Phenological, morphological and molecular characterization of some promising walnut (Juglans regia L) genotypes in Konya

(1)

ORIGINAL ARTICLE

https://doi.org/10.1007/s10341-018-0411-9

Phenological, Morphological and Molecular Characterization of Some

Promising Walnut (Juglans regia L) Genotypes in Konya

Muzaffer Ipek1_{· Şeyma Arıkan}1_{· Lütfi Pırlak}1_{· Ahmet Eşitken}1

Received: 17 September 2017 / Accepted: 9 November 2018 / Published online: 10 December 2018 © Springer-Verlag GmbH Deutschland, ein Teil von Springer Nature 2018

Abstract

This study was carried out between 2010 and 2013 in three districts (Ak¸sehir, Bey¸sehir, and Hüyük) of Konya. During survey study, about 2500 walnut genotypes were observed for different walnut pomological characteristics such as shelled nut weight (g) and percentage of the kernel (%). The promising genotypes were selected among these walnut populations. In 2010 and 2012, 300 walnut genotypes were selected and these selected walnut genotypes number were decreased from 300 to 8 genotypes in 2013. Eight genotypes were found promising due to features of shelled nut and kernel. The shelled fruit weight ranged from 8.70 to 14.34 g. The percentage of kernel rates were determined between 40.15 and 63.21%. Genotype “BE-14” had the highest oil content (65.72%). Selected walnut genotypes were compared with Turkish and foreign walnuts cultivars by ISSR DNA marker system for genetic similarity. The highest similarity was found between “AK2” and “AK3” (89.10%) while the lowest similarity was found between ‘Franquette’ and ‘Chandler’ (49.10%).

Keywords ISSR marker · Juglans regia L. · Phenology · Pomology

Phänologische, morphologische und molekulare Charakterisierung vielversprechender Walnuss-Genotypen (Juglans regia L.) in Konya

Schlüsselwörter ISSR Marker · Juglans regia L. · Phänologie · Pomologie

Introduction

The genus Juglans is taxonomically classified into four groups; these are Rhysocaryon (Black walnuts of Amer-ica), Cardiocaryon (Japanese, Manchurian, and Chinese walnuts), Trachycaryon (butter nut of Eastern North Amer-ica) and Juglans, which is comprised of single species “Juglans regia L.” (Khan et al.2010). The Juglans species spread throughout the World. The species mostly grown in South and North America, West Indies, from Southeast-ern Europe to EastSoutheast-ern Asia and Japan (Manning 1978). The Juglans regia L. is the most economically important cultivated species in Juglans genus (Do˘gan et al. 2014; Karimi et al.2014). The world walnut production is about 3.5 million tons per year and China is the biggest walnut

Muzaffer Ipek

mipek@selcuk.edu.tr

1 _{Department of Horticulture, Selçuk University, Konya,}

Turkey

producer with 1.7 million tons per year. The Iran, USA, and Turkey are the other biggest walnut producer with yearly 453,988 tons, 420,000 tons and 190,000 tons re-spectively (Anonymous2013,2015). The Turkey has more than 13 million walnut trees, more than 7 million trees in yielding (Anonymous 2015). The most of these trees in production are non-grafted so they are important for walnut breeding programs and plant breeders. Although seedling population causes non-uniform fruit production for mar-kets, it provides good opportunities for genetic diversity. In this respect, Turkey has a rich genetic potential but nu-merous genotypes possessing fruit characteristics have been destroyed through timber use. This situation has caused a large decrease in valuable germplasm and exhaustion of walnut genotypes. Although Turkey has very rich walnut populations, selection studies related to desirable charac-ters have been poorly conducted. If high yielding and qual-ity fruit phenotypes were selected and then propagated, walnut production could be increased. In order to select promising genotypes, morphologic, isozyme and molecu-lar markers must be used. Morphological markers are few

(2)

and might have epistatic effects. Isozyme markers are also few in number and may be affected by the environment. Therefore, DNA markers which are not affected by the en-vironment, have been suggested for the determination of genetic similarity among the genotypes. Nowadays, DNA-based markers have been used quite extensively because of their advantages over traditional morpho-biochemical markers (Bernatzky et al.1989; Bhattacharya and Ranade

2001; Gepts 1993). DNA markers such as random am-plified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction fragment length polymorphism (RFLP), simple sequence repeat (SSR), sequence-charac-terized amplified regions (SCAR) and amplified fragment length polymorphism (AFLP) have advantages in produc-ing large amounts of markers and can be used for various purposes such as fingerprinting, marker-assisted selection, genetic mapping, etc. (Yıldırım and Kandemir2001). The ISSR markers were used to study the molecular character-ization of selected walnut genotypes and cultivars. ISSR has been the most common marker system used in walnuts. Furthermore, in walnuts, ISSR has been successfully used to study the genetic divergence among selected genotypes and cultivars. These studies proved that molecular markers are useful in delineating the genetic relationships among closely related walnut genotypes and cultivars. Thus, ISSR were used to investigate the genetic relationships among se-lected walnut genotypes and cultivars in the present study.

Material and Methods

Material

The present study was conducted in three provinces (Ak¸se-hir, Bey¸se(Ak¸se-hir, and Hüyük) of Konya in 2010–2013. During survey study, about 2500 walnut genotypes were observed for some walnut characteristics. In 2010 and 2012, fruit samples were collected from 300 walnut genotypes. The se-lected walnut genotypes number were decreased from 300 to 8 genotypes by features of nut and kernel in 2013. Methods

In this study, firstly, walnut genotypes were selected ac-cording to nut weight. About 300 genotypes having nuts which were more than 8.50 g/nut were selected. After se-lection kernel percentage (%) was calculated. The kernel percentage of genotypes which was more than 40% were selected among 300 genotypes. The eight genotypes left at the end of this selection and selection of walnut genotypes was ended. After the selection process, tree characteristics, phenological properties, nut and kernel characteristics and genetic similarity were investigated.

Determination of Walnut Tree Characteristics

The selected walnut trees were investigation in terms of tree characteristics such as tree age (year), tree height (m), diameter of tree crown (m), circumference of trunk (cm), periodicity (absent/present) and susceptibility to anthrac-nose disease (susceptible/tolerant) (Anonymous1999).

Determination of Phenological Characteristics of Genotypes

In walnut trees, time of bud burst, time of leaf bud burst, time of open male flower, time of open female flower, fruit set in the lateral branch, fruit number on a peduncle and time of leaf drop characteristics were observed and saved data (Anonymous1999).

Determination of Nut and Kernel Characteristics

In terms of nut and kernel, nut diameter (mm), shell thick-ness (mm), nut weight (g), kernel weight (g), percentage of kernel (%), shell color, kernel color, kernel removal, shell surface texture, shell hardness and shell seal were studied (Anonymous1999).

Molecular Characterization of Walnut Genotypes

Total genomic DNA was extracted from fully expanded leaves of actively growing shoots, with CTAB methods (Doyle 1991). The leaves of Turkish and foreign walnut cultivars were obtained from the Turkish Fruit Research Institute. The required dilution of the DNA was done by quantification of the DNA on 0.8% agarose gel followed by staining with UViewTM_{loading dye (Bio-Rad) using}λ DNA as a standard.

Seventeen polymorphic ISSR primers were selected for this study based on the results of earlier studies. PCR am-plifications of genomic DNA were conducted according to Zietkiewicz et al. (1994). In the PCR amplification was used 20 µl of reaction mixture containing 75 mM Tris-HCl, 20 mM (NH4)2SO4, 0.1% Tween-20, 2.0 mM MgCl2; 100 µM each of dGTP, dATP, dCTP, and dTTP; 100 pM Primer, 1 U Taq polymerase and 10 ng genomic DNA. The PCR sched-ule followed was 94 ºC for 2 min followed by 40 cycles of 94 ºC for 1 min, 40–60 ºC for 1 min, 72 ºC for 2 min and a fi-nal incubation at 72 ºC for 7 min. The PCR products were separated on a 1.5% agarose gel as mentioned elsewhere (Do˘gan et al.2014).

Only distinct, reproducible, well-resolved fragments were scored as present (1) or absent (0) for each of the ISSR markers. The polymorphic index (PIC) of each marker was calculated using a modified form of the original formula

(3)

Table 1 The tree characteristics of walnut genotypes

Genotypes T.A. T.H. D.T.C. T.C. P S.A. Coordinate

AK-2 15–20 10 8 110 Absent Susceptible 38°2000900N–31°28052700W

AK-3 15–20 10 7 95 Absent Susceptible 38°2000800N–31°28051600W

BE-1 12–15 8 6 97 Absent Susceptible 37°34025300N–31°48042600W

HÜ-5 10–15 8 6 75 Absent Susceptible 37°57090300N–31°33075200W

T.A Tree Age (year), T.H Tree Height (m), D.T.C Diameter of Tree Crown (m), T.D Trunk Circumference (cm), P Periodicity, S.A. Susceptibility

of Anthracnose

PIC = 1 −XP i2

where Pi is the band frequency of the ith allele (Smith et al.

1997). Resolving power of primers was calculated formula RP = ˙I b

Ib = 1 − .2xj0.5 − pj/

where p is the rate of I band in all genotypes (Prevost and Wilkinson1999). Genetic distance estimated by Jac-card coefficient between pairs and dendrogram based on the unweighted pair group method with arithmetical aver-ages (UPGMA) were analyzed using NTSYSpc 2.2 (Rohlf

2000).

Determination of Total Oil Content of Selected Genotypes

The total oil content of selected genotypes was analyzed by Soxhlet extraction method according to a Gravimetric method of AOAC 948.22 (Horwitz2000).

Protein Content of Selected Genotypes

The protein content of genotypes was determined by the Kjeldahl method according to the AOAC 950.48 (Horwitz

2000). The determined nitrogen value was multiplied 5.3 for % protein values of genotypes.

Result and Discussion

Tree Characteristics

The all walnut genotypes’ age ranged from 10 to 70 years. The oldest walnut tree was genotype BE-14 (60–70 years). The tree height of walnut genotypes was measured between 8 and 14 meters. The highest tree height was obtained from BE-14 (14 m). Similarly, the highest diameter of tree crown (18 m) and trunk circumference (210 cm) was measured in

BE-14 genotype. The younger genotypes had a lower diam-eter of tree crown and trunk circumference. The periodicity was not shown in all walnut genotypes. In terms of suscep-tibility to Anthracnose disease, all walnut tree showed to susceptible (Table1).

Phenological Characteristics

The phenological characteristics of genotypes showed vari-ability in three provinces Ak¸sehir, Bey¸sehir and Hüyük. In contrast to nut traits, the phenological traits may be affected by tree age (McGranahan and Forde 1985; Sharma and Sharma2001). The late leafing in walnut is valuable char-acteristic, because of escape the early spring frost. There is a negative correlation between late leafing and lateral bud fruitfulness. The lateral bud fruitfulness is fairly cor-related with early leafing (Germain 1995). The vegetative bud break was between 01 and 17 April. About ten days af-ter bud break, leaf occurred (10–27 April). The genotypes of Bey¸sehir province opened leaf later than Ak¸sehir and Hüyük province. All walnut genotypes showed andromo-noecious characteristics. The male flowers opened at the end of April (24–30 April) except HÜ-5 (15–21 April). The female flower opened a week after male flower. The fruit set in lateral branches rates ranged from 40 to 60%. The lateral bud fruitfulness ratio of ‘Chandler’, ‘Fernor’ and ‘Fernette’ are 80–90%. The Turkish walnut cultivars has lower lateral bud fruitfulness ranged from 20 to 40% (Akça and Tosun 2005). Aslanta¸s (2006) reported that se-lected genotypes from Çoruh Valley had 40–63% lateral bud fruitfulness. All genotypes had two nuts on a peduncle. The leaf drop was observed varied from 01 to 17 November (Table2).

Nut and Kernel Characteristics

Nowadays, the consumer prefers to clean, uniformly light in color, thin shell, easily removable kernel and high kernel rate at least 50% (McGranahan and Leslie2012). However,

(4)

Table 2 The phenological characteristics of walnut genotypes

V. B.B. L.B.B. O.M.F. O.F.F. F.S.L.B. F.N.P. L.F.

AK-2 1–7 April 15–20 April 29–30 April 2–5 May 40 2 12–17 November

AK-3 1–7 April 15–20 April 29–30 April 2–5 May 40 2 12–17 November

BE-1 13–17 April 20–27 April 24–30 April 1–5 May 60 2 1–7 November

BE-5 13–17 April 20–27 April 24-30 April 1–5 May 60 2 1–7 November

HÜ-5 1–7 April 10–15 April 15–21 April 21–27 April 60 2 12–17 November

B.B Vegetative Bud Break, L.B.B Time of Leaf Bud Burst, O.M.F Time of Open Male Flower, O.F.F Time of Open Female Flower, F.S.L.B Fruit

Set in Lateral Branch (%), F.N.P Fruit Number on Peduncle, L.F Time of Leaf Fall

Table 3 Nut and kernel characteristics

N.D. S.T. N.W. K.W. K.P. TOC TPC S.C. K.C. K.R. S.S1 _S.H. _S.S2

AK-2 34.16 1.74 14.09 7.27 51.59 57.08 19.93 Dark Dark

Yellow

Easy Grooved Hard Close

AK-3 33.89 2.13 14.34 6.60 46.03 55.82 21.90 Light Light

Yellow

Easy

Inter-mediate

Hard

Inter-mediate

BE-1 30.51 1.81 13.05 5.24 40.15 57.19 14.55 Light Light

Yellow

Easy

Inter-mediate

Hard Close

BE-3 27.71 2.22 10.89 6.86 62.99 50.78 9.50 Light Light

Yellow

Easy

Inter-mediate

Hard

Inter-mediate

BE-5 27.60 1.87 9.49 4.52 47.62 51.93 21.83 Light Light

Yellow

Easy

Inter-mediate

Hard Close

BE-13 31.62 1.69 9.97 5.03 50.45 58.52 12.74 Light Light

Yellow

BE-14 31.02 1.44 8.70 5.50 63.21 65.72 9.59 Light Light

Yellow Easy Inter-mediate Hard Close HÜ-5 32.21 1.69 11.52 5.74 49.82 57.49 22.35 Dark Dark Yellow

W.D Nut Diameter (mm), S.T Shell Thickness (mm), N.W Nut Weight (g), K.W Kernel Weight (g), K.P Kernel Percentage (%), TOC Total Oil

Content (%), TPC Total Protein Content (%), S.C Shell Color, K.C Kernel Color, K.R Kernel Removal, S.S1_{Shell Surface, S.H Shell Hardiness,}

S.S2_{Shell Seal}

there is an enormous variability in walnut traits e. g., nut sizes (small to very large), shape, shell thickness (very thin to very thick), the degree of shell seal, the color of kernels, and the taste and appearance of kernels (Rashid 1998). Nut and kernel quality is strongly affected by genotype, environment, and their interaction (McGranahan and Leslie

2012). The all genotypes which were observed showed high variability in measured traits (Table1,2and3). The mean of walnut diameter of genotypes were varied from 27.60 to 34.16 mm. The highest walnut diameters were measured in AK-2 while it was the lowest in genotype BE-5. The promising walnut genotypes should have a thin shell. Zhadan and Strukov (1977) reported that shell thick-ness should be between 0.70–1.50 mm. Nenjuhin (1971) suggested the optimum shell thickness of 0.92 mm. The shell thickness ranged from 1.44 to 2.22 mm. The genotype BE-14 (1.44 mm) stayed in Zhadan and Strukov (1977)’ shell thickness values. The shell thickness may affect ker-nel rate like genotype BE-14. The nut weight was found

between 14.34 and 8.70 g. The genotype AK-3 had the highest nut weight and genotype BE-14 was the lowest. In contrast to nut weight, BE-14 had the highest kernel ratio (63.21%). The Turkish cultivar ’ ¸Sebin’ has 9.40 g nut weight and 63% kernel rate. In addition, there are some reports on nut weight which varied from 7.82 g to 18.74 g in Turkey (Akca et al. 2015; Akçay and Tosun 2005; Aslanta¸s2006). The kernel weight of genotypes was mea-sured between 4.52 g and 7.27 g, while kernel percentage ranged from 40.15 to 63.21%. The high kernel percentage is desirable for commercially for processing efficiency and transportation but a high kernel ratio indicates thin shell with weak shell strength (Akca and Ozongun2004). The highest kernel rate was found with 63.21% and this ratio of kernel is closer data reported by Zeneli et al. (2005) (63.80%) and Aslanta¸s (2006) (67.14%) than Cosmulescu and Botu (2012) (71.70%) and Khadivi-Khub et al. (2015) (72.22%). Germain (1995) reported that the genotypes having a higher kernel rate than 50% are desirable for

(5)

wal-Table 4 Level of polymorphism and discriminating capacity of the ISSR primers

Sequence (50–30) B.P. A.T (°C) TBN PBN PR (%) PIC RP

UBC807 AGA GAG AGA GAG AGA GT 17 52 4 0 0.00 0.00 0.00

UBC813 CTC TCT CTC TCT CTC TT 17 50 5 4 80.00 0.33 0.30

UBC814 CTC TCT CTC TCT CTC TA 17 50 5 5 100.00 0.37 0.35

UBC815 CTC TCT CTC TCT CTC TG 17 52 5 3 60.00 0.57 0.19

UBC819 GTG TGT GTG TGT GTG TA 17 50 4 3 75.00 0.87 0.32

UBC834 AGA GAG AGA GAG AGA GYT 18 52 4 2 50.00 0.81 0.38

UBC836 AGA GAG AGA GAG AGA GYA 18 52 5 2 40.00 0.31 0.14

UBC840 GAG AGA GAG AGA GAG AYT 18 52 4 1 25.00 0.96 0.08

UBC844 CTC TCT CTC TCT CTC TRC 18 54 6 6 100.00 0.43 0.43

UBC846 CAC ACA CAC ACA CAC ART 18 54 4 3 75.00 0.47 0.32

UBC851 GTG TGT GTG TGT GTG TYG 18 54 5 5 100.00 0.27 0.30

UBC855 ACA CAC ACA CAC ACA CYT 18 52 6 3 50.00 0.27 0.13

UBC856 ACA CAC ACA CAC ACA CYA 18 52 4 2 50.00 0.28 0.08

UBC860 TGT GTG TGT GTG TGT GRA 18 52 6 4 66.67 0.36 0.23

UBC868 GAA GAA GAA GAA GAA GAA 18 48 6 3 50.00 0.22 0.27

ISSR28 GTG TGT GTG TGT GTG TYA 18 50 3 2 66.67 0.26 0.31

ISSR43 AGA GAG AGA GAG AGA GY 17 50 6 3 50.00 0.28 0.24

Total 82 51 – – –

Means 4.82 3.00 61.08 0.42 0.24

BP Base Pairs, A.T Annealing Temperature, TBN Total Band Number, PBN Polymorphic Band Number, PR Polymorphism Rate, PIC Polymorphism Information Content, RP Resolving Power

nut breeding programs. The four genotypes proved to be promising for a new variety having higher kernel ratio i. e., >50%. The genotype BE-14 had the highest kernel percent-age (63.21%), followed by genotype BE-3 (62.99%), AK-2 (51.59%) and BE-13 (50.45%). According to reports of McGranahan and Leslie (1991) and Akça (1999), the kernel percentages of BE14, BE3, AK2 and BE13 was higher than many international varieties such as ’Payne’ (50%), ’Hart-ley’ (46%), ’Franquette’ (45%), ’Chico’ (46%), ’Sunland’ (57%), ’Vina’ (49%), ‘Chandler’ (50%), ’Pedro’ (46%), ’Cisco’ (46%) and Turkish varieties like ’Kaplan’ (40%), ’Yalova-2’ (50.37%) and ’Bilecik’ (56%). It has been re-ported that fruit characteristics are not affected by tree age (Sharma and Sharma2001). The selected genotypes ages varied from 10 to 70 years old. There are no blank nuts in most of studies genotypes, the present study that included all genotypes having no blank nuts accordance with earlier studies. Shell color showed cohesion with kernel color. Two genotypes (AK-2 and HÜ-5) had dark shell color while the others had light. Similar results were showed in kernel color for these genotypes. The dominant kernel color was light amber in 8 genotypes but AK-2 and HÜ-5 genotypes’ kernel color was amber. The kernel removal of genotypes was determined easy to all genotypes. In generally, the walnut breeders have used kernel removal and kernel color to select promising genotypes in walnut breeding programs (Aslanta¸s 2006; Cosmulescu and Botu 2012; Kazankaya et al.2001; Sharma and Sharma2001; Yarilgac et al.2001;

Zeneli et al. 2005). The shell surface texture was smooth in three genotypes and the other genotypes was interme-diate. The shell seal was close in six genotypes and two had intermediate (Table 3). Generally, thin shell showed poor seal at suture that insect and dirt can easily enter to nuts, sometimes nuts with thick shell can show weak seal. Also, nuts having poor seal easily is broken longitudinally (Khadivi-Khub et al.2015). In addition, the chemical prop-erties of walnuts were investigated. The average oil content was found between 50.78–65.72%. The highest oil content was calculated from genotype “BE-14”, while the lowest was genotype “BE-3”. This result showed to accordance with Simsek (2010) and Akca et al. (2015). The protein content of genotypes varied from 9.50–22.35% (Table 3). The genotype “HÜ-5” had higher protein content than the other genotypes and it was similarly to result of Akca et al. (2015) and Simsek (2010).

DNA Polymorphism Among the Walnut Genotypes All ISSR primers selected for this study generated DNA polymorphism among 8 selected walnut genotypes and 9 walnut cultivars. A total of 82 ISSR markers were gener-ated by the 17 primers; 51 of these markers were polymor-phic, indicating 61.08% DNA polymorphism among the genotypes and cultivars (Table4). The size of the amplified products ranged from 500 to 1500 bp, with 3–6 bands per primer.

(6)

b le 5 Genet ic si m il ari ty am ong th e 9 genot ypes and 8 cul ti v ars of w al nut real ized from ISSR m ark ers 13 1 .0 0 0 –– ––– ––– – – – – – – – – 5 0. 782 1. 000 – – – – – – – – – – – – – – – 14 0. 836 0. 800 1. 000 – – – – – – – – – – – – – – 3 0. 836 0. 727 0. 855 1. 000 – – – – – – – – – – – – – 1 0. 873 0. 764 0. 818 0. 855 1. 000 – – – – – – – – – – – – 3 0. 800 0. 727 0. 782 0. 855 0. 745 1. 000 – – – – – – – – – – – 2 0. 764 0. 764 0. 818 0. 891 0. 818 0. 782 1. 000 – – – – – – – – – – 5 0. 782 0. 745 0. 800 0. 873 0. 836 0. 800 0. 873 1. 000 – – – – – – – – – 0. 855 0. 745 0. 764 0. 800 0. 764 0. 836 0. 727 0. 818 1. 000 – – – – – – – – 0. 782 0. 673 0. 727 0. 764 0. 691 0. 836 0. 691 0. 782 0. 818 1. 000 – – – – – – – O 0. 764 0. 727 0. 709 0. 745 0. 709 0. 782 0. 709 0. 800 0. 836 0. 836 1. 000 – – – – – – ˙ IN 0. 655 0. 691 0. 636 0. 636 0. 673 0. 636 0. 709 0. 691 0. 691 0. 655 0. 745 1. 000 – – – – – ˙ ILE C ˙ IK 0. 673 0. 673 0. 691 0. 691 0. 691 0. 727 0. 727 0. 745 0. 709 0. 709 0. 727 0. 618 1. 000 – – – – 0. 764 0. 727 0. 636 0. 709 0. 709 0. 745 0. 709 0. 727 0. 800 0. 727 0. 782 0. 782 0. 727 1. 000 – – – U ETTE’ 0. 636 0. 600 0. 618 0. 618 0. 582 0. 618 0. 582 0. 564 0. 600 0. 600 0. 618 0. 691 0. 564 0. 691 1. 000 – – H A N -0. 636 0. 636 0. 655 0. 618 0. 618 0. 727 0. 618 0. 600 0. 673 0. 636 0. 618 0. 618 0. 709 0. 618 0. 491 1. 000 – ¸S 0. 618 0. 545 0. 600 0. 673 0. 636 0. 636 0. 636 0. 655 0. 618 0. 618 0. 600 0. 600 0. 582 0. 636 0. 655 0. 618 1. 000 BE-13 HÜ-5 BE-14 AK-3 BE-1 BE-3 A K-2 BE-5 K AMAN ‘FERNOR’ PEDR O ¸SEB ˙ IN B ˙ ILE C ˙ IK ¸SEN-2 ‘FRAN- QU E T T E ’ ‘C H A N -DLER’ MARA ¸S -18

(7)

Genetic Similarity Among the Walnut Genotypes Genetic similarity was estimated from the ISSR markers by following the methods of Jaccard. The maximum genetic similarity (0.891) was recorded between AK-2 and AK-3 while the minimum (0.491) was found between ‘Chandler’ and ‘Franquette’. Further, the average genetic similarity was calculated as 0.688 (Table5). The similarity matrix based on ISSR datasets is graphically represented as a dendrogram using the UPGMA method shown in Fig.1. The dendro-gram of ISSR grouped the 8 walnut genotypes and 9 culti-vars into six major clusters, showing great genetic diversity among them. The genetic similarity coefficients revealed a substantial amount of genetic similarity among the walnut genotypes, though the genotypes were collected from the different region of varied climatic conditions. Genotypes from the same geographic regions show closer genetic sim-ilarity than those from geographically distant regions, but we found closer relatedness between BE-5 and AK-2 with AK-3. This situation could have been occurred due to seed transferred between two provinces. All genotypes were sep-arated from all cultivars on dendrogram (Fig.1).

Fig. 1 ISSR dendrogram derived from UPGMA cluster analysis

Conclusion

The Turkey is one of the most important centers of rich wal-nut germplasm. This rich walwal-nut genetic resource formed by seed propagation. The walnut seedlings showed diversity in the natural population. Because of diversity, walnut has unisexual flowers and is a cross pollinated. The gene flow increases the chance of gene recombination and genetic diversity. The present research was conducted in Ak¸sehir, Bey¸sehir and Hüyük province where has highly diversified walnut population formed a seedling. The eight promis-ing walnut genotypes were selected by some phenological, morphological and tree characteristics in these provinces. These genotypes can help to improve economically and sustainable walnut production.

Conflict of interest Each named author has substantially contributed

to conducting the underlying research and drafting this manuscript and the named authors have no conflict of interest.

References

Akça Y (1999) A study on the determination of performance of some walnut cultivars in ecological condition of Tokat (1997–1998 di-vision). In: 3rd Nat Hort Plant Congress Turkey, pp 14–17 Akça Y, Ozongun S (2004) Selection of late leafing, late flowering,

lat-erally fruitful walnut (Juglans regia) types in Turkey. N Z J Crop Hortic Sci 32:337–342

Akça Y, Bilgen Y, Ercisli S (2015) Selection of superior persian walnut (Juglans regia L.) from seedling origin in Turkey. Acta Sci Pol Hortorum Cultus 14:103–114

Akçay ME, Tosun ˙I (2005) Bursa ili III. alt bölgesinde (Gemlik, Orhangazi, ˙Iznik ve Mudanya) yeti¸stirilen ceviz tiplerinin selek-siyonu. Bahçe Ceviz 34:57–62

Anonymous (1999) Guidelines For The Conduct Of Tests For Distinct-ness, Uniformity And Stability in Walnut. UPOV, TG/125/6, Gen-ova

Anonymous (2013) http://faostat3.fao.org/home/E (Access to Date

11/10/2016)

Anonymous (2015) https://biruni.tuik.gov.tr/bitkiselapp/bitkisel.zul

(Access to Date 11/10/2016)

Aslanta¸s R (2006) Identification of superior walnut (Juglans regia) genotypes in north-eastern Anatolia, Turkey. N Z J Crop Hortic Sci 34:231–237

Bernatzky R, Tanksley S, Brown A, Frankel O, Marshall D, Williams J (1989) Restriction fragments as molecular markers for germplasm evaluation and utilization. In: The use of plant genetic resources, pp 353–362

Bhattacharya E, Ranade SA (2001) Molecular distinction amongst va-rieties of mulberry using RAPD and DAMD profiles BMC. Plant Biol 1:1

Cosmulescu S, Botu M (2012) Walnut biodiversity in south-western Romania resource for perspective cultivars. Pak J Bot 44:307–311 Doyle J (1991) DNA protocols for plants. In: Molecular techniques in

taxonomy. Springer, Berlin Heidelberg, pp 283–293

Do˘gan Y, Kafkas S, Sütyemez M, Akça Y, Türemi¸s N (2014) As-sessment and characterization of genetic relationships of walnut (Juglans regia L.) genotypes by three types of molecular markers. Sci Hortic 168:81–87

Gepts P (1993) The use of molecular and biochemical markers in crop evolution studies. In: Evolutionary biology. Springer, Berlin Hei-delberg, pp 51–94

(8)

Germain E (1995) Genetic improvement of the Persian walnut (Juglans regia L.). In: III International Walnut Congress 442, pp 21–32 Horwitz W (2000) Fat (crude) in nuts and nut products.

Gravimet-ric method (AOAC Official Method 948.22) Officials methods of analysis of AOAC International, 17th edn. AOAC International, Gaithersburg, MD.

Karimi R, Ershadi A, Nia AE, Sharifani M, Rasouli M, Ebrahimi A, Vahdati K (2014) Morphological and molecular evaluation of per-sian walnut populations in northern and western regions of Iran. J Nuts 5:21–31

Kazankaya A, Koyuncu MA, Koyuncu F, Yarilgac T, Sen SM (2001) Some nut properties of walnuts (Juglans regia L.) of edremit coun-try. In: International Society for Horticultural Science (ISHS)

Leuven, pp 97–100 https://doi.org/10.17660/ActaHortic.2001.

544.11

Khadivi-Khub A, Ebrahimi A, Mohammadi A, Kari A (2015) Char-acterization and selection of walnut (Juglans regia L.) genotypes from seedling origin trees. Tree Genet Genomes 11:54

Khan MW, Khan IA, Ahmad H (2010) Estimation of genetic diversity in walnut. Pak J Bot 42:1791–1796

Manning WE (1978) The classification within the Juglandaceae. Ann Missouri Bot Gard. 65(4):1058–1087

McGranahan G, Forde H (1985) Relationship between clone age and selection trait expression in mature walnuts. J Am Soc Hortic Sci 110:692–696

McGranahan G, Leslie C (1991) Walnuts (Juglans). In: International Society for Horticultural Science (ISHS) Leuven, pp 907–974 https://doi.org/10.17660/ActaHortic.1991.290.20

McGranahan G, Leslie C (2012) Walnut. In: Fruit breeding. Springer, Berlin Heidelberg, pp 827–846

Nenjuhin V (1971) Selection of plus trees of the walnut in Ukraine. Plant Breed Abstr 41(1):187

Officials methods of analysis of AOAC International, 17th edn AOAC International, Gaithersburg, MD

Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98(1):107–112

Rashid A (1998) Temperate Fruit Genetic Diversity in Pakistan. In: World conference on horticulture research

Rohlf F (2000) NTSYS-pc, Numerical taxonomy and multivariate analysis system, version 2.2. Exeter Publishing, Setauket Sharma O, Sharma S (2001) Genetic divergence in seedling trees of

Persian walnut (Juglans regia L.) for various metric nut and kernel characters in Himachal Pradesh. Sci Hortic 88:163–171 Simsek M (2010) Selection of walnut types with high fruit bearing and

quality in Sanliurfa population. Int J Phys Sci 5:992–996 Smith J et al (1997) An evaluation of the utility of SSR loci as

molec-ular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theor Appl Genet 95:163–173

Yarilgac T, Koyuncu F, Koyuncu MA, Kazankaya A, Sen SM (2001) Some promising walnut selections (Juglans regia L.). In: Interna-tional Society for Horticultural Science (ISHS) Leuven, pp 93–96 https://doi.org/10.17660/ActaHortic.2001.544.10

Yıldırım A, Kandemir N (2001) Genetic markers and analysis meth-ods. Plant biotechnology-genetic engineering applications Selcuk Universitesi Vakfı Yayınları (in Turkish)

Zeneli G, Kola H, Dida M (2005) Phenotypic variation in native walnut populations of Northern Albania. Sci Hortic 105:91–100 Zhadan V, Strukov M (1977) Breeding walnut for fruit size. Plant

Breed Abstr 11:10805

Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reac-tion amplificareac-tion. Genomics 20:176–183