Morphological variability of wild pomegranate (Punica granatum L.) accessions from natural habitats in the Northern parts of Iran

Scientia Horticulturae 264 (2020) 109165

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Morphological variability of wild pomegranate (Punica granatum L.) accessions from natural habitats in the Northern parts of Iran

T

Ali Khadivi*, Farhad Mirheidari, Younes Moradi, Simin Paryan Department of Horticultural Sciences, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349, Arak, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Germplasm Breeding Fruit quality Aril color Pomegranate

Wild pomegranates (Punica granatum L.) are considered as functional food with significant health-promoting properties. The objective of the current research was to determine morphological and pomological variability among the wild pomegranate accessions from the Northern parts of Iran. Analysis of variance (P < 0.01) revealed meaningful differences among the accessions studied based on all the attributes recorded. Fruit weight ranged from 19.20–185.00 g with an average of 59.89. Fruit peel color showed high variability and ranged from yellow to brown-red. In addition, fresh weight of 100 arils ranged between 5.75 and 27.55 g with an average of 11.77. High range of diversity was observed among the accessions based on aril color including glassy, pale milk, pink, white-red, red, and dark red. Seed hardness was predominantly hard, although some accessions had soft and semi-soft seeds (14 and 48 accessions, respectively). The range of total soluble solids was 9.30-27.00% with an average of 17.09 and fruit taste was sour-sweet in 12 accessions and also sweet in 11 accessions. Principal component analysis (PCA) revealed that fruit-related characters were important factors in differentiating the accessions. Cluster analysis based on Ward method showed two different major clusters among all the accessions studied. Most of the accessions studied could be selected as a parent in breeding programs for the improvement of juice quality, seed softness, and aril and fruit appearance. The obtained data here could be used to design sampling strategies, create core collections, and establish pomegranate breeding studies.

1. Introduction The genus Punica belongs to the family Punicaceae and consists of two species including Punica granatum L. and P. protopunica Balf. f. However, some authors classified the ornamental dwarf pomegranate (P. nana L.) as a distinct species (Melgarejo and Martinez, 1992). The P. protopunica is endemic to Socotra Island, Yemen (Guarino et al., 1990). The pomegranate is probably originated from Iran (Simmonds, 1976; Levin, 1994) and from there it diversified to other regions like Mediterranean countries, India, China, Pakistan, and Afghanistan, possibly through ancient trade routes. It is one of the oldest known edible fruits (Damania, 2005). Wild pomegranate is well adapted to a wide range of climatic conditions and therefore has a wide geographical distribution. The wild types of pomegranate in central Asia vary widely in fruit size, sweetness, ripening time, juiciness, and the proportions of seeds and flesh (IBPGR, 1986). In Iran, pomegranate grows wild in the Northern parts of the country. It also occurs wild in some parts of Afghanistan, Pakistan (Nasir and Ali, 1972), and Western Himalayan regions in India (Pandey et al., 2008).

⁎

Wild pomegranate fruits have been noted with much smaller arils, thicker rinds, and higher acidity than cultivated ones (Kher, 1999). In recent years, the demand for its value-added products got increased due to its recognition as a great source of natural antioxidants and healthpromoting constituents like organic acids, anthocyanins, phenolics, vitamins, and minerals (Thakur et al., 2010, 2011). Wild pomegranates are characterized by different types of phytonutrients in the form of anthocyanins, phenolics, and flavonoids that impart antioxidant properties in addition to color (Sharma and Thakur, 2016). The bioactive components are mainly concentrated in the juicy arils of the fruit and the presence of a sufficient amount of the organic acids, vitamins, and minerals have led to categorize it as a functional food with nutraceutical properties. Considering the nutritional and nutraceutical properties of the wild pomegranate, its industrialization in the form of different commercial processed products is very important in fulfilling the nutrient requirements of the people in developing countries particularly as a cheaper source of bioactive components as compared with cultivated one. Identifying and preserving genetic diversity is an important factor in any crop improvement program. Parental varieties identified on the

Corresponding author. E-mail address: [email protected] (A. Khadivi).

https://doi.org/10.1016/j.scienta.2019.109165 Received 25 November 2019; Received in revised form 24 December 2019; Accepted 27 December 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Corporation, Tokyo, Japan), in ˚Brix.

basis of divergence for any breeding program would be more promising. Grouping or classification of genotypes based on a suitable scale is quite imperative to understand the usable variability (Arunachalam, 1981). Morphological characterization of native germplasm accessions is the first step for their rational introduction in modern and efficient production chains and a standardized description of phenotypic traits based on the adoption of specific descriptor lists is a pre-requisite for this purpose (Frankel, 1970). Studies on genetic diversity of wild pomegranate can illustrate the various facets of the domestication process and reasons for its diversification (Langlie et al., 2014). Despite its potential, studies on genetic resources of this wild plant species are almost lacking. Thus, the objective of the current research was to determine the phenotypic variability that exists among the wild pomegranate accessions in natural habitats of Mazandaran and Gilan provinces from the Northern parts of Iran and to understand the degree of polymorphism of the morphometric characteristics among the accessions.

2.3. Statistical analysis Data were analyzed using the mean values and then the conclusions were drawn. The mean, variances, correlations, and genetic diversity were calculated to deduce the genetic similarity/dissimilarity. Significant differences between the traits were revealed using one-way analysis of variance (ANOVA) by SAS (SAS Institute, Cary, NC, USA, 1990). Coefficients of variation (CV) were determined as indicators of variability. Correlations between the traits were determined using the Pearson correlation coefficient with SPSS® software version 16 (SPSS Inc., Chicago, IL, USA, Norusis, 1998). Relationships among the accessions were investigated with principal component analysis (PCA) using SPSS® software. Cluster analysis was performed using Euclidean distance coefficient by Ward method by PAST software (Hammer et al., 2001). The calculation of distances was done after standardization using Z-scores. Furthermore, scatter plot was created according to the first two principal components (PC1 and PC2) using PAST software.

2. Materials and methods

3. Results and discussion

2.1. Plant material

3.1. Morphological characterizations

Phenotypic variation of 204 wild accessions of pomegranate (P. granatum) from natural habitats of Mazandaran and Gilan provinces in the North of Iran was assessed using morphological and pomological characteristics. Geographical coordinates and altitude corresponding to each surveyed area are shown in Table 1. To avoid the possibility of sampling and collecting clones of the selected accessions, at least 200 m distance was considered between the accessions in each collection site.

The ANOVA (P < 0.01) revealed meaningful differences among the accessions studied based on all the attributes recorded. The range of CV was from 9.28 (in seed length) to 210.00% (in leaf serration depth) with an average of 51.20% (Table 2). In addition, 60 out of 71 characters measured showed CVs more than 20.00% confirming the revealed differences by ANOVA among the accessions. Tree growth habit was predominantly spreading (111 accessions), although other growth habit types were observed including weeping (6), open (33), semi-erect (39), and erect (15). The tree skeleton-related characters such as tree growth vigor, tree height, branching, branch density, branch flexibility, canopy density, and leaf density were predominantly high. Tendency to suckering was intermediate in the majority of accessions (88) (Table 3). Three forms of leaf shape were observed including elliptic-lanceolate (59 accessions), lanceolate (112), and broad elliptic (33). Acute leaf apex shape was predominant (101 accessions). In addition, 150 out of 204 accessions studied had none leaf serration. Leaf length ranged from 19.09–77.80 mm and leaf width varied from 6.43 to 22.84 mm. The range of petiole length was 1.62–11.55 mm and petiole diameterwas 0.42–1.60 mm. Thorns were presented in 201 out of 204 accessions studied (Table 3). Thorn length and thickness on annual shoots of those 201 accessions ranged from 2.52 to 41.02 mm and 0.53–2.98 mm, respectively. Fruits in most of the accessions (102) were ripened in late September. Maturity and harvesting are the factors that affect the quality of fruits. Wild pomegranate is a non-climacteric fruit that is harvested at the ripe stage. At maturity, the surface of green immature wild pomegranate fruit is turned to yellowish-green, whereas, the green-purple rind color of immature fruit of cultivated pomegranate is turned to deep pink with reddish and yellowish patches at maturity (Bhat, 2007; Dhaygude, 2010). The milky white color of arils at the early stage turns to creamy pearl white when fruit matures and with the advancement of fruit maturity, the percentage of arils increases, while that of seed, rind and the thickness of rind decreases gradually in pomegranate (Khodade et al., 1990). The optimum harvesting date ensures to access the maximum nutritional properties of cultivated as well as wild pomegranates. Fruit yield was intermediate in the majority of accessions (112) and followed by high (65). The accessions were clustered into five groups based on fruit shape including round (61), ovate (2), oval (52), elliptical (20), and oblate (69). In addition, fruit base shape was truncate (73 accessions), covex (101), and angular (30). Furthermore, fruits in

2.2. The characters evaluated Phenotypic variability among the studied accessions was determined using 71 morphological and pomological characters (Table 2). A total of 50 leaves and 50 fruits per accession were randomly selected to evaluate the characters related to those organs. The traits including leaf length, leaf width, petiole length, petiole diameter, thorn length on annual shoot, thorn thickness on annual shoot, fruit length, fruit diameter, sepal length, sepal base width, fruit calyx length, fruit calyx diameter, fruit stalk length, aril length, aril width, seed length, and seed width were measured using a digital caliper. Fruit weight, fruit peel weight, and 100 arils fresh weight were measured using an electronic balance with 0.01 g precision. The qualitative characters (Table 3) were measured based on rating and coding on the basis of the method developed by Mars and Marrakchi (1999). In addition, the total soluble solids (TSS) were determined by a refractometer (pocket PAL-1 ATAGO Table 1 Geographical description for collection areas of wild pomegranate accessions investigated from Mazandaran and Gilan provinces in Iran. No.

Province

Area

Latitude (N)

Longitude (E)

Altitude (m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mazandaran Mazandaran Mazandaran Mazandaran Mazandaran Mazandaran Mazandaran Mazandaran Gilan Gilan Gilan Gilan Gilan Gilan Gilan

Polsefid Sooteh Shirgah Ghajarkhil Hasanabad Vachklaye Mashlabad Shirood Limesara Sarvlat Daroogar Korfmehale Kalamsar Poonel Ardeh

36°07′03″ 36°46′03″ 36°18′30″ 36°46′30″ 36°48′35″ 36°54′28″ 36°40′14″ 36°50′37″ 36°56′41″ 36°57′16″ 37°06′37″ 37°07′02″ 37°08′47″ 37°31′53″ 37°32′16″

53°03′22″ 53°06′44″ 52°53′08″ 53°03′14″ 53°08′12″ 50°36′16″ 51°06′28″ 50°49′22″ 50°32′23″ 50°32′52″ 50°20′58″ 50°15′07″ 49°50′57″ 49°01′06″ 48°49′48″

565 −27 223 −10 −21 209 112 −19 258 66 −10 −10 39 869 972

2

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Table 2 Descriptive statistics for morphological traits utilized in the studied wild pomegranate accessions. No.

Character

Unit

Min.

Max.

Mean

SD

CV (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71

Tree growth habit Tree growth vigor Shoot color Tree height Branching Branch density Branch flexibility Trunk type Trunk diameter Canopy density Tendency to suckering Leaf density Leaf shape Leaf apex shape Leaf length Leaf width Leaf upper surface color Leaf lower surface color Leaf serration presence Leaf serration depth Leaf serration shape Leaf margin form Petiole length Petiole diameter Petiole color Thorn presence on shoot Thorn length on annual shoot Thorn thickness on annual shoot Ripening time Yield Fruit length Fruit diameter Fruit shape Fruit base shape Fruit symmetry Pacifier presence on fruit Stamen density in fruit calyx Sepal number Sepal length Sepal base width Fruit calyx form Fruit calyx length Fruit neck presence Fruit calyx diameter Fruit weight Fruit stalk length Fruit peel color Fruit peel necrotic presence Fruit peel sunburn presence Fruit peel cracking presence Internal peel color Fruit peel thickness Fruit peel weight Internal layer color Internal layer thickness Septum color Septum thickness Septum transparency Aril adherence to internal peel Aril shape Aril color 100 aril fresh weight Aril length Aril width Seed length Seed width Seed hardness Fruit juice color Fruit taste Fruit quality Total soluble solids

Code Code Code Code Code Code Code Code Code Code Code Code Code Code mm mm Code Code Code Code Code Code mm mm Code Code mm mm Date Code mm mm Code Code Code Code Code Number mm mm Code mm Code mm g mm Code Code Code Code Code Code g Code Code Code Code Code Code Code Code g mm mm mm mm Code Code Code Code %

1 1 1 1 1 1 1 1 1 1 1 1 1 1 19.09 6.43 1 1 0 0 1 1 1.62 0.42 1 0 0.00 0.00 Mid-Sep 1 27.20 30.98 1 1 0 0 1 3 6.36 4.08 1 3.00 0 4.85 19.20 3.25 1 0 0 0 1 1 5.21 1 1 1 1 1 1 1 1 5.75 5.66 3.08 4.82 1.92 1 1 1 1 9.30

9 5 9 5 5 5 5 7 5 5 5 5 5 5 77.80 22.84 5 5 5 5 5 3 11.55 1.60 25 1 41.02 2.98 Early Oct 5 63.27 71.40 9 5 1 5 5 8 13.91 9.11 5 14.71 1 23.05 185.00 16.80 21 5 5 5 5 5 53.28 5 5 7 5 5 5 7 11 27.55 10.22 8.73 8.00 7.03 5 7 9 7 27.00

4.47 3.85 6.63 3.57 3.96 3.92 4.23 4.19 3.25 3.51 2.96 4.25 2.75 2.85 44.53 13.89 3.25 2.09 0.75 0.59 1.60 1.74 4.13 0.83 8.93 0.99 11.54 1.58 3.82 3.37 43.16 47.48 5.33 2.58 0.68 0.57 3.62 6.11 10.30 6.27 3.37 7.73 0.98 12.69 59.89 8.16 6.40 1.62 0.96 0.83 2.43 2.11 20.73 1.77 2.11 3.93 1.94 3.25 2.18 4.02 6.85 11.77 7.97 5.38 6.43 3.18 4.25 3.90 4.81 3.48 17.09

2.08 1.36 2.08 1.46 1.17 1.26 1.09 1.83 1.32 1.45 1.51 1.01 1.32 1.92 12.01 3.05 1.22 1.11 1.56 1.24 1.06 0.97 1.39 0.19 7.04 0.12 6.52 0.57 1.15 1.29 7.03 7.50 3.25 1.36 0.47 1.17 1.63 0.73 1.43 1.01 1.11 1.73 0.14 2.66 27.47 2.62 4.62 1.77 1.66 1.53 0.97 1.49 8.97 1.00 1.45 2.10 1.23 1.50 1.43 1.79 3.01 3.39 0.84 0.92 0.60 0.86 1.22 1.79 1.53 2.23 3.01

46.55 35.32 31.33 40.76 29.42 32.17 25.79 43.58 40.68 41.31 51.05 23.81 48.07 67.51 26.96 21.97 37.63 53.16 207.73 210.00 66.13 55.57 33.74 23.13 78.82 12.22 56.49 36.12 30.18 38.40 16.28 15.79 60.99 52.75 68.97 205.96 45.14 11.91 13.85 16.13 33.03 22.33 14.18 20.99 45.86 32.14 72.11 108.95 172.50 183.86 39.84 70.52 43.29 56.33 68.63 53.31 63.40 46.09 65.46 44.58 43.94 28.81 10.50 17.13 9.28 27.03 28.73 45.87 31.87 63.94 17.62

3

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Table 3 Frequency distribution for the measured qualitative morphological characters in the studied wild pomegranate accessions. Frequency (no. of accessions) Character Tree growth habit Tree growth vigor Shoot color Tree height Branching Branch density Branch flexibility Trunk type Trunk diameter Canopy density Tendency to suckering Leaf density Leaf shape Leaf apex shape Leaf upper surface color Leaf lower surface color Leaf serration presence Leaf serration depth Leaf serration shape Leaf margin form Thorn presence on shoot Ripening time Yield Fruit shape Fruit base shape Fruit symmetry Pacifier presence on fruit Stamen density in fruit calyx Fruit calyx form Fruit neck presence Fruit peel necrotic presence Fruit peel sunburn presence Fruit peel cracking presence Internal peel color Fruit peel thickness Internal layer color Internal layer thickness Septum color Septum thickness Septum transparency Aril adherence to internal peel Aril shape Seed hardness Fruit juice color Fruit taste Fruit quality

0 – – – – – – – – – – – – – – – – Absent Absent – – Absent – – – – Absent Absent – – Absent Absent Absent Absent – – – – – – – – – – – – –

(150) (150)

(3)

(66) (145)

(4) (70) (141) (143)

1 Weeping (6) Low (22) Gray (9) Low (33) Low (9) Low (15) Low (6) Single (26) Low (33) Low (34) Low (60) Low (2) Elliptic lanceolate (59) Acute (101) Light green (27) Light green (99) Low (25) Low (29) Entire (150) Smooth (129) Present (201) Mid-September (9) Low (27) Round (61) Truncate (73) Present (138) Present/Short (37) Low (46) Close (16) Present (200) Low (67) Low (16) Low (22) Cream (61) Low (122) Cream (126) Low (119) Glassy (57) Low (121) Low (46) Low (111) Oval (37) Soft (14) Colorless (34) Bitter-sour (6) Low (69)

3 Spreading (111) Intermediate (73) Gray brown (6) Intermediate (80) Intermediate (88) Intermediate (80) Intermediate (67) Multi-trunk/Low (65) Intermediate (112) Intermediate (84) Intermediate (88) Intermediate (73) Lanceolate (112) Obtuse (18) Green (125) Green (99) Intermediate (9) Intermediate (17) Lobate (47) Curly (75) – Late September (102) Intermediate (112) Ovate (2) Covex (101) – Present/Medium (15) Intermediate (49) Semi-open (134) – Intermediate (36) Intermediate (28) Intermediate (24) Yellow (140) Intermediate (51) Yellow (77) Intermediate (57) Milky (24) Intermediate (70) Intermediate (87) Intermediate (66) Stretched (45) Semi-soft (48) Pink (66) Very sour (41) Intermediate (55)

5 Open (33) High (109) Light brown (60) High (91) High (107) High (109) High (131) Multi/Intermediate (79) High (59) High (86) High (56) High (129) Broad elliptic (33) Rounded (85) Dark green (52) Dark green (6) High (20) High (8) Undulate (7) – – Early October (93) High (65) Oval (52) Angular (30) – Present/Long (7) High (109) Open (54) – High (31) High (19) High (15) Light brown (3) High (31) Brown (1) High (28) Cream (94) High (13) High (71) High (27) Triangular (103) Hard (142) Red (82) Sour (134) High (42)

7 Semi-erect (39) – Brown (68) – – – – Multi/High (34) – – – – – – – – – – – – – – – Elliptical (20) – – – – – – – – – – – – – Yellow (29) – – – Prismatic (19) – Crimson (22) Sour-sweet (12) Very high (38)

9 Erect (15) – Dark brown (61) – – – – – – – – – – – – – – – – – – – – Oblate (69) – – – – – – – – – – – – – – – – – – – – Sweet (11) –

Thakur et al. (2010) observed an average fruit weight as 64.00 g. Fruit calyx length ranged from 3.00–14.71 mm and fruit calyx diameter varied from 4.85 to 23.05 mm. The number of sepals was 3–8, while sepal length varied from 6.36 to 13.91 mm and sepal base width ranged from 4.08–9.11 mm. Stamen density in fruit calyx was predominantly high (109). Fruit calyx form was predominantly semi-open was (134 accessions), although close (16) and open (54) forms were observed. Fruit neck was observed in most of the accessions (200). Fruit peel weight ranged from 5.21–53.28 g with an average of 20.73. Fruit peel thickness was low in most of the accessions (122). The peel thickness is among the main selection criteria; fruits with thin peel are intended for processing, while those with a thick peel that provides resistance to transport and storage are selected for fresh consumption (Khadivi et al., 2018). Furthermore, fruits with thick peels might have the ability to resist peel cracks. A thick peel enclosing the edible arils protects the fruits from pests and pathogens that enter the fruits via these cracks (Jalikop et al., 2006, 2005). Thus, the accessions with thick peel might be used to breed for varieties that resist cracking. Fruit peel color showed high variability and included yellow (27 accessions), light green (59), green (23), green-red (44), red (3), redyellow (17), yellow-red (10), brown (15), brown-green (3), brown-

138 out of 204 accessions investigated were symmetric (Table 3). Fruit length ranged between 27.20 and 63.27 mm, while fruit diameter varied between 30.98–71.40 mm (Table 2). In several studies on wild pomegranates from India, the range of fruit length has been recorded as follows: 53.10–75.30 mm (Pant, 1995) and 46.90–62.80 mm (Thakur et al., 2011). In addition, several studies on wild pomegranates from India reported the range of fruit diameter as follows: 42−66 mm (Parmar and Kaushal, 1982), 43.50–65.00 mm (Pant, 1995), and 44.60–54.20 mm (Thakur et al., 2011). In another study, the average fruit length and fruit diameter have been reported as 58.90 mm and 46.40 mm, respectively (Thakur et al., 2010). Moreover, Sharma and Thakur (2016) recorded the average fruit length and fruit diameter of wild pomegranate fruits as 55.99 mm and 43.26 mm, respectively. Fruit size is an important trait attracting consumers for the fresh market. Fruit weight is the most pertinent criteria used during the sorting process. Fruit weight ranged from 19.20–185.00 g with an average of 59.89. In several studies on wild pomegranates from India, the range of fruit weight has been reported as follows: 80.50–85.17 g (Parmar and Kaushal, 1982; Kher, 1999), 59.77–101.00 g (Pant, 1995), and 55.10–83.50 g (Thakur et al., 2011). In addition, Singh and Kingsley (2008) reported an average fruit weight as 63.00 g. Similarly, 4

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Fig. 1. The pictures of leaves, fruits, and arils of wild pomegranate accessions studied.

traits to pomegranate consumers and also is an important quality attribute in pomegranate marketing (Janick and Moore, 1975; Drogoudi et al., 2005; Ashton et al., 2006). Aril length ranged from 5.66 to 10.22 mm and aril width varied from 3.08 to 8.73 mm. In addition, fresh weight of 100 arils ranged between 5.75 and 27.55 g with an average of 11.77. In a study on wild pomegranates from India, Kher (1999) reported an average weight of 100 arils as 12.65 g. In another study, an average weight of 100 arils

yellow (2), and brown-red (1). In addition, internal peel color was cream (61), yellow (140), and light brown (3). Fruit peel color is a very important indicator of the quality of fresh fruits. It also helps in estimating the stage of maturity of fruit. The main pigments of fruits which affect the fruit peel color are chlorophylls, carotenoids, and anthocyanins. Different fruit peel colors were reported in wild pomegranates from India (Parmar and Kaushal, 1982; Kher, 1999; Thakur et al., 2011; Sharma and Thakur, 2016). Red fruit peel is one of the most attractive 5

Character Tree growth habit Tree growth vigor Shoot color Tree height Branching Branch density Branch flexibility Trunk type Trunk diameter Canopy density Tendency to suckering Leaf density Leaf shape Leaf apex shape Leaf length Leaf width Leaf upper surface color Leaf lower surface color Leaf serration presence Leaf serration depth Leaf serration shape Leaf margin form Petiole length Petiole diameter Petiole color Thorn presence on shoot Thorn length on annual shoot Thorn thickness on annual shoot Ripening time Yield Fruit length Fruit diameter Fruit shape Fruit base shape Fruit symmetry Pacifier presence on fruit Stamen density in fruit calyx Sepal number Sepal length

0.02 0.11 0.00 −0.03 −0.01 0.00 0.17 0.06 −0.09

0.07

−0.13

−0.05 0.44 0.80** 0.81** −0.12 0.21 0.42 0.69** 0.29

0.69** 0.10 0.05 −0.04 0.00 0.03

−0.09

0.06

−0.04

−0.01

−0.02 0.10 0.03 0.18 0.21

−0.19

−0.02

0.20 0.24 0.10 0.05 0.10 −0.02 0.17 −0.04

0.09

0.07 0.06

0.09 0.10 0.08 0.07 −0.02 −0.09 0.07 0.05 −0.14

0.05 0.19 0.06 −0.04 0.18 0.01

−0.02

−0.05

−0.04

6

−0.05

−0.10 0.01 0.15 −0.05 0.10

−0.05

0.13

−0.16 0.15 0.83** 0.90** 0.25 0.04 0.07 0.04

0.05

−0.06 0.49

−0.02 −0.09

0.12

−0.16

0.13 0.01

0.04 0.02 −0.09 −0.03 0.05 −0.08 −0.14 −0.12

0.03

0.07

0.07 −0.15 0.09 −0.03 0.09

0.90**

0.88**

0.85**

0.05 −0.02

−0.01

0.08 −0.22 0.02 0.03 −0.01 0.04 0.04 0.02

0.07

0.00

0.06 0.69** 0.68** 0.01 −0.01

0.03

0.05

0.08

0.03

0.04 −0.01 −0.24 0.83** 0.75** 0.04

−0.01 0.03 −0.12 0.14 0.14 −0.01

0.02

0.04 0.13 0.04 −0.10 0.04 0.17 −0.02 0.08 0.09

0.05

5 0.02

0.05 0.16 −0.13 0.02 −0.22 −0.07 0.17 0.05 −0.03

0.07

4 0.13

0.06 −0.06 0.17 0.17 0.01 −0.03 0.04 0.01

−0.10 0.00 −0.07 0.06 −0.21

0.03

0.05

0.17

0.00

0.09 0.07 −0.10 −0.01 0.04 −0.02

0.13

0.58**

0.10

3 −0.09

2 −0.08

1 0.09

Component

−0.03 0.19

−0.08

−0.13 0.04 0.01 0.02 −0.04 −0.04 −0.16 0.04

0.18

−0.03

−0.14 0.07 0.05 0.11 0.05

0.03

0.06

−0.05

−0.02

−0.04 0.04 0.01 0.04 −0.09 −0.02

0.07 0.06

−0.05

0.00 0.17 0.20 0.14 0.11 0.00 0.07 0.19

−0.17

−0.07

0.23 0.02 −0.01 0.08 0.15

−0.02

−0.01

0.05

0.07

−0.13 0.14 0.09 −0.04 0.07 0.14

−0.05 0.25 −0.12 −0.01 −0.10 0.00 0.10 −0.05 −0.09

0.16

−0.18 0.04 −0.24 0.05 −0.03 0.10 −0.10 −0.11 −0.09 −0.11

7 0.02

6 −0.13

−0.01 0.18

−0.02

0.11 0.03 −0.04 −0.05 −0.01 0.17 0.04 0.11

−0.04

0.05

−0.14 −0.02 −0.04 0.01 0.09

−0.03

0.02

0.09

0.03

−0.01 −0.13 0.00 0.08 0.01 −0.08

0.03 0.22 −0.16 −0.12 −0.31 −0.05 0.14 0.16 0.00

0.10

8 −0.03

−0.01 −0.04

−0.11

0.24 0.10 0.08 0.06 −0.12 −0.01 0.04 0.03

0.72**

0.69**

0.09 0.14 −0.07 0.02 0.29

−0.01

0.08

0.02

0.05

−0.17 0.09 0.39 −0.03 0.04 0.03

0.00 0.40 −0.10 −0.04 −0.12 0.00 0.30 −0.01 −0.05

0.35

9 0.15

−0.04 0.26

0.01

−0.07 0.28 −0.01 −0.08 0.15 0.10 0.08 0.25

0.10

−0.16

−0.04 0.09 0.00 −0.23 0.03

0.02

−0.07

−0.05

0.04

0.12 −0.07 −0.11 0.16 −0.01 −0.05

0.01 0.02 0.07 0.11 0.38 0.74** 0.05 0.15 0.75**

−0.01

10 0.02

0.10 0.20

0.03

0.56** −0.47 −0.04 0.01 0.03 0.11 −0.23 0.13

0.03

0.02

0.18 0.14

0.05

−0.19 −0.03 0.10 −0.13 −0.57** 0.78** 0.19 0.31

0.04

0.05

0.22 0.08 −0.08 0.01 0.25

0.02

−0.02 −0.15 0.01 0.08 −0.05 0.13

−0.03

−0.11

0.07

0.00 −0.01 0.19 0.06 −0.02 0.08

0.18 −0.22 −0.01 0.00 0.24 0.00 −0.22 0.04 0.00

−0.23

12 −0.01

0.09

−0.02

−0.01

0.11 0.12 −0.12 −0.01 0.06 0.07

0.68** −0.03 0.02 0.03 −0.28 0.06 0.00 −0.21 −0.11

0.00

11 0.11

0.10 0.05

0.03

−0.09 −0.06 0.07 0.06 0.22 0.02 −0.10 0.04

−0.01

−0.04

0.00 −0.12 0.11 0.02 0.00

−0.04

0.01

−0.02

0.02

0.05 0.65** 0.50 −0.23 0.41 0.00

−0.03 0.23 −0.10 0.08 0.20 −0.06 0.09 −0.02 −0.02

0.18

13 0.03

−0.09 −0.17

−0.10

0.17 0.25 0.07 0.01 −0.08 −0.03 0.05 0.08

−0.01

−0.01

−0.64** 0.02 −0.11 0.07 0.01

0.00

−0.01

0.06

−0.01

−0.14 −0.01 −0.07 0.04 0.06 −0.01

0.19 0.14 −0.05 −0.06 0.15 0.07 0.17 0.11 0.10

0.07

14 0.10

Table 4 Eigenvalues of the principal component axes from the PCA of morphological characters in the studied wild pomegranate accessions.

−0.10 −0.02

−0.13

0.24 −0.08 0.01 0.02 −0.04 0.15 0.01 −0.06

0.07

0.08

−0.05 0.06 0.06 −0.14 −0.11

0.00

0.00

0.01

0.83**

−0.07 0.05 0.00 −0.02 0.03 0.81**

0.01 −0.11 0.05 0.00 0.13 −0.01 −0.02 0.05 −0.03

−0.19

15 −0.03

0.10 −0.14

0.09

−0.02 0.04 0.06 0.00 0.11 0.01 −0.23 0.19

0.02

−0.11

−0.09 0.00 −0.07 0.04 0.00

−0.02

0.00

0.01

0.00

−0.18 0.26 −0.02 0.02 0.10 −0.01

0.03 0.26 −0.08 −0.04 −0.23 0.06 0.32 0.14 −0.06

0.22

16 −0.06

0.06 0.31

−0.01

−0.09 −0.14 0.02 −0.01 0.11 0.06 −0.12 0.16

−0.20

−0.17

−0.01 0.15 −0.10 −0.08 −0.60**

0.01

−0.02

−0.07

0.09

−0.13 0.08 0.04 −0.04 −0.01 −0.06

−0.13 0.11 0.03 −0.06 0.00 −0.07 0.14 0.01 0.10

0.04

17 0.02

0.79** −0.08

0.00

0.01 0.18 −0.21 0.07 0.09 0.10 0.18 −0.58**

0.03

−0.05

−0.01 0.10 0.02 −0.05 0.05

0.02

0.01

0.01

−0.10

0.07 0.07 0.08 −0.02 −0.05 0.03

0.08 −0.03 −0.01 0.06 0.03 −0.11 0.29 0.01 −0.07

−0.09

18 0.06

−0.03 0.08

0.14

0.04 −0.03 0.06 −0.04 0.09 −0.01 0.18 0.04

−0.03

−0.19

−0.12 −0.05 0.16 0.01 −0.18

−0.05

−0.02

−0.01

0.08

0.10 0.13 0.30 −0.07 0.07 0.01

−0.12 −0.05 −0.07 0.07 −0.13 0.00 −0.05 −0.01 −0.01

0.03

19 0.06

0.04 0.13

0.12

0.09 −0.18 0.04 −0.01 0.08 0.04 0.25 0.05

0.10

0.00

−0.30 −0.08 0.14 0.06 0.06

0.03

0.03

0.03

−0.08

−0.02 0.12 0.01 −0.04 0.04 0.06

0.01 0.05 0.06 0.02 0.01 −0.03 0.07 −0.21 0.02

0.02

20 0.78**

0.01 −0.07

0.72**

0.11 0.06 −0.02 −0.04 −0.11 0.01 −0.07 0.10

−0.11

−0.05

−0.01 0.16 −0.32 0.01 −0.08

−0.02

0.06

0.04

−0.13

0.00 −0.06 0.13 0.00 0.06 0.03

0.05 0.07 0.06 0.02 0.02 0.07 0.04 −0.02 −0.13

0.11

22 0.10

0.03 −0.01

0.08

0.26 −0.02 0.02 −0.02 0.23 0.09 −0.15 0.19

0.01

0.02

0.00 −0.17 0.11 0.76** 0.09

0.01

0.03

−0.05

−0.09

0.06 −0.04 0.12 0.08 0.01 −0.01

−0.11 −0.09 0.17 0.00 0.11 −0.20 −0.06 0.03 −0.06

−0.09

23 0.12

(continued on next page)

0.07 −0.11

−0.02

0.13 −0.11 0.03 0.08 0.05 0.11 0.41 −0.01

−0.10

0.15

−0.06 −0.08 −0.10 −0.04 −0.12

0.01

0.06

0.02

−0.08

−0.18 −0.11 0.07 0.09 −0.08 0.09

−0.01 −0.07 0.06 0.15 0.01 −0.02 −0.12 −0.10 0.04

0.01

21 −0.03

A. Khadivi, et al.

Scientia Horticulturae 264 (2020) 109165

7

0.02

0.02

0.04 −0.34

−0.05

0.11

0.89** 0.03

−0.22

−0.13 0.84** 0.23

0.24 −0.02 0.02 −0.25 −0.01 0.87** 0.17 0.72** 0.62**

−0.27

−0.07 0.02 0.01

−0.07 0.06 −0.12 0.00 −0.08 0.04 0.14 0.01 0.16

4.48 6.31 16.06

0.04

0.29 0.17 0.53**

0.23 0.32 0.18 0.04 0.11 0.20 0.37 0.26 0.05

6.92 9.75 9.75

3.69 5.19 21.25

0.04 0.02 −0.13

−0.06 −0.12 0.02

−0.02 0.12 −0.15

0.09

−0.05

0.03

3.63 5.11 26.37

0.04 0.03 −0.04 −0.04 0.04 0.12 0.13 0.02 0.05

0.08 0.11 0.01

−0.02

−0.02 0.04 −0.05

0.05

2.73 3.85 30.21

0.01 −0.10 0.06 0.01 0.03 −0.02 0.00 −0.06 0.06

−0.02 −0.01 −0.05

0.00

−0.07 0.09 −0.07

0.08

0.09 −0.15

−0.04 0.02

0.08 0.19

−0.02

0.09

−0.22

0.02

−0.07

−0.13

−0.06

0.18

−0.15

−0.05

0.15

0.01

0.10

−0.23

0.07

0.00

0.07 0.05 0.03 0.04

−0.03 0.07 −0.06 0.05

0.18 −0.07 0.00 −0.06

0.08 −0.08 −0.05 −0.08

0.91** −0.02 −0.02 0.32

−0.09 0.13 0.22 −0.02 0.08

−0.03

−0.03

0.55**

−0.01 0.07 −0.01 0.07

0.02

−0.18 0.17 −0.27 0.09

0.06 0.15 0.06 −0.02

0.42 −0.24 −0.13 −0.08

** Eigenvalues are significant ≥ 0.53.

Sepal base width Fruit calyx form Fruit calyx length Fruit neck presence Fruit calyx diameter Fruit weight Fruit stalk length Fruit peel color Fruit peel necrotic presence Fruit peel sunburn presence Fruit peel cracking presence Internal peel color Fruit peel thickness Fruit peel weight Internal layer color Internal layer thickness Septum color Septum thickness Septum transparency Aril adherence to internal peel Aril shape Aril color 100 aril fresh weight Aril length Aril width Seed length Seed width Seed hardness Fruit juice color Fruit taste Fruit quality Total soluble solids Total % of Variance Cumulative %

Component

Table 4 (continued)

2.58 3.64 33.85

0.06 0.19 0.11 0.01 0.03 0.09 −0.13 −0.06 0.09

−0.18 0.09 0.16

0.03

0.80** 0.71** −0.84**

0.07

0.12 −0.03

0.19

0.14

−0.02

0.03

2.38 3.35 37.20

0.81** 0.51 0.74** 0.15 0.12 0.12 −0.09 0.16 −0.17

0.03 0.15 0.52

−0.06

0.04 0.21 −0.07

−0.02

0.08 −0.07

−0.12

0.02

−0.05

−0.18

0.17 0.06 −0.10 −0.05

−0.09

−0.02 0.02 −0.11 0.21 −0.03

−0.03 −0.02 0.00 0.08

0.05 −0.22 −0.14 −0.10

2.26 3.18 40.38

−0.07 −0.09 0.06 0.04 −0.22 −0.04 −0.43 −0.24 0.06

−0.02 0.01 −0.22

0.35

0.02 0.27 0.00

0.65**

0.16 0.30

0.69**

0.06

−0.12

−0.01

−0.05 −0.01 −0.11 0.29

0.21

0.23 −0.03 −0.05 −0.09

2.12 2.98 43.36

−0.06 −0.17 0.00 −0.18 0.24 −0.03 −0.07 −0.06 0.06

0.01 −0.01 −0.09

0.09

−0.04 0.11 −0.04

0.14

0.04 −0.03

−0.12

−0.13

0.02

−0.02

0.03 0.05 −0.06 0.06

−0.02

−0.15 −0.11 −0.03 0.14

1.97 2.78 46.14

−0.03 0.03 −0.07 0.02 0.13 −0.06 −0.12 0.03 0.04

0.25 −0.09 0.03

0.09

−0.06 −0.03 0.07

−0.10

−0.08 0.24

−0.04

0.01

−0.01

−0.09

−0.09 0.16 0.01 0.03

0.00

0.19 −0.17 −0.12 −0.03

1.94 2.73 48.87

−0.04 0.05 −0.09 −0.10 0.16 0.08 −0.05 −0.02 −0.18

−0.11 0.07 −0.01

0.31

0.12 −0.04 0.11

0.08

−0.03 0.08

0.00

−0.07

0.12

0.04

−0.05 0.06 −0.04 −0.32

0.28

0.41 0.01 0.01 0.13

1.72 2.43 51.29

−0.01 −0.04 −0.01 0.11 −0.01 0.02 0.08 −0.06 −0.05

−0.02 0.01 −0.06

−0.03

−0.05 0.04 −0.01

0.16

0.00 −0.19

0.15

−0.06

0.07

0.08

−0.11 0.12 −0.05 0.04

0.11

0.02 0.01 0.20 0.15

1.65 2.33 53.62

−0.04 −0.11 0.15 −0.04 −0.02 −0.02 0.10 0.01 0.09

−0.11 −0.09 0.02

0.16

0.09 −0.01 −0.01

0.12

0.03 −0.04

−0.09

0.01

−0.03

−0.10

0.02 0.07 0.06 0.06

0.29

0.01 0.13 0.12 −0.63**

1.61 2.26 55.88

−0.09 0.07 0.02 −0.11 0.32 0.04 0.04 −0.09 −0.06

−0.09 0.03 −0.13

−0.11

0.01 0.01 0.04

−0.06

0.07 −0.28

0.18

−0.04

0.16

0.33

0.03 0.72** −0.08 0.06

−0.11

−0.12 −0.11 0.13 −0.09

1.43 2.02 57.90

0.08 0.02 0.18 −0.03 −0.04 −0.02 −0.09 0.07 −0.07

−0.04 −0.01 0.00

−0.17

0.09 −0.16 0.04

−0.15

−0.02 0.12

0.03

0.11

0.04

−0.12

0.01 −0.03 0.02 0.10

−0.05

0.00 −0.10 0.01 0.02

1.43 2.01 59.91

−0.05 −0.04 −0.03 −0.06 −0.08 0.06 0.05 −0.16 −0.14

0.08 0.10 −0.15

−0.06

0.10 0.00 −0.10

−0.20

0.01 −0.09

0.02

0.01

0.15

0.44

0.00 −0.09 0.76** 0.26

0.02

−0.22 0.15 −0.14 0.14

1.41 1.99 61.90

−0.03 0.26 −0.01 0.77** 0.31 −0.07 0.16 0.04 0.03

−0.16 −0.09 0.12

−0.17

−0.02 0.07 0.02

0.09

−0.03 0.08

−0.02

0.00

0.03

0.04

−0.06 −0.12 −0.10 0.02

−0.03

0.00 −0.10 −0.05 0.13

1.40 1.97 63.86

−0.04 0.00 0.04 0.02 0.15 0.02 −0.14 0.02 0.07

0.11 0.08 −0.02

−0.02

0.10 −0.09 0.11

−0.25

0.02 0.19

0.07

−0.02

0.07

0.04

0.02 −0.14 0.04 −0.16

0.16

−0.38 −0.11 0.06 0.00

1.33 1.87 65.73

−0.01 −0.04 0.03 −0.12 0.20 −0.01 0.06 −0.17 0.02

0.00 0.04 0.07

0.27

0.03 0.10 −0.01

−0.05

−0.05 0.01

0.10

0.74**

−0.10

0.27

−0.03 −0.14 −0.02 −0.19

−0.37

−0.16 0.37 0.31 0.16

1.17 1.65 67.38

−0.06 −0.01 0.05 −0.02 −0.50 −0.05 −0.05 0.02 0.00

0.07 −0.08 0.03

−0.07

−0.06 0.03 0.07

0.06

0.04 −0.30

−0.03

0.06

−0.10

−0.12

0.01 −0.06 −0.02 −0.01

−0.01

−0.04 −0.27 −0.18 0.09

1.16 1.63 69.01

0.05 −0.20 0.03 −0.05 0.07 −0.05 −0.14 −0.13 0.09

−0.33 −0.03 −0.15

0.32

−0.05 0.05 0.00

−0.05

0.06 −0.03

−0.10

−0.05

0.70**

0.13

0.03 0.09 0.06 −0.07

0.15

−0.03 0.14 −0.29 −0.04

1.10 1.54 70.55

0.00 0.03 −0.04 −0.01 0.05 0.02 0.09 −0.02 −0.25

−0.44 −0.01 −0.05

−0.10

−0.05 0.02 0.01

−0.11

0.04 0.09

0.10

0.12

0.05

−0.27

0.00 −0.05 0.09 0.20

−0.04

0.02 −0.17 0.30 −0.07

1.07 1.51 72.06

0.01 −0.03 0.09 −0.04 0.26 0.08 −0.05 0.06 −0.22

0.16 0.06 0.04

−0.05

0.06 0.03 −0.01

−0.04

0.02 0.29

0.05

0.00

0.00

0.04

−0.06 0.07 0.04 0.30

0.04

−0.05 0.11 −0.09 −0.02

A. Khadivi, et al.

Scientia Horticulturae 264 (2020) 109165

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Fig. 2. Scatter plot for the studied wild pomegranate accessions based on PC1/PC2. The symbols represent the accessions of each area in the plot including Ardeh (A), Daroogar (D), Ghajarkhil (G), Hasanabad (H), Kalamsar (Ka), Korfmehale (K), Limesara (L), Mashlabad (M), Polsefid (P), Poonel (P), Sarvlat (Sa), Shirgah (Sh), Shirood (Shi), Sooteh (S), and Vachklaye (V).

sour-sweet in 12, sweet in 11, and very sour in 41 accessions. The accessions with sweet and sour-sweet fruit taste are acceptable for pomegranate fresh consumption. High TSS content is highly desirable industrial traits in pomegranate fruit juice making as it associates with sweetness and flavor especially if it combined low juice acidity and tannin concentration (Shwartz et al., 2009). The range of TSS was 9.30-27.00% with an average of 17.09. In several studies on wild pomegranates from India, the range of TSS has been reported as follows: 12.00-17.00% (Sharma and Sharma, 1990), 10.60-14.00% (Pant, 1995), and 15.40-16.50% (Thakur et al., 2011). In addition, Parmar and Kaushal (1982) and Singh and Kingsley (2008) reported an average of 15.50 and 16.40% for TSS in wild pomegranates, respectively. Moreover, an average of 20.08% TSS has been recorded by Thakur et al. (2010) and 18.00% by Sharma and Thakur (2016) in wild pomegranates. The accessions with soft seeds, big arils, red fruit peel, pink or red arils, high juice content, thin peel, and no sourness are suitable for the extraction of arils (Zavala and Cozza, 2012). Thus, based on the above quality-related attributes, fruit quality was high in 42 and very high in 38 accessions. The pictures of leaves, fruits, and arils of the wild pomegranate accessions studied are shown in Fig. 1.

was reported as 15.34 g (Thakur et al., 2010). Total aril weight per fruit varied from 8.93–133.07 g and thus edible portion ranged from 40.85 to 78.82%. Thakur et al. (2011) reported a range of 33.00–48.70 g for weight of arils per fruit. Parmar and Kaushal (1982) reported an average of edible portion as 64.00% in wild pomegranates, while edible portion in cultivated pomegranate cultivars has been recorded in the range of 61.00-68.00% (Sood et al., 1982; Khodade et al., 1990; Singh and Sethi, 2003; Jashkaran et al., 2005). Aril shape showed four types among the accessions including oval (37), stretched (45), triangular (103), and prismatic (19) (Fig. 1). A high range of diversity was observed among the accessions based on aril color including glassy (19 accessions), pale milk (20), pink (40), whitered (27), red (65), and dark red (33). The genotypes showing pink or red arils are targets for pomegranate breeders (Zavala and Cozza, 2012; Fawole and Opara, 2013). In a study on wild pomegranates from India, Parmar and Kaushal (1982) observed various colors for arils that ranged from pink to blood red. Furthermore, Bhat (2007) and Sharma and Thakur (2016) recorded pink color for arils, whereas Thakur et al. (2011) reported red-purple color for arils of wild pomegranate fruits. Seed length ranged from 4.82 to 8.00 mm and seed width varied from 1.92 to 7.03 mm. Seed hardness was predominantly hard (142 accessions), although some accessions had soft and semi-soft seeds (14 and 48 accessions, respectively). The soft and semi-soft seeded accessions are suitable in this important trait to be used for cultivation and in breeding programs for decreasing seed hardness in the commercial pomegranate cultivars. In pomegranates with soft seeds, the testa width is thinner and the ratio of testa weight to total seed yield is lower (Prohit, 1985). High juice content is a desirable attribute in pomegranate production and other fruits, and it is the most important character from an industrial point of view (Maestre et al., 2000). The soft-seeded pomegranates have been recommended as parents for developing high juice cultivars due to their significantly higher content of the juice (Jalikop and Kumar, 1998). Fruit juice color was red in the majority of accessions (82), although pink (66 accessions) and crimson (22) colors were observed. Fruit taste was sour in most of the accessions (134). In addition, fruit taste was

3.2. Correlations among the measured characters The correlation coefficient is used to measure the strength of a linear association between two variables, where the value r = 1 means a perfect positive correlation and the value r = -1 means a perfect negative correlation. Correlation coefficients showed meaningful correlations between some characters recorded (data not shown). Tree growth vigor showed positive and significant correlations with tree height (r = 0.77), branching (r = 0.31), branch density (r = 0.44), trunk diameter (r = 0.51), canopy density (r = 0.38), leaf density (r = 0.28), and leaf shape (r = 0.22) and was in line with the previous findings (Khadivi-Khub et al., 2015; Khadivi et al., 2018). Leaf length showed positive and significant correlations with leaf width (r = 0.54), petiole length (r = 0.49), and petiole diameter (r = 0.46), in agreement 8

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

with the previous results (Khadivi-Khub et al., 2015; Khadivi et al., 2018). Fruit yield was significantly and positively correlated with tree growth vigor (r = 0.21), tree height (r = 0.19), branch density (r = 0.23), trunk diameter (r = 0.30), and canopy density (r = 0.28). Fruit weight showed positive and significant correlations with tree growth vigor (r = 0.20), tree height (r = 0.21), leaf width (r = 0.24), fruit length (r = 0.84), fruit diameter (r = 0.92), fruit shape (r = 0.26), sepal length(r = 0.35), sepal base width (r = 0.25), and fruit calyx diameter (r = 0.45) and corresponded with other findings (Karimi and Mirdehghan, 2013; Khadivi-Khub et al., 2015; Khadivi et al., 2018). In addition, 100 arils fresh weight was significantly and positively correlated with fruit length (r = 0.52), fruit diameter (r = 0.54), fruit shape (r = 0.21), sepal length (r = 0.43), fruit weight (r = 0.60), aril color (r = 0.31), aril length (r = 0.66), aril width (r = 0.60), and seed length and was in line with the previous findings (Khadivi-Khub et al., 2015; Khadivi et al., 2018). The strong correlation between fruit weight and both fruit dimensions (length and diameter) and fruit components indicated that selection for larger fruits will lead to higher aril weight and vice versa. Fruit taste was significantly and positively correlated with fruit length (r = 0.28), fruit diameter (r = 0.29), fruit weight (r = 0.34), aril color (r = 0.16), 100 arils fresh weight (r = 0.30), and fruit juice color (r = 0.21). Fruit quality showed positive and significant correlations with fruit length (r = 0.33), fruit diameter (r = 0.38), fruit weight (r = 0.38), aril color (r = 0.60), 100 arils fresh weight (r = 0.53), aril length (r = 0.36), aril width (r = 0.22), fruit juice color (r = 0.65), fruit taste (r = 0.25), and TSS (r = 0.37). The TSS showed positive and significant correlations with aril color (r = 0.42) and fruit juice color (r = 0.43). 3.3. Principal component analysis (PCA) The PCA is a technique used to emphasize variation and bring out strong patterns in a dataset. It is often used to make data easy to explore and visualize. The PCA simplifies the complexity of high-dimensional data while retaining trends and patterns. It does this by transforming the data into fewer dimensions, which act as summaries of features. High-dimensional data are very common in biology and arise when multiple features. The PCA reduces data by geometrically projecting them onto lower dimensions called principal components (PCs), with the goal of finding the best summary of the data using a limited number of PCs. The first PC is chosen to minimize the total distance between the data and their projection onto the PC. For PCA, components with eigenvalues of more than 1.00 were retained to uphold reliability of the final output. Thus, 23 PCs were observed which contributed 72.06% of total variance (Table 4). Values above 0.53 were considered to be significant for the studied traits. The PC1 explained 9.75% of total variance and was represented by fruit length (0.83), fruit diameter (0.90), fruit calyx diameter (0.55), fruit weight (0.91), fruit peel weight (0.89), and 100 arils fresh weight (0.53) with positive correlations. The PC2 explained 6.31% of total variance and was constituted by tree growth vigor (0.53), branching (0.80), branch density (0.81), canopy density (0.69), and leaf density (0.69) with positive correlations. The PC3 explained 5.19% of total variance and was represented by aril color (0.84), fruit juice color (0.87), fruit quality (0.72), and TSS (0.62) with positive correlations. These characters were the most effective traits for separating and identifying the studied genotypes. In addition, these traits are economically important and can also be applied as a useful tool for selecting genotypes or new cultivars with superior traits. It has been reported that fruit-related characters were important factors in differentiating and analyzing breeding materials dealing with the morphological characterization of cultivated pomegranates (KhadiviKhub et al., 2015). In addition, a dispersion bi-plot prepared according to PC1 and PC2

Fig. 3. Ward cluster analysis of the studied wild pomegranate accessions based on morphological traits using Euclidean distances.

9

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

Fig. 4. Bi-plot for the studied areas of wild pomegranates based on morphological characters.

among all the characters analyzed, those related to fruit were what had the highest power of discrimination, and are therefore the most useful for genetic characterization studies of pomegranate germplasm. In general, knowledge on the extent of the genetic diversity found in the collection is essential for germplasm management. In this study, these data may help in the developing of strategies for pomegranate germplasm management and may allow for more efficient use of this germplasm in future pomegranate breeding programs. Most of the accessions studied here could be selected as parents in breeding programs for the improvement of juice quality, seed softness, and aril and fruit appearance as major recent breeding objectives for pomegranate breeding programs (Crites et al., 2014).

reflected the relationship between the accessions in terms of phenotypic similarity. The accessions were distributed into four sides of the plot and showed significant variations (Fig. 2). By starting from negative toward positive values of PC1, the accessions showed gradual increases in fruit length, fruit diameter, fruit calyx diameter, fruit weight, fruit peel weight, and 100 arils fresh weight. Furthermore, by starting from negative to positive values of PC2, the accessions indicated gradual increases in tree growth vigor, branching, branch density, canopy density, and leaf density. Bi-plot is a type of exploratory graph used in statistics, a generalization of the simple two-variable scatterplot. A biplot allows information on both samples and variables of a data matrix to be displayed graphically. Samples are displayed as points while variables are displayed either as vectors, linear axes or nonlinear trajectories. In the case of categorical variables, category level points may be used to represent the levels of a categorical variable.

4. Conclusion Evaluation of genetic diversity in the wild pomegranates might be useful to identify new germplasm sources that would result in both qualitatively as well as quantitatively enhanced yields when crossed with existing varieties. The genetic resources of wild pomegranate offer great scope for utilization in the pomegranate improvement programs. The gene pool needs to be assembled from areas of its occurrence and further evaluated for biotic and abiotic traits. The wild forms of pomegranate have been found as rich as pomegranate fruits because of the presence of various chemical components that have potential beneficial physiological activities. The presence of valuable bioactive components makes this fruit highly nutritious. There have been increasing the interest of utilization of wild and underutilized fruits as food because of their health-promoting benefits. Using low-cost value-added products of this fruit can ultimately offer society a way of utilizing the broad health benefits of this fruit as it has been reported to be a very rich source of nutritional components by various research workers in their previous studies. The current findings provided the data within the germplasm that could be used for the selection of appropriate accessions as breeding material. For instance, one of the main objectives of pomegranate breeding programs is the production or improvement of cultivars with soft-seed fruits. In the current study, 14 wild accessions including Shirgah-7, Shirgah-12, Hasanabad3, Hasanabad-9, Hasanabad-12, Vachklaye-2, Shirood-8, Shirood-10, Sarvlat-7, Sarvlat-20, Sarvlat-21, Daroogar-9, Poonel-5, and Ardeh-4 had fruits with soft-seed that are applicable as a parent in breeding programs for improving this trait. The obtained data here could be used to design sampling strategies, create core collections, and establish pomegranate breeding studies.

3.4. Cluster analysis Cluster analysis is a technique to group similar observations into a number of clusters based on the observed values of several variables for each individual. Cluster analysis is similar in concept to discriminant analysis. The group membership of a sample of observations is known upfront in the latter while it is not known for any observation in the former. Cluster analysis based on Ward method showed two different major clusters among all the accessions studied (Fig. 3). The first cluster (I) contained 57 accessions. The second cluster (II) consisted of the majority of the accessions studied and was divided into two sub-clusters with high diversity. Sub-cluster II-A consists of 57 accessions, while sub-cluster II-B contained 96 accessions. Furthermore, the association between the natural habitats studied was visualized in greater detail according to the morphological characters. Based on the bi-plot created using PCA of population analysis, the 15 natural habitats studied formed four groups (Fig. 4). The areas including Korfmehale and Poonel were placed into the first group. Mashlabad, Polsefid, and Shirood areas were placed in the second group. In addition, four areas including Limesara, Daroogar, Ardeh, and Kalamsar formed the third group, while the fourth group consisted of rest the areas including Ghajarkhil, Hasanabad, Sarvlat, Shirgah, Sooteh, and Vachklaye. Broad phenotypic diversity existed among the wild pomegranate accessions. Beside significant variations among the accessions of different collection areas, great variability was found within the same varietal group. The considerable phenotypic variation observed here indicated that such germplasm is valuable genetic resources for pomegranate improvement. Therefore, it has become imperative to establish strategies for preserving wild pomegranate germplasm and conserving these genetic resources. As a result of the present study,

Author contributions section Farhad Mirheidari, Younes Moradi, and Simin Paryan performed the 10

Scientia Horticulturae 264 (2020) 109165

A. Khadivi, et al.

experiments and collected data; Ali Khadivi guided all aspects of the research project, analyzed data, and wrote the manuscript. All authors approved the final manuscript.

identification of the promising selections. Sci. Hortic. 238, 234–245. Khadivi-Khub, A., Kameli, M., Moshfeghi, N., Ebrahimi, A., 2015. Phenotypic characterization and relatedness among some Iranian pomegranate (Punica granatum L.) accessions. Trees 29, 893–901. Kher, R., 1999. Note on the physico-chemical characters of wild pomegranate (Punica protopunica L.). Ann. Biol. 15 (2), 231–232. Khodade, M.S., Wavhal, K.N., Kale, P.N., 1990. Physico-chemical changes during growth and development of pomegranate fruit. Indian J. Hortic. 47 (1), 21–27. Langlie, B.A.S., Mueller, N.G., Spengler, R.N., Fritz, G.J., 2014. Agricultural origins from the ground up: archaeological approaches to plant domestication. Am. J. Bot. 101, 1601–1617. Levin, G.M., 1994. Pomegranate (Punica granatum L.) plant genetic resources in Turkmenistan. Plant Genet. Resour. Newsl. 97, 31–36. Maestre, J., Melgarejo, P., Tomas-Barberan, F.A., Garcia-Viguera, C., 2000. New food products derived from pomegranate. In: Melgarejo-Moreno, P. (Ed.), Production, Processing and Marketing of Pomegranate in the Mediterranean Region: Advances in Research and Technology. Mars, M., Marrakchi, M., 1999. Diversity of pomegranate (Punica granatum L.) germplasm in Tunisia. Genet. Resour. Crop Evol. 46, 461–467. Melgarejo, P., Martinez, R., 1992. El Granado. Ediciones Mundi-Prensa Libros, S. A., Madrid. Nasir, E., Ali, S.I., 1972. Flora of West Pakistan. Fakhri Pring Press, Karachi, pp. p. 501. Norusis, M.J., 1998. SPSS/PC Advanced Statistics. SPSS Inc., Chicago. Pandey, A., Tomer, A.K., Bhandari, D.C., Pareek, S.K., 2008. Towards collection of wild relatives of crop plants in India. Genet. Resour. Crop Evol. 55, 187–202. Pant, K.S., 1995. Variation Studies and Provenance Evaluation of Wild Pomegranate (Punica granatum L.). PhD Thesis. Dr YS Parmar UHF, Nauni Solan, HP. Parmar, C., Kaushal, M.K., 1982. Wild Fruits of Sub-himalayan Region. Kalyani Publishers, New Delhi, pp. 74. Prohit, A.G., 1985. Soft-seedness of commercial pomegranate varieties. Indian J. Agric. Sci. 55, 367–368. SAS® Procedures, 1990. Version 6, 3rd ed. SAS Institute, Cary, NC. Sharma, A., Thakur, N.S., 2016. Influence of active packaging on quality attributes of dried wild pomegranate (Punica granatum L.) arils during storage. J. Appl. Nat. Sci. 8 (1), 398–404. Sharma, S.D., Sharma, V.K., 1990. Variation for chemical characters in some promising strains of wild pomegranate (Punica granatum L.). Euphytica 49, 131–133. Shwartz, E., Glazer, I., Bar-Ya’akov, I., Matityahu, I., Bar-Ilan, I., Holland, D., Amir, R., 2009. Changes in chemical constituents during the maturation and ripening of two commercially important pomegranate accessions. Food Chem. 115, 965–973. Simmonds, N.W., 1976. Evolution of Crop Plants. Longman, London, UK. Singh, D., Sethi, V., 2003. Screening of pomegranate genotypes for the preparation of quality grade anardana. J. Food Sci. Technol. 40 (2), 236–238. Singh, D.B., Kinglsley, A.R.P., 2008. Effect of convective drying on quality of anardana. Indian J. Hortic. 65 (4), 413–416. Sood, D.R., Dhindra, K.S., Wagle, D.S., 1982. Studies on the nutritive value of pomegranate. Haryana J. Hortic. Sci. 11, 175–178. Thakur, N.S., Bhat, M.M., Rana, N., Joshi, V.K., 2010. Standardization of pre-treatments for the preparation of dried arils from wild pomegranate. J. Food Sci. Technol. 47 (6), 620–625. Thakur, N.S., Dhaygude, G.S., Gupta, A., 2011. Physico-chemical characteristics of wild pomegranate fruits in different locations of Himachal Pradesh. Int. J. Farm Sci. 1 (2), 37–44. Zavala, M.F., Cozza, F., 2012. The Argentinean experience in the cultivation of 1000 ha of pomegranates (5 provinces) test of varieties and management of crop. In: Melgarejo, P., Valero, D. (Eds.), The Second International Symposium on the Pomegranate. International Centre for Advanced Mediterranean Agronomic Studies, Madrid, Spain, pp. 47–50 October 19-21.

Declaration of Competing Interest None. References Arunachalam, G., 1981. Genetic distances in plant breeding. Indian J. Genet. 41, 226–236. Ashton, R., Baer, B., Silverstein, D., 2006. The Incredible Pomegranate Plant and Fruit. Third Millennium Publishing, USA. Bhat, M.M., 2007. Standardization of Drying Technology of Wild Pomegranate (Punica granatum L.) for Product Development. MSc Thesis, Dr YS Parmar U H F, Nauni, Solan, H P, India. Crites, A.M., Robison, G.D., Mills, L., 2014. Growing Pomegranates in Southern Nevada. Damania, A.B., 2005. The pomegranate: its origin, folklore, and efficacious medicinal properties. In: Nene, Y.L. (Ed.), Agriculture Heritage of Asia-Proceedings of the International Conference. Asian Agri History Foundation, Secunderabad, India. pp. 175–183. Dhaygude, G.S., 2010. Value Added Products From Wild Pomegranate (Punica granatum L.). MSc Thesis, Dr. YS Parmar UHF, Nauni, Solan, HP, India. Drogoudi, P.D., Tsipouridis, C., Michailidis, Z., 2005. Physical and chemical characteristics of pomegranates. HortScience 40, 1200–1203. Fawole, O.A., Opara, U.L., 2013. Changes in physical properties, chemical and elemental composition and antioxidant capacity of pomegranate (cv. Ruby) fruit at five maturity stages. Sci. Hortic. 150, 37–46. Frankel, O.H., 1970. Evaluation and utilization introductory remarks. In: Frankel, O.H., Bennett, E. (Eds.), Genetic Resources in Plants. Their Exploration and Conservation. Blackwell, Oxford, UK, pp. 395–492 pp. 554. Guarino, L., Miller, T., Baazara, M., Obadi, N., 1990. Socotra: the island of Bliss revisited. Diversity 6 (3/4), 28–31. Hammer, Ø, Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4 (1), 9. http:// palaeoelectronica.org/2001_1/past/issue1_01.htm. IBPGR, 1986. Genetic Resources of Tropical and Sub-Tropical Fruits and Nuts (Excluding Musa). International Board for Plant Genetic Resources, Rome, pp. 97–100. Jalikop, S.H., Kumar, P.S., 1998. Use of soft, semi-soft and hard-seeded types of pomegranate (Punica granatum) for improvement of fruit attributes. Indian J. Agric. Sci. 68, 87–91. Jalikop, S.H., Kumar, P.S., Rawal, R.D., Ravindra, K., 2006. Breeding pomegranate for fruit attributes and resistance to bacterial blight. Ind. J. Hortic. 63, 352–358. Jalikop, S.H., Rawal, R.D., Kumar, R., 2005. Exploitation of sub-temperate pomegranate Daru in breeding tropical varieties. Acta Hortic. 696, 107–112. Janick, J., Moore, J., 1975. Advances in Fruit Breeding. Purdue University Press, Purdue, USA. Jashkaran, S., Singh, A.K., Singh, H.K., 2005. Evaluation of pomegranate (Punica granatum L.) varieties for nectar making. Beverage and Food world 32 (5), 42–43. Karimi, H.R., Mirdehghan, S.H., 2013. Correlation between the morphological characters of pomegranate (Punica granatum L.) traits and their implications for breeding. Turk. J. Bot. 37, 355–362. Khadivi, A., Ayenehkar, D., Kazemi, M., Khaleghi, A., 2018. Phenotypic and pomological characterization of a pomegranate (Punica granatum L.) germplasm collection and

11