IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

Study of genetic diversity in pomegranate germplasm of province of

Mehdi Zahravi1*, Mohammad Reza Vazifeshenas2

1Department of Genetics and National Plant Gene Bank of Iran, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization (AREEO). P. O. Box: 31359-33151, Karaj, Iran. 2Agriculture and Natural Resources Research Center of Yazd, Agricultural Research, Education and Extension Organization (AREEO), Yazd, Iran. *Corresponding author, Email: [email protected]. Tel: +98-26-32701260. Fax: +98-26-32716793.

Abstract INTRODUCTION A total of 117 pomegranate genotypes collected Pomegranate (Punica granatum L.) is a member of from different areas of of Iran were Punicaceae family and is considered for its edible fruits, studied for genetic variation by evaluating 23 medicinal properties and application in food industry morphological traits according to the international (Martinez-Nicolas et al., 2016). Pomegranate is descriptor. Similar diversity pattern of the originated from Iran and neighboring countries (Stover measured characteristics was observed in three and Mercure, 2007). The importance of this product in types of sweet, sweet-sour and sour varieties. The non-oil exports made it to be considered as an export traits shape of fruit base, suckering tendency, vigor commodity in the agricultural sector (Anonymous, of tree, fruit shape and aril color had the highest 2009). Iran is one of the largest pomegranate producers power of discrimination. The results of evaluation in the world (Sarkhosh et al., 2006). About 5% of the of genetic distances revealed discrepancies in the production of horticultural products and about 15% of pairs of genotypes with similar denominations. semi-tropical fruits production of Iran are allocated to The sites of Taft and Yazd were located in the pomegranates. Approximately 10% of the cultivation same group in the dendrogram of origins of the area of the subtropical fruits of Iran is dedicated to germplasm, while and together were pomegranate (Anonymous, 2009). placed in another group separated from group Pomegranate is able to grow in a wide range of of Ashkezar and . Abarhuk and Khatam climatic conditions and is suitable for a variety of regions also made single-membered groups. soils. However, it is mainly cultivated in the marginal The results of discriminant analysis revealed areas of the desert, which is not suitable for the that a large number of genotypes were assigned economic cultivation of many fruit trees (Sarkhosh to their original collecting locations with low et al., 2006). Considering that the marginal areas of probabilities indicating a high degree of genetic the desert cover the vast surface of Iran, pomegranate exchanges. The genotypes were separated in 11 cultivation is very important in this unfavorable different groups by K means clustering method situation (Mirjalili, 2003). Yazd province is located with defined characteristics. The supplement of in the central plain of Iran. This province is ranked characterization of the germplasm was suggested fifth in terms of cultivation area and production of through evaluating commercial traits and by pomegranates after the provinces of Fars, Markazi, molecular markers. Khorasan Razavi and Isfahan (Anonymous, 2009). Key words: Gene Bank, Gene Pool, Genetic Yazd province is characterized by the distribution of Resources, Genetic Variation. its pomegranate orchards in most of the provinces so

20 Zahravi and Vazifeshenas

that it can be said that most of the agricultural land stage, Zarei and Azizi (2010) concluded that Faroogh is suitable for the production of this horticultural crop cultivar had more considerable advantages than the (Behzadi Sharebabaki, 1991). Pomegranate cultivation other evaluated cultivars and was recommend to be in the province faces restrictions such as cold weather used as either for export and internal consumption in winter and early spring. The land of pomegranate or as processed forms in fruit juice factories. This orchards in this province is subjected to destruction cultivar had the highest aril and content of reducing and erosion. Many of the old orchards are a mixture sugar and lowest fruit weight and peel percentage. of different varieties of high quality and poor quality Evaluation of 26 quantitative and qualitative fruit traits with different characteristics, and some orchards do in eleven commercial pomegranate cultivars showed not have enough fruiting, and therefore, it is necessary that they were significantly different for all evaluated to take systematic steps to improve and replace these traits, except peel and aril percentage (Tatari et al., orchards (Behzadi Sharebabaki, 1991; Vazifeshenas, et 2011). The results of diversity analysis of 49 wild and al., 2009). commercial pomegranate genotypes by 26 qualitative Identifying genetic diversity and classifying genetic and quantitative traits of fruits and leaves showed that resources is a fundamental issue for the successful besides commercial genotypes, wild genotypes also design of breeding programs and has a significant had desired traits, so the authors insisted on preventing role in facilitating the management of conservation these genotypes from extinction and also on using them of genetic collections (Martinez-Nicolas et al., 2016). for pomegranate improvement and establishment of Iran has a rich pomegranate germplasm. More than collections as a basic step for the preservation purpose 760 pomegranate genotypes from different provinces (Adabi Firouz Jaei et al., 2013). of Iran have been collected in Yazd collection. Due This research was conducted to investigate the to the long history of cultivation and diversity, genetic diversity of pomegranate genotypes in Yazd pomegranate cultivars have similarity of names or province and to identify distinctive traits. similar genotypes in different regions. Studies have been carried out to identify pomegranate genotypes, MATERIALS AND METHODS properties and diversity in some regions of Iran with limited morphological characteristics (Behzadi Plant materials of this research included pomegranate Sharebabaki, 1991) as well as molecular features in germplasms previously collected from different the past (Moslemi et al., 2010; Zeinalabedini et al., regions within Yazd province of Iran (Table 1). These 2012; Nemati et al., 2012; Kazemi Alamuti et al., comprised 117 out of more than 760 pomegranate 2013). Properties such as geometric mean diameter, genotypes which are maintained at Yazd pomegranate volume, true density and packaging coefficient collection in Agriculture and Natural Resources values, projected and surface areas, terminal velocity, Research Centre of Yazd (35°31′N, 51°67′E, 918 m dropping time and drag and buoyancy forces which alt.). The studied trees were 25 years old and planted are necessary to the design of transportation, grading in four replicates. A total of 23 traits were evaluated and processing equipment of the pomegranate varieties according to international descriptor of International have been investigated in commercial pomegranate Union for the Protection of New Varieties of Plants varieties (Dadashi et al., 2001). Investigation on (2011) by obtaining thirty samples from each replicate genetic relations among some closely related Iranian in the 2011-2012 growing season. These traits included pomegranate genotypes with similar names, from Yazd fruit shape, shape of fruit base, shape of fruit apex, and Isfahan areas showed that despite the low level of fruit symmetry, length of fruit crown, skin color, aril detected polymorphism by AFLP marker, differences color, petiole color, apical nectar gland, leaf length to existed among genotypes in one cultivar as well as width ratio, relative length of petiole to leaf middle between the two geographical regions (Rahimi et al., vein, position of flowers, bearing habit, regularity 2005). Investigation of physicochemical properties of of flowering, growth habit, vigor of tree, suckering ten varieties of Yazd pomegranate seeds showed that tendency, wood surface, thorness, color of one-year- the highest quantities of minerals belonged to Mg, K old shoot, shape of leaf blade, shape of leaf apex and and Na (Samadloiy et al., 2006). In this research, the intensity of leaf green color. Descriptive statistics quantities of ash and protein in pomegranate seeds including minimum, maximum, median and mode were 1.81-2.35% and 6.63- 12.95%, respectively. By were calculated. Shannon index (Shannon, 1948) was studying some of the major physical and chemical used as the criterion for the variability of the evaluated properties of six pomegranate cultivars at the ripening traits within each group of sweet, sweet-sour and sour

21 IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017 Table Table 1 . Pomegranate. Yazd of germplasm province the studyfor genetic used diversity of Table 1. 1 1 13 12 11 1 17 1 9 2 1 o 19 1 7 3

.

Gabri Ardakan Gabri Tab Mehriz Bazri Marvast Yazdi agh Marvast Shekari Genotype agh Marvast Mehriz Marvast agh Yazdi Shahvar Goroch Yazdi Shirin Shahvar Ghermez Taft Ghermez Daneh Souski Mehriz Ghermez Daneh Souski Mehriz Abnabati Abrand Khatouni Aminah Abrand Poust Sefid Torsh Mehriz arati Ghermez Daneh Mehriz Marvast Meykhosh Mehriz Marvast Khormaei Yazdi Gardan Togh Abad Poust SiahAbrand Yazdi Malas Pomegranate germplasm of Yazd province used for the study of genetic diversity. - o

- arz Mehrmahi arz

- - Abad Abad

-

Yazd rigin Yazd Yazd Yazd Mehriz Ardakan Khatam Taft Mehriz Mehriz Yazd Yazd Mehriz Khatam Khatam Yazd Yazd Yazd Mehriz

3 37 3 3 3 33 32 3 3 29 2 27 2 2 2 2 22 21 2 o 1 3

.

Tab Mehriz Saryazd asibi Mehriz Attar Khaeh Aghda Shirin Khatouni Taft Gl Peivandi Taft Gl Dabbehi Mehriz Saryazd agh Ardakan Siah Poust Taft Khatouni Aminah Ardakan abati Taft Gl Mamouli Taft Sahourehi Genotype arabaran Poust Ghermez Torsh Mehriz arabaran agh Dorosht Mehriz Mehrmahi Ardakan Aghda Shahvar Siah Taft Daneh Souski Ardakan Aghdaei Meykhosh Taft Gabri Gl - o - arz Abanmahi

Yazd Mehriz Mehriz Ardakan Taft Taft Mehriz Taft Ardakan Taft Taft rigin Khatam Khatam Mehriz Ardakan Taft Ardakan Taft Ardakan

7 3 2 1 9 7 3 2 1 39 o

.

Kermani Torsh Bafq Torsh Kermani Golabi Gardan Genotype azok Poust Goroch Dadashi Abarkuh Pou Shirin Taft Gl Magasi Bafq Khoshk Poust Sefideh Dar Dadashi Shahvar Behabad Shahvar Malas Abarkuh Poust Sefid Torsh Abarkuh PoustKoloft Meykhosh Bafq Pou Kouti Poust Koloft Dadashi arabaran Kadouei imouli ararbaan Gl Torsh Gazi arabaran Sefid Khani Daneh arabaran Riz imouli Mehriz arat Barik Galou arabaran Gl Torsh Gazi arat Koloft Poust Shirin aeh 2

st azok azok st st azok azok st

Abarkuh Bafq rigin Ashkezar Abarkuh Taft Bafq Ashkezar Bafq Abarkuh Abarkuh Bafq Ashkezar Khatam Khatam Khatam Khatam Mehriz Khatam Khatam

22 Zahravi and Vazifeshenas Continue. 71 73 7 7 72 7 9 7 3 2 1 77 9 7

Malas Malas Siah Bafq Daneh Gl Torsh Mamouli Taft Mamouli Gl Torsh Shirin Ashkezar arak Bafq Porbar Kouti Post Sefid Khoshk Bafq Khoshk Sefid Post Ghors Galou Boland Boland Galou Ghors Meybodi Post Ghermez Malas Ashkezar 1 Daraeh Goroch Ashkezar Post Ghermez Ashkezar Shirin Shahvar Behabad Koloft Post Shahvar Malas Abarkuh Koloft Poust Shirin Ashkezar Peivandi Malas Bafq Koutah Galou Ghors Ashkezar agh PoustSefid Bafq Ghermez Daneh Redki Abarkuh Poust azok Torsh Bafq Koloft Post Shahvar Malas 2 Daraeh Shahvar Goroch Abarkuh Meykhosh

aghi aghi

Bafq Taft Ashkezar Bafq Bafq Ashkezar Ashkezar Ashkezar Ashkezar Bafq Abarkuh Ashkezar Bafq Ashkezar Bafq Abarkuh Bafq Yazd Abarkuh Bafq

97 9 9 9 93 92 91 9 9 7 3 2 1 79 7

Abanmahi Abrandabad Abanmahi Moushi Taft Tokhm Bafq aghi Kouti Shirin Shireh Sefideh Bafq Torsh arak Abarkuh Gereh Malas Ashkezar Torsh arak Torsh Poust Koloft Torsh Behabad Koloft Poust Shirin Ardakan Chak Chak Poust Ghermeze 1 Saghand Daraeh Shahvar Shirin Saghand Sorkh Taneh Shrin Saghand azok Poust Shour Marvast azok Poust Shirin 1 Daraeh Dadashi Shahvar Abarkuh azok Poust Meykhosh Bafq Sefid Daneh Redki Ashkezar Malas Ashkezar Poust abati Sefid Ashkezar Peivandi Dadashi

Gardan Boland Boland Gardan

Bafq Yazd Taft Bafq Bafq Abarkuh Ashkezar Ardakan Bafq Ardakan Ardakan Ardakan Ardakan Khatam Yazd Ab Bafq Ashkezar Ashkezar Ashkezar arkuh

117 11 11 11 113 112 111 11 19 1 17 1 1 1 13 12 11 1 99 9

Seh Anboli Taft Anboli Seh Saih Abrandabad Gabri Torsh Poust azok Poust azok Torsh Sagha Poust Ghermez agh Saghand Mamouli Meykhosh Ardakan Chak Torsh azok Poust Dadashi Goroch Bafq azok Poust Shahvar Malas Saghan Koloft Post Shour Saghand Poust Koloft Torsh Saghand Poust azok Bafti Torsh Saghand BaftiPoust Koloft Saghand Ghermez Daneh Malas Saghand 2 Daraeh Sorkh Shirin Tah Abrandabad Sourati Gabri Mehriz Marvast Tafti Torsh Saghand Koloft Poust Shirin Saghand Poust Koloft Torsh Mahriz Marvast Malas Shirin Saghand azok Poust Shirin

Poust Sefid Chak Chak Poust Sefid nd

d

Yazd Taft Ardakan Ardakan Ardakan Ardakan Ashkezar Bafq Ardakan Ardakan Ardakan Ardakan Ardakan Ardakan Yazd Mehriz Ardakan Ardakan Khatam Ardakan

23 IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017 Table 2. Descriptive statistics of the studied traits in the pomegranate germplasm of Yazd province, Iran. Table 2. Descriptive statistics of the studied traits in the pomegranate germplasm of Yazd province, Iran.

Trait Minimum Maximum Median Mode Shannon Fruit shape 1 3 2 1 1. Shape of fruit base 1 2 1 1.2 Shape of fruit apex 1 2 2 2 .9 Fruit symmetry 2 .99 ength of fruit crown 1 3 1 1 .3 Skin color 3 3 3 .93 Aril color 3 7 3 1.3 Petiole color 1 1 1 .7 Apical nectar gland 1 3 1 1 . eaf length to width ratio 7 .9 Relative length of petiole to leaf middle vein 1 3 2 2 .92 Position of flowers 1 3 1 1 .93 Bearing habit 1 3 1 1 .2 Regularity of flowering 1 3 1 1 .9 Growth habit 1 3 3 3 .7 Vigour of tree 1 3 2 3 1.7 Suckering tendency 3 9 3 3 1.9 ood surface 1 3 3 3 . Thorness 1 7 1 1 .79 Color of one-year-old shoot 2 3 2 2 .1 Shape of leaf blade 1 3 1 1 .9 Shape of leaf apex 1 3 3 3 .29 Intensity of leaf green color 3 3 3 .7

varieties as well as in total germplasm. The score of RESULTS genotypes for each class within the assayed traits The distribution mode of the studied traits within the were converted to 0 or 1, representing the absence or germplasm was examined by descriptive statistics presence of the related characteristics, respectively, (Table 2). The values of mode indicated that the for the statistical analyses. Rogers method (Rogers, traits spheroid fruit shape (1), truncate shape of fruit 1992) was used for estimating the genetic similarity base (1), regular shape of fruit apex (2), non-angular and genetic distances among genotypes. Variability symmetrical shape of fruits (5), short fruit crown (1), of distances of origins of the studied germplasm was reddish yellow skin of fruit (3), light pink aril color displayed by boxplot. Dendrogram of germplasm (3), red petiole (1), absence of apical nectar gland (1), origins was constructed based on genetic distances. medium ratio of leaf length to width ratio (5), short Discriminant analysis of principal components (Jombart length of petiole relative to leaf middle vein (2), lateral et al., 2010) was performed to investigate assignment position of flowers or inflorescence (1), predominant degree of the genotypes to their original collecting distribution of flower buds on young shoots (1), regular site. In order to make a better distinction within the habit of flowering (1), upright growth habit of tree (3), germplasm population, K means clustering approach strong vigor of tree (3), low suckering tendency (3), was conducted based on principal components. In coarse wood surface (3), absence of thorns (1), green this procedure, the dimension of data is reduced color of one-year-old shoot with pink stripes (2), by a principal component analysis (PCA) and the obtuse shape of leaf blade (1), acute shape of leaf apex transformed data is subjected to successive K-means (3) and low intensity of leaf green color (3) were the analyses with an increasing number of clusters (k). most prevalent characteristics among the investigated For each model, Bayesian information criterion (BIC) genotypes. is computed as a statistical measure of goodness of fit, which allows to choose the optimal k (Jombart The traits shape of fruit base and shape of leaf et al., 2010). Features of the clusters were described blade had the highest and lowest diversities based on by examining frequent characteristics through mode Shannon index, respectively (Table 2). Three groups of statistics. Statistical analysis and drawing the plots sweet, sour and sweet-sour genotypes showed a similar were performed by R software 3.2.2. trend in diversity pattern of the traits based on Shannon

24 Zahravi and Vazifeshenas

index (Figure 1). Sweet genotypes had higher Shannon A values for the traits fruit shape, shape of fruit base, fruit symmetry, fruit skin color, frequency of apical nectar glands, position of flowers, wood surface than sour and sweet-sour genotypes. Diversity of the traits length of crown, suckering tendency, leaf apex shape and intensity of leaf green color were highest in sweet- sour genotypes. Sour genotypes possessed the highest amount of Shannon index for the traits shape of fruit apex, aril color, petiole color, leaf length to width ratio, relative length of petiole to leaf middle vein, regularity of flowering, growth habit, vigor of tree, thorness, and color of one-year-old shoots as well as shape of leaf blade. B Figure 1. Barplot of Shannon index for the Based on genetic distances, genotypes ‘Dadashi studied traits of Yazd pomegranate germplasm in Poust Koloft’ (46) and ‘Khatouni Shirin Aghda’ (31), each of sweet (a), sweet-sour (b) and sour (c) and ‘Narak Shirin Ashkezar’ (67) and ‘Shirin Poust varieties. Koloft Behabad’ (96) had in pairs the highest similarity and genotypes ‘Shirin Poust Nazok Marvast’ (90) fsh: fruit shape, fba: shape of fruit base, fap: and ‘Nimouli Kadouei Harabarjan’ (45), and ‘Shirin shape of fruit apex, fsy: fruit symmetry, flc: Poust Nazok Marvast’ (90) and ‘Shirin Poust Koloft length of fruit crown, sc: skin color, sec: seed Abarkuh’ (68) had in pairs the highest distance within color, pco: petiole color, ang: apical nectar sweet germplasm (Figure 2). Genotypes with highest gland, lr: leaf length to width ratio, lpv: relative similarity in sweet-sour germplasm were in pairs ‘Malas length of petiole to leaf middle vein, position Shahvar Behabad’ (50) and ‘Zagh Dorosht Harabarjan pfi: Mehriz’ (37), and ‘Malas Shahvar Behabad’ (50) and of flowers, bha: bearing habit, rfl: regularity of

‘Gardan Golabi Abarkuh’ (51), and ‘Malas Gardan flowering, gha: growth habit, vtr: vigour of tree, Boland Ashkezar’ (85) and ‘Malas Shahvar Behabad’ Figure suckering 1. Barplot tendency, of Shannon wood index surface, for the C Figure 1. Barplot of Shannon index for theste: su: thr: (50) (Figure 2). studiedthorness, traits shc: of Yazdcolor pomegranateof one-year-old germplasm shoot, ash: in studied traits of Yazd pomegranate germplasm in The highest distance in sweet-sour germplasm eachshape of of sweet leaf blade, (a), sweet las: shape-sour (b)of leafand apex,sour lic:(c) each of sweet (a), sweet-sour (b) and sour (c) belonged in pairs to genotypes ‘Gabri Sourati varieties.intensity of leaf green color. Abrandabad’ (103) and ‘Zagh Yazdi’ (2), and ‘Gabri varieties. fsh: fruit shape, fba: shape of fruit base, fap: Sourati Abrandabad’ (103) and ‘Nabati Poust Sefid fsh: fruit shape, fba: shape of fruit base, fap:shape of fruit apex, fsy: fruit symmetry, flc: Ashkezar’ (83) (Figure 2). Genotypes ‘Narak Torsh shape of fruit apex, fsy: fruit symmetry, flc:length of fruit crown, sc: skin color, sec: seed Ashkezar’ (78) and ‘Bazri Marvast Mehriz’ (5), ‘Redki color, petiole color, apical nectar Daneh Ghermez Bafq’ (62) and ‘Gl Gabri Taft’ (23), ‘Gl length of fruit crown, sc: skin color, sec: seed pco: ang: Torsh Mamouli Taft’ (75) and ‘Nimouli Riz Harabarjan’ color, pco: petiole color, ang: apical nectargland, lr: leaf length to width ratio, lpv: relative (42), ‘Redki Daneh Sefid Baq’ (86) and ‘Nimouli gland, lr: leaf length to width ratio, lpv: relativelength of petiole to leaf middle vein, pfi: position Riz Harabarjan’ (42), ‘Torsh Poust Sefid Chak Chak of flowers, bearing habit, regularity of length of petiole to leaf middle vein, pfi: position bha: rfl: Ardakan’ (114) and ‘Gl Gazi Torsh Harabajan’ (44), flowering, gha: growth habit, vtr: vigour of tree, ‘Torsh Poust Koloft Saghand’ (100) and ‘Gl Magasi of flowers, bha: bearing habit, rfl: regularity of ste: suckering tendency, su: wood surface, thr: Taft’ (54), and ‘Torsh Poost Koloft Saghand’ (97) and Figure 1.flowering, Barplot of gha: Shannon growth index habit, for thevtr: studiedvigour traitsof tree, thorness, shc: color of one-year-old shoot, ash: ‘Gl Torsh Mamouli Taft’ (75) were in pairs the most of Yazd pomegranateste: suckering germplasm tendency, in su:each woodof A: surface,sweet, B: thr: similar and genotypes ‘Koutji Poust Nazok Bafq’ (47) sweet-sour and C: sour varieties. shape of leaf blade, las: shape of leaf apex, lic: fsh: fruit shape,thorness, fba: shc:shape color of fruit of base,one-year fap:- oldshape shoot, of fruit ash: and ‘Torsh Poust Ghermez Harabarjan’ (38), ‘Shour intensity of leaf green color. apex, fsy:shape fruit symmetry, of leaf blade, flc: length las: shapeof fruit crown,of leaf skc:apex, skin lic: Poust Nazok Saghand’ (91) and ‘Torsh Poust Ghermez color, sec: aril color, pco: petiole color, ang: apical nectar Harabarjan’ (38), ‘Torsh Poust Ghermez Harabarjan’ gland, lwr:intensity leaf length of leaf to greenwidth color.ratio, lpv: relative length (38) and ‘Koutji Poust Nazok Bafq’ (47), ‘Kermani of petiole to leaf middle vein, pfi: position of flowers, bha: Torsh Bafq’ (57) and ‘Koutji Poust Nazok Bafq’ (47), bearing habit, rfl: regularity of flowering, gha: growth habit, ‘Shour Poust Nazok Saghand’ (91) and ‘Kermani vtr: vigour of tree, ste: suckering tendency, wsu: wood surface, thr: thorness, shc: color of one-year-old shoot, ash: Torsh Bafq’ (57), ‘Shour Poust Nazok Saghand’ (91) shape of leaf blade, las: shape of leaf apex, lic: intensity of and ‘Goroch Darajeh 1 Ashkezar’ (72) were in pairs leaf green color.

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IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

A B C

Figure 2. Gray-scaled heat map of similarity matrices between pomegranate genotypes of Yazd (Iran) in each of sweet (a), sweet-sour (b) and sour (c) classes.

Figure 2. Gray-scaled heat map of similarity matrices between pomegranate genotypes of Yazd (Iran) in each of A: sweet, B: sweet-sour and C: sour classes.

Figure 2. FigureGray-scaled 2. Gray heat-scaled map heatof map of A B similarity similaritymatrices matricesbetween between

pomegranatepomegranate genotypes ofgenotype Yazd (Iran)s of Yazd (Iran) in each of sweetin each (a), of sweet sweet-sour (a), (b)sweet and-sour (b) and

sour (c) classes.sour (c) classes.

C

Figure 3. Dendrogram of cluster analysis Figure 3. DendrogramFigure of 3cluster. Dendrogram analysis of cluster analysis of pomegranateof pomegranate sweet sweet (a),of pomegranatesweet (a), sweet-sour -(b) soursweet (b) (a), sweet-sour (b) and sourand sour(c) varieties(c) varietiesand of sourYazd, of (c)Yazd, Iran varieties, Iran, of Yazd, Iran, throughthrough complete complete linkage throughlinkage method completemethod based based linkage method based

on Rogerson Rogers (1992) (1992) distance donistance sRogers. s. (1992) distances.

Figure 3. Dendrogram of cluster analysis of pomegranate A: sweet, B: sweet-sour and C: sour varieties of Yazd, Iran, through complete linkage method based on Rogers (1992) distances.

the least similar in sour germplasm. The genotypes Taft and Abarkuh were located higher in the boxplot were divided into eight, seven and eight groups by indicating higher distances to other origins. These dendrogram of cluster analysis within each of sweet, regions along with Yazd had the shortest length of sweet-sour and sour germplasm, respectively (Figure 3). boxes showing lower variation in the distances of Genetic distances of the studied germplasms were these regions to other places. Ardakan had the highest also investigated based on their origin and the results box length which indicates that this region had varied were shown by boxplot (Figure 4). The boxes of distances with other origins being very close to some

26

Figure . Genetic distances of pomegranate genotypes with Yazd origin. FigureFigure . Genetic . Genetic distances distances of pomegranate of pomegranate genotypes genotypes with Yazd with Yazdorigin. origin.

Figure 3. Dendrogram of cluster analysis of pomegranate sweet (a), sweet-sour (b) and sour (c) varieties of Yazd, Iran, through complete linkage method based on Rogers (1992) distances.

Zahravi and Vazifeshenas

Figure 4. Genetic distances of pomegranate genotypes with Yazd origin. Figure . Genetic distances of pomegranate genotypes with Yazd origin.

Figure 5. Comparison of dendrogram and geographical origins of pomegranate genotypes of Yazd, Iran.

of them while far from the others. Dendrogram of the Three regions of Abarkuh, Taft and Mehriz were origins of the studied germplasm located Taft and Yazd differentiated from five other origins in the distribution regions in the same group while Bafq and Ardakan plot of individuals based on the first two discriminant together were placed in another group distinct from functions (Figure 6). Discrimination of Taft was group of Ashkezar and Mehriz. Each of Abarhuk and mainly based on the first discriminant function, with Khatam regions also made single-membered groups. the highest contribution of the traits thorness (thr.5 and (Figure 5). thr.7), petiole color other than red and yellow (pco.3), shape of leaf blade (ash.1 and ash.3), very short length Discriminant analysis was performed to study the of petiole relative to leaf middle vein (lpv.1) and lateral origin relationship of the genotypes more precisely. position of flowers (pfi.1) (Figure 7).

27 IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

Figure 6. Distribution of pomegranate germplasm of Yazd (Iran) in biplot of the first two discriminant functions.

Figure 7. Presentation loadings of the traits in the first two discriminant functions in the pomegranate germplasm of Yazd, Iran.

thr: thorness, ste: suckering tendency, pco: petiole color, ash: shape of leaf blade, lpv: relative length of petiole to leaf middle vein, pfi: position of flowers. Figure 7. Presentation loadings of the traits in the first two discriminant functions in the pomegranate germplasm of Yazd, Iran.

The other regions were differentiated along with very short length of petiole relative to leaf middle vein axis of the second discriminant function. The loading (lpv.1) had the main contribution in variation of second plot revealed that the traits very high and high discriminant function. suckering tendency (ste.7 and ste.9), shape of leaf Membership probability of assignment of the blade (ash.1 and ash.3), very high thorness (thr.7), and individuals to the eight germplasm origin was plotted

28

Figure 7. Presentation loadings of the traits in the first two discriminant functions in the pomegranate germplasm of Yazd, Iran. ton, cn tndnc, tol colo, a of laf lad, lat lnt of tol ton, cn tndnc, Zahravi tol andcolo, Vazifeshenas ato laf of ddl laf lad,n, oton lat of lntflo of tol to laf ddl n, oton of flo

A B

ton, cn tndnc, tol colo, a of laf lad, lat lnt of tol to laf ddl n, oton of flo

Figure . Membership probability of assignment of individuals to the germplasm origins (a) and groups developed through K means clustering approach (b) after performing discriminant function analysis on pomegranate genotypes of Yazd, Iran. FigureFigure 8. Membership . Membership probability ofprobability assignment of of assignmentindividuals to A: of the individuals germplasm toorigins the andgermpl B: groupsasm developedorigins (a) through K means clustering approach after performing discriminant function analysis on pomegranate genotypes of Yazd, Iran. Populationand 1 groupsto eight aredeveloped Abarkuh, Ardakan, through Ahkezar, K Bafq,means Khatam, clustering Mehriz,Population Taftapproach and 1 toYazd, eight respectively.(b) are afterAbarkuh, performing Ardakan, Ahkezar, Bafq, Khatam, Mehriz, Taft and Yazd, discriminant function analysis on pomegranate genotypesrespectively. of Yazd, Iran.

Population 1 to eight are Abarkuh, Ardakan, Ahkezar, Bafq, Khatam, Mehriz, Taft and Yazd, (Figure 8). The results revealed a high level of admixture in ther studiedespectively. germplasm. K means clustering approach was performed to detect the structures in the population with the highest discrimination and to compare the resulting differentiation pattern with that resulted from origins of the genotypes.Figure . MembershipThe values ofprobability BIC showed of assignment of individuals to the germplasm origins (a) that with 11 clusters in the population, the highest and groups developed through K means clustering approach (b) after performing discrimination is attainable (Figure 9). Plotting the number of clusterdiscriminant members against function that analysis of individuals on pomegranate genotypes of Yazd, Iran. related to their origin revealed that some clusters comprised individualsPopulation of certain1 to eight origins are Abarkuh, exclusively Ardakan, Ahkezar, Bafq, Khatam, Mehriz, Taft and Yazd, (Figure 10). Clusterrespectively. 11 made the most exclusive group comprising only individuals belonging to Ardakan,

Taft and Bafq while clusters 4 and 10 generally included individuals from all origins. Clusters 5, 7 and 9 comprised mostly of sweet varieties. Sweet-sour varieties were mostly included in clusters 1, 3, 4, 6, Figure 9. Variance values explained by principal components and the trend of 8 and 10, and sour varieties were mainly lodged in Bayesian information criterion (BIC) on selecting different number of clusters through clusters 2 and 11. Clusters 2 and 10 had the highest similarity while clusters 7 and 10 had the maximum K means clustering approach in the study of pomegranate genotypes of Yazd, Iran. distance (Table 3).

ModeFigure statistic 9. wasVariance used to valuesdetermine explained the features by principal components and the trend of of eachBayesian cluster (Table information 4). With thiscriterion approach (BIC) the ontraits selecting different number of clusters through ovoid fruit shape (3), opened shape of fruit base (4), long Kfruit means apex clustering(1), symmetrical approach pentagon in the shape study of of pomegranate genotypes of Yazd, Iran. fruits (2), short fruit crown (1), reddish yellow skin of fruit (3), dark red aril (sec), red petiole (1), absence of apical nectar gland (1), high ratio of leaf length Figure 9. Variance values explained by principal components and the trend of Bayesian information criterion (BIC) on to width ratio (7), medium length of petiole relative selecting different number of clusters through K means to leaf middle veinFigure (3), 9lateral. Variance position values of flowers explained or clustering by principal approach components in the study ofand pomegranate the trend genotypes of inflorescence (1),Bayesian predominant information distribution criterion (BIC) of flower onof selecting Yazd, Iran. different number of clusters through

29 K means clustering approach in the study of pomegranate genotypes of Yazd, Iran.

IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

Figure 1. Membership distribution of pomegranate genotypes of Yazd (Iran) with different origins within groups developed by K means clustering approach. Figure 10. Membership distribution of pomegranate genotypes of Yazd (Iran) with different origins within groups developed by K means clustering approach.

Table 3. Distance matrix of different groups within pomegranate germplasm of Yazd Table(Iran) 3. Distancedeveloped matrix by of differentK means groups clustering within pomegranate approach. germplasm of Yazd (Iran) developed by K means clustering approach.

Clusters 2 3 7 9 1 11 1 .33 .292 .7 .311 .2 . . .37 .2 .397 2 .39 .3 .3 .31 .27 .1 .12 .29 .31 3 .32 .31 .2 .29 . .279 .3 .32 .3 .3 .7 .2 .391 .2 .27 .39 .1 .3 .11 .32 .23 .329 .3 .3 .2 .31 7 .39 . .9 .39 .37 .9 .22 9 .377 .33 1 .7

buds on young shoots (1), irregular habit of flowering (5), short fruit crown (1), red skin of fruit (4), light red (rfl), upright growth habit of tree (gha), strong vigor aril (5), red petiole (1), absence of apical nectar gland of tree(vtr), low suckering tendency (3), coarse wood (1), high ratio of leaf length to width ratio (7), short surface (3), absence of thorns (1), green color of one- length of petiole relative to leaf middle vein (2), lateral year-old shoot with pink stripes (2), obtuse shape of position of flowers or inflorescence (1), predominant leaf blade (1), acute shape of leaf apex (3) and low or distribution of flower buds on young shoots (1), medium intensity of leaf green color (3-5) were known irregular habit of flowering (3), upright growth habit as the major features of cluster 1. of tree (3), strong vigor of tree (3), low suckering Cluster 2 was characterized with the traits ellipsoid tendency (3), coarse wood surface (3), absence of fruit shape (2), truncate shape of fruit base (1), long thorns (1), green color of one-year-old shoot with pink fruit apex (1), non-angular symmetrical shape of fruits stripes (2), obtuse shape of leaf blade (1), acute shape

30 Zahravi and Vazifeshenas

Table . Features of the groups in pomegranate germplasm of Yazd (Iran) Tabledeveloped 4. Features by of K the means groups inclustering pomegranate approach germplasm basedof Yazd (Iran)on the developed values by of K meansmode clustering statistics approach. based on the values of mode statistics.

Cluster Trait 1 2 3 7 9 1 11 tst 2 3 2 2 1 2 1 2 1 2 3 fsh 3 2 2 1 3 1 1 1 2 2 1 fba 1 1 3 2 2 2 3 1 1 3 fap 1 1 2 2 1 2 2 2 2 1 2 fsy 2 2 3 3 3 2 flc 1 1 1 2 1 1 1 3 1 1 3 skc 3 3 3 3 3 3 3 sec 7 3 3 3 7 3 3 3 3 pco 1 1 1 1 1 1 1 3 1 1 1 ang 1 1 1 1 1 1 3 1 1 1 1 lwr 7 7 7 7 7 lpv 3 2 2 2 2 2 1 2 2 2 3 pfi 1 1 1 3 1 1 2 1 3 3 1 bha 1 1 1 1 1 1 3 3 1 1 1 rfl 3 3 1 1 3 3 3 1 1 1 3 gha 3 3 3 3 3 3 3 3 1 3 1 vtr 3 3 3 2 2 3 2 1 1 2 3 ste 3 3 3 3 3 3 7 3 7 3 7 wsu 3 3 1 1 1 1 1 3 3 3 1 thr 1 1 1 1 1 1 1 1 1 1 1 shc 2 2 2 2 2 2 2 2 2 2 2 ash 1 1 1 1 1 1 1 1 1 1 1 las 3 3 3 3 3 3 3 3 3 3 3 lic *3- 3 3 3 3 3 3 3 3 *3-

1 22 2 7 19 3 1 1 2 12 11 13 1 3 3 27 21 7 1 2 17 3 9 9 1 9 9 23 77 29 1 3 2 9 33 1 32 1 31 2 17 3 11 71 2 7 3 2 3 2 1 3 11 73 7 9 7 9 1 39 111 37 99 2 9 9 7 3 2 113 Members 13 72 1 12 79 11 1 19 11 91 92 7 7 112 11 3 7 97 93 7 117 9 1 1 *The case of multi-modes tst: type of varieties based on taste, fsh: fruit shape, fba: shape of fruit base, fap: shape of fruit apex, fsy: fruit symmetry, flc: length of fruit crown, skc: skin color, sec: aril color, pco: petiole color, ang: apical nectar gland, lwr: leaf length to width ratio, * The case of multi-modes lpv: relative length of petiole to leaf middle vein, pfi: position of flowers, bha: bearing habit, rfl: regularity of flowering, gha: growth habit, vtr: vigour of tree, ste: suckering tendency, wsu: wood surface, thr: thorness, shc: color of one-year-old shoot, ash:tst: shapetype of of varietiesleaf blade, based las: shape on taste, of leaf fsh: apex, fruit lic: shape,intensity fba: of leaf shape green of color. fruit base, fap: shape of fruit apex, fsy: fruit symmetry, flc: length of fruit crown, skc: skin color, sec: seed color, pco: petiole color, ang: apical nectar gland, lwr: leaf length to width ratio, lpv: relative length of petiole to leaf middle vein, pfi: position of flowers, bha: bearing habit, rfl: regularity of flowering, gha: growth habit, vtr: vigour of tree, 31 IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

of leaf apex (3) and low intensity of leaf green color of fruit base (2), regular size of fruit apex (2), (3). asymmetrical shape of fruits (3), short fruit crown (1), reddish yellow skin of fruit (3), dark red aril (7), red Major characteristics of Cluster 3 included ellipsoid petiole (1), absence of apical nectar gland (1), high ratio fruit shape (2), truncate shape of fruit base (1), regular of leaf length to width ratio (7), short length of petiole size of fruit apex (2), symmetrical pentagon shape of fruits (2), short fruit crown (1), reddish yellow skin relative to leaf middle vein (2), lateral position of of fruit (3), light pink aril color (3), red petiole (1), flowers or inflorescence (1), predominant distribution absence of apical nectar gland (1), medium ratio of leaf of flower buds on young shoots (1), irregular habit of length to width ratio (5), short length of petiole relative flowering (3), upright growth habit of tree (3), strong to leaf middle vein (2), lateral position of flowers or vigor of tree (3), low suckering tendency (3), smooth inflorescence (1), predominant distribution of flower wood surface (1), absence of thorns (1), green color of buds on young shoots (1), regular habit of flowering one-year-old shoot with pink stripes (2), obtuse shape (1), upright growth habit of tree (3), strong vigor of tree of leaf blade (1), acute shape of leaf apex (3) and low (3), low suckering tendency (3), smooth wood surface intensity of leaf green color (3) were majorly assigned (1), absence of thorns (1), green color of one-year-old to the cluster 6. shoot with pink stripes (2), obtuse shape of leaf blade Characteristics spheroid fruit shape (1), covex (ash) (1), acute shape of leaf apex (3) and low intensity shape of fruit base (2), regular size of fruit apex (2), of leaf green color (3). asymmetrical shape of fruits (3), short fruit crown (1), Cluster 4 could be recognized mainly with the traits reddish yellow skin of fruit (3), light pink aril (3), red spheroid fruit shape (1), covex shape of fruit base (3), petiole (1), moderately developed apical nectar gland regular size of fruit apex (2), non-angular symmetrical (3), high ratio of leaf length to width ratio (7), very short shape of fruits (5), medium fruit crown (2), red skin length of petiole relative to leaf middle vein (1), both of fruit (4), light pink aril color (3), red petiole (1), lateral and terminal position of flowers or inflorescence absence of apical nectar gland (1), medium ratio of (pfi) (2), predominant distribution of flower buds on leaf length to width ratio (5), short length of petiole old shoots (bha) (3), irregular habit of flowering (rfl) relative to leaf middle vein (2), terminal position of (3), upright growth habit of tree (gha) (3), medium flowers or inflorescence (3), predominant distribution vigor of tree(vtr) (2), low suckering tendency (ste) (7), of flower buds on young shoots (1), regular habit of smooth wood surface (wsu) (1), absence of thorns (thr) flowering (1), upright growth habit of tree (3), medium (1), green color of one-year-old shoot with pink stripes vigor of tree (2), low suckering tendency (3), smooth (shc) (2), obtuse shape of leaf blade (ash) (1), acute wood surface (1), absence of thorns (1), green color of shape of leaf apex (las) (3) and low intensity of leaf one-year-old shoot with pink stripes (2), obtuse shape green color (lic) (3) were major in Cluster 7 of leaf blade (1), acute shape of leaf apex (3) and low Cluster 8 was characterized with the traits spheroid intensity of leaf green color (3). fruit shape (1), angular shape of fruit base (3), regular Cluster 5 had the features of ovoid fruit shape (3), size of fruit apex (2), non-angular symmetrical shape covex shape of fruit base (2), long fruit apex (1), of fruits (5), long fruit crown (3), reddish yellow skin asymmetrical shape of fruits (3), short fruit crown (1), of fruit (3), light pink aril color (3), petiole color other dark red skin of fruit (skc) (5), light pink aril color than red or yellow (3), absence of apical nectar gland (3), red petiole (1), absence of apical nectar gland (1), (1), medium ratio of leaf length to width ratio (5), short medium ratio of leaf length to width ratio (5), short length of petiole relative to leaf middle vein (2), lateral length of petiole relative to leaf middle vein (2), lateral position of flowers or inflorescence (1), predominant position of flowers or inflorescence (1), predominant distribution of flower buds on old shoots (3), regular distribution of flower buds on young shoots (1), habit of flowering (1), upright growth habit of tree irregular habit of flowering (3), upright growth habit (3), weak vigor of tree (1), low suckering tendency of tree (3), medium vigor of tree (2), low suckering (3), coarse wood surface (3), absence of thorns (1), tendency (3), smooth wood surface (1), absence of green color of one-year-old shoot with pink stripes (2), thorns (1), green color of one-year-old shoot with pink obtuse shape of leaf blade (1), acute shape of leaf apex stripes (2), obtuse shape of leaf blade (1), acute shape (3) and low intensity of leaf green color (3). of leaf apex (3) and low intensity of leaf green color The traits ellipsoid fruit shape (2), truncate shape of (3). fruit base (1), regular size of fruit apex (2), symmetrical The traits spheroid fruit shape (1), covex shape pentagon shape of fruits (2), short fruit crown (1),

32 Zahravi and Vazifeshenas

reddish yellow skin of fruit (3), light pink aril color which was lower than the third quartile of similarity (3), red petiole (1), absence of apical nectar gland (1), values (0.64), but also very close to the first quartile medium ratio of leaf length to width ratio (5), short (0.44). In other words, despite their names being length of petiole relative to leaf middle vein (2), terminal the same, these two genotypes did not show close position of flowers or inflorescence (3), predominant resemblance. Genotype 97 and 108 also had the same distribution of flower buds on young shoots (1), regular names as ‘Torsh Poust Koloft Saghand’. Similarity habit of flowering (1), spreading growth habit of tree value for these two genotypes was estimated 0.57 (1), weak vigor of tree (1), high suckering tendency which is lower than the third quartile of similarity (7), coarse wood surface (3), absence of thorns (1), values (0.64) and close to the median (0.52). green color of one-year-old shoot with pink stripes (2), Therefore these two genotypes also appeared to be obtuse shape of leaf blade (1), acute shape of leaf apex different despite the same designation. There were (3) and medium intensity of leaf green color (5) were two genotypes (14 and 24) with the name of ‘Aminah known as the major features of cluster 9. Khatouni’, which one of them was originated form Cluster 10 could be recognized mainly with the Taft and the other from Abrand-Abad. Based on the traits ellipsoid fruit shape (2), truncate shape of fruit value of similarity (0.39) these two genotypes also had base (1), long fruit apex (1), non-angular symmetrical low resemblance. Genotypes 60 and 69 (from Bafq shape of fruits (5), short fruit crown (1), red skin of and Bahabad, respectively) both were named ‘Malas fruit (4), light red aril color (5), red petiole (1), absence Shahvar Post Koloft’ and with a similarity value (0.61) of apical nectar gland (1), medium ratio of leaf length not higher than the third quartile. Genotypes 78 and 80 to width ratio (5), short length of petiole relative to with common name of ‘Narak Torsh’ and originated leaf middle vein (2), terminal position of flowers or from Ashkezar and Bafq, respectively, also appeared inflorescence (3), predominant distribution of flower to have low resemblance with the similarity value buds on young shoots (1), regular habit of flowering of 0.39. Two genotypes 68 and 96 shared the name (1), upright growth habit of tree (3), strong vigor of tree of ‘Shirin Poust Koloft’ and were from Abarkuh and (2), high suckering tendency (3), coarse wood surface Bahabad, respectively, and had medium similarity (3), absence of thorns (1), green color of one-year-old of 0.52. There were also two genotypes (55 and 90) shoot with pink stripes (2), obtuse shape of leaf blade with the name of ‘Shirin Poust Nazok’, collected (1), acute shape of leaf apex (3) and low intensity of from Abarkuh and Marvast, respectively, with a low leaf green color (3). similarity value of 0.42. The name of ‘Souski Daneh Ghermez’ was assigned to genotypes 18 from Mehriz Members of mainly express the traits of Cluster 11 and 19 from Taft, while these two genotypes had a spheroid fruit shape (1), angular shape of fruit base (3), medium similarity value of 0.61. Two genotypes 13 regular size of fruit apex (2), non-angular symmetrical (from abarkuh) and 49 (from Abrand-Abad) with the shape of fruits (5), long fruit crown (3), reddish yellow common name of ‘Torsh Poust Sefid’ showed a low skin of fruit (3), light pink aril color (3), red petiole similariy value (0.43). Therefore, as it is evident, the (1), absence of apical nectar gland (1), high ratio of pairs of genotypes which shared the common names leaf length to width ratio (7), medium length of petiole within the studied germplasm did not appear to have relative to leaf middle vein (3), lateral position of similar phenotypic features. Grouping varieties with flowers or inflorescence (1), predominant distribution different agronomic characteristics under the same of flower buds on young shoots (1), irregular habit of designation have been reported before. Melgarejo and flowering (3), spreading growth habit of tree (1), strong Salazar (2003) observed that there were varieties with vigor of tree (3), high suckering tendency (7), smooth different agronomic characteristics under common wood surface (1), absence of thorns (1), green color of name of ‘Mollar de Elche’’. one-year-old shoot with pink stripes (2), obtuse shape of leaf blade (1), acute shape of leaf apex (3) and low Identification of the distinctive traits is necessary or medium intensity of leaf green color (lic) (3-5). for better characterization and analysis of genetic diversity of germplasm collection (Martinez-Nicolas et al., 2016). Shannon index for the traits shape of fruit DISCUSSION base, suckering tendency, vigor of tree, fruit shape The population under study contained a few pairs of and aril color was higher than one. So these traits genotypes with similar nominations. Two genotypes had the capability of making higher distinction in the of 40 and 44 had the same name of ‘Gl Gazi Torsh studied germplasm. Traits fruit symmetry, position of Harabarjan’, however they had similarity value of 0.48 flowers or inflorescence, skin lorco and relative length

33 IRANIAN JOURNAL of GENETICS and PLANT BREEDING, Vol. 6, No. 2, Oct 2017

of petiole to leaf middle vein also had a high value of discrimination and are suggested for the characterization Shannon index (higher than 0.9). Therefore, these traits of pomegranate germplasm collections. Similarity of could be considered as differentiating characteristics of phenotypic characteristics in genotypes with common pomegranate germplasm in the second order. Martinez- denomination was investigated and the results indicated Nicolas et al. (2016) suggested parameters related high discrepancies. It is suggested that characterization to fruit and seed size as well as the juice’s acidity of the germplasm is continued by supplementing and pH as the most useful for pomegranate genetic commercial traits and molecular marker evaluations. characterization since they had the highest power of discrimination which was opposed to leaf and flower REFERENCES characteristics with low power of differentiation. Adabi Firouz Jaei Z., Zamani M., and Fatahi Moghadam M. 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