.6 ± 120.1 437.0 ± 81.1 420.4 ± 94.8 468.5 ± 75.4 621.3 ± 124.0 738.1 ± 131.4 655.8 ± 121.4 549.5 ± 108.3 883.1 ± 243.7 683.5 ± 122.7 428.0 ± 114.2 638.8 ± 121.4 442.5 ± 94.3 417.3 ± 72.0 896.6 ± 216.5 32.258

(mM) 6.739 ± 1.26 5.375 ± 0.79 6.809 ± 1.33 5.936 ± 1.07 7.141 ± 0.98 3.903 ± 0.79 6.054 ± 1.42 6.229 ± 1.45 5.664 ± 1.16 5.495 ± 0.95 8.030 ± 1.06 4.325 ± 0.69 3.674 ± 0.61 6.883 ± 1.39 6.204 ± 1.60 4.800 ± 1.25 4.192 ± 0.82 4.208 ± 0.85 4.269 ± 0.91 5.867 ± 1.43 5.641 ± 0.89 6.013 ± 1.44 5.019 ± 0.98 7.410 ± 1.65 5.888 ± 1.43 4.786 ± 1.13 6.161 ± 1.03 4.354 ± 1.10 4.173 ± 0.89 7.392 ± 1.29 0.302

(mg/L) 194.1 ± 48.3 128.1 ± 24.5 209.3 ± 33.3 155.6 ± 32.5 169.0 ± 30.5 186.8 ± 51.9 257.1 ± 36.1 183.6 ± 37.0 175.2 ± 45.9 175.0 ± 41.0 195.8 ± 41.2 201.6 ± 32.1 189.1 ± 78.9 143.4 ± 33.3 149.4 ± 35.5 160.1 ± 29.9 135.5 ± 41.2 169.3 ± 64.7 162.2 ± 29.3 123.2 ± 36.0 131.4 ± 64.5 141.0 ± 40.2 170.9 ± 40.4 135.4 ± 77.5 194.0 ± 58.7 318.9 ± 47.3 192.4 ± 43.1 133.9 ± 11.0 214.1 ± 90.9 166.9 ± 65.6 13.256

44.0 ± 65.8 22.6 ± 42.2 38.6 ± 52.0 1.772

685.3 ± 232.2 529.3 ± 155.8 645.4 ± 179.3 10.229

5.460 ± 1.49 4.996 ± 1.41 6.287 ± 1.65 0.096

146.2 ± 66.4 -±190.8 ± 51.6 3.442

Eta η2 0.564 0.247 0.454

***significant effect at the 0.001 level.

the historical migration of germplasm (Ophir et al., 2014), human selection in different geographical regions exerted a crucial role in shaping genetic diversity (Parvaresh et al., 2012). Lacks of an official framework for the production and distribution of certified pomegranate propagative material has encouraged Cyprus farmers to select cuttings individually from trees carrying desirable traits. This practice likely curbed the erosion of local genetic variability further reinforced by the persistent preference of local markets for fruits of familiar varietal character. The ten SSRs revealed 21 distinct genotypes. Jaccard coefficients ranged from 0.3 to 1. The average value was 0.63, which was lower than that reported by Jian et al., 2012. Cluster analysis grouped the accessions into six major sub-clusters (Fig. 3B). The most genetically distinct accession was 'Marinouda1'. The majority of Cypriot accessions (18/29) grouped into sub-cluster IIIb, with most (13/18) being also phenotypically close (Fig. 3A). According to Villa et al. (2005) ” landrace is a dynamic population(s) of a cultivated plant that has historical origin, distinct identity and lacks formal crop improvement, as well as often being genetically diverse, locally adapted and associated with traditional farming systems”. It can be inferred that this set of thirteen

accessions constitutes the core of a Cypriot landrace. Similarly, the widely grown in the Mediterranean basin varieties 'Wonderful' and 'Mollar de Elche', actually constitute groups of genotypes presenting limited genetic and phenotypic variation (Holland and Bar- Ya’akov, 2018). Introduced variety 'Wonderful' constituted sub-cluster IVa along with accessions 'Mazotos1' and 'Mazotos2'. The genetic similarity of 'Wonderful' to 'Mazotos1' (GD = 1.0) suggests they likely represent the same clonal selection. 'Wonderful'-type germplasm presumably originated in the Mediterranean basin (Ophir et al., 2014), but although this variety predominates in neighboring countries (Holland and BarYa’akov, 2018) the core of the Cypriot pomegranate landrace remains genetically and phenotypically divergent from it. However, given the extensive geneflow between different regions reported for this species (Luo et al., 2018; Zarei and Sahraroo, 2018), it can be speculated that hybridization of local germplasm with introduced material also occurred in Cyprus. For example, the accession 'Mazotos2' was genetically close to the introduced variety 'Wonderful' (GD = 0.7), however phenotypically it was closer to the core of the local germplasm. 12

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Fig. 3. A. Dendrogram of 30 pomegranate accessions based on squared Euclidean distances calculated on standardized mean phenotypic data across years. B: UPGMA dendrogram of 30 pomegranate accessions based on Jaccard genetic similarity.

4. Conclusions

Appendix A. Supplementary data

The long history of pomegranate in Cyprus, proximity to the center of origin and geographical isolation shaped a unique genetic diversity. Twenty-one distinct local genotypes were identified, the majority of which formed a core sub-cluster constituting the Cypriot pomegranate landrace. These genotypes were genetically and phenotypically divergent from commercial variety Wonderful and shared similar phenotypic characteristics of moderate fruit weight, thin rind, moderate seed hardness and high juice content low in anthocyanins, phenolics and antioxidant capacity. Accessions highly suitable for cultivation under Mediterranean pedoclimatic conditions include 'Sotirkatiko', 'Chocolata', 'Sotira', 'Finikaria' and 'Kato Mylos1-3' distinguished for their very high juice content. They may also serve as breeding material for improving the juice content of 'Wonderful'. 'Mazotos2' constitutes promising breeding material for improving the Cypriot pomegranate landrace in terms of color and earliness, and soft-seeded 'Marinouda1 and 3' for imparting improved aril texture. Constituent signature traits for pomegranate accessions were those related to juice acidity, including titratable acidity and the citrate/ malate ratio. These traits demonstrated very limited yearly variation. Aril weight, seed hardness, fruit gross weight and fruit shape were the physical traits determined mainly by genotype. Cypriot germplasm had similar SSC range to other germplasm collections. Glucose and fructose were the predominant juice sugars. Total sugars were generally higher in large-fruited accessions with darker juice. Cypriot germplasm was characterized overall by low acidity. Based on the maturity index (SSC/ TA), six accessions were sweet-sour and 23 accessions were sweet. Juice antioxidant capacity was associated primarily with total phenolics and less so with juice color and anthocyanin content.

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 13

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