Genetic Diversity Based on ISSR Markers of Apple Genotypes in Ardahan , Turkey

Within the present study, it was conducted a genetic diversity analysis using ISSR markers for some apple genotypes grown in Ardahan region, Turkey. Total genomic DNA (gDNA) isolation from apple leaves was performed using commercial kits. Five ISSR primers were used to determine the genetic diversity among the genotypes studied. Polymerase Chain Reaction (PCR) was performed with all gDNA samples to produce bands to score. PCR products were run in agarose gel and visualized under UV light. Bands on the gels were scored as “1”, while no bands at the corresponding positions were scored as “0”, to generate the matrix file. Five ISSR primers produced a total of 35 bands, and 21 of them were polymorphic. The polymorphic bands rated approximately 60%. Phylogenetic relationships and genetic distances between the genotypes were calculated by using the PAUP [Phylogenetic Analysis Using Parsimony (and Other Methods)] program. According to the PAUP data, the closest genetic distance was 0.03704 between ‘Kaburga’ and ‘Japon Apple’ genotypes, while the furthest genetic distance was 0.48148 between ‘Karanfil Apple’ and ‘Sisli Uruset’. The phylogenetic analysis obtained using UPGMA algorithm produced a phylogenetic tree with two clades. The results suggest that ISSR markers are useful tools for determining genetic relationships among apple genotypes.


Introduction
Rosaceae is one of the most diversified and large plant families, including economically important fruit trees.This family consists of more than 100 genera and 3,000 species and it is the third most important plant family in terms of economic significance in mild climate regions (Zarei et al., 2017).The family, which generally includes taxa in tree and bush forms, also comprises herbaceous taxa.The family members, which have cosmopolite characteristics, are mostly spread in the Northern hemisphere (Serdar et al., 2014).
Apple (Malus domestica) of the Rosaceae family is one of the most important cultivated fruit trees in the mild climate regions of the world (Mahmood et al., 2016).Apple has been reported to be distributed to different gene centers in the world, primarily Europe, Anatolia, Himalaya, China, Japan, Korea and North America with its 48 species (Dziubiak, 2004;Ercişli, 2004).Turkey is one of the most important producers of apples, with an estimated production of 2,925,828 tons in 2016 (Fao, 2016).There are over 460 local apple genotypes in Turkey, all with different qualities (Ertürk and Akçay, 2010).Apple is one of the most important fruit species, commonly preferred by consumers due to its taste, nutritional content and economic value; its nutritive components include dietary fiber, as well as rich antioxidant active ingredients, carbohydrates and essential minerals (Wiseman, 2001;Sadik et al., 2003).It is a good source of phenolics and antioxidants for humans (Wolfe et al., 2003;Wolfe and Liu, 2003;Abaci and Sevindik, 2014).
Molecular marker methods are based on the principle of determining polymorphic regions in DNA molecules.Molecular markers are commonly used with the purpose of detecting genetic polymorphism, genetic identification, hybrid plant identification for hybridization development, genetic mapping and marker-assisted selection (Jiang, 2013;Sesli and Yegenoglu, 2017).One of the techniques is ISSR (Inter Simple Sequence Repeat) which offers excellent means to investigate genetic diversity of plants (Arslan and   Tamkoç, 2011 the ISSR technique is frequently used for determining the genetic diversity of apple genotypes ( 2001;Korbin 2016).Development of the ISSR technique rapid use of the organisms in the studies of genetic variability.The ISSR is based on the PRC amplification of DNA fragments between two reversed, simple sequential recurrence regions of expandable distances (Zietkiewicz al., 1994).
In the present diversity using ISSR markers for some apple genotypes grown in the Ardahan region of Turkey

Materials and M
Plant samples and genomic DNA isolation Leaf samples of apple genotypes used in the st collected from certain regions in Ardahan (Turkey) between July and August, 2015 (Fig. 1) DNA (gDNA) samples were extracted using DNeasy Plant Mini Kit (GeneMark).The gDNA samples were stored at 20 °C when not at use.

Plant samples and genomic DNA isolation
Leaf samples of apple genotypes used in the st collected from certain regions in Ardahan (Turkey) between July and August, 2015 (Fig. 1) DNA (gDNA) samples were extracted using DNeasy Plant Mini Kit (GeneMark).The gDNA samples were stored at 20 °C when not at use.

Fig. 1. Location of Ardahan Province
Sevindik Deif et al., 2013).In the present day, the ISSR technique is frequently used for determining the genetic diversity of apple genotypes (Goulao and Oliveira, 2002;He et al., 2011; Development of the ISSR technique rapid use of the organisms in the studies of genetic variability.The ISSR is based on the PRC amplification of DNA fragments between two reversed, simple sequential recurrence regions of expandable distances (Zietkiewicz study, it was investigat diversity using ISSR markers for some apple genotypes grown in the Ardahan region of Turkey.

Plant samples and genomic DNA isolation
Leaf samples of apple genotypes used in the st collected from certain regions in Ardahan (Turkey) between July and August, 2015 (Fig. 1) DNA (gDNA) samples were extracted using DNeasy Plant Mini Kit (GeneMark).The gDNA samples were stored at  ).In the present day, the ISSR technique is frequently used for determining the Goulao and Oliveira, 2011; Uzun Development of the ISSR technique has ensured the rapid use of the organisms in the studies of genetic variability.The ISSR is based on the PRC amplification of DNA fragments between two reversed, simple sequential recurrence regions of expandable distances (Zietkiewicz investigated the genetic diversity using ISSR markers for some apple genotypes Plant samples and genomic DNA isolation Leaf samples of apple genotypes used in the study were collected from certain regions in Ardahan (Turkey) between July and August, 2015 (Fig. 1).Total genomic DNA (gDNA) samples were extracted using DNeasy Plant Mini Kit (GeneMark).The gDNA samples were stored at PCR reactions and their Tm degrees

Data analysis
The pictures were used for the evaluation of the results in the analysis formed at the end of the amplification, only the bright bands were taken into consideration.
The presence (1) and absence (0) of bands were specified to construct the data matrix.PAUP [Phylogenetic Analysis Using (Swofford, 2002) was used to perform phylogenetic analyses using ISSR data.
).In the present day, the ISSR technique is frequently used for determining the Goulao and Oliveira, Uzun et al., has ensured the rapid use of the organisms in the studies of genetic variability.The ISSR is based on the PRC amplification of DNA fragments between two reversed, simple sequential recurrence regions of expandable distances (Zietkiewicz et genetic diversity using ISSR markers for some apple genotypes udy were collected from certain regions in Ardahan (Turkey) Total genomic DNA (gDNA) samples were extracted using DNeasy Plant Mini Kit (GeneMark).The gDNA samples were stored at -

Data analysis
The pictures were used for the evaluation of the results in the analysis of ISSR formed at the end of the amplification, only the bright bands were taken into consideration.
The presence (1) and absence (0) of bands were specified to construct the data matrix.PAUP [Phylogenetic Analysis Using Parsimony (and Other Methods)] Version 4.0b10 (Swofford, 2002) was used to perform phylogenetic analyses using ISSR data.
The pictures were used for the evaluation of the results of ISSR-PCR.In the reading of the bands formed at the end of the amplification, only the bright bands were taken into consideration.
The presence (1) and absence (0) of bands were specified to construct the data matrix.PAUP [Phylogenetic Analysis Parsimony (and Other Methods)] Version 4.0b10 (Swofford, 2002) was used to perform phylogenetic analyses In order to visualize gDNA samples, 0.8% standard agarose gel electrophoresis procedure was performed.For PCR amplification, five ISSR primers were used (Table 1).ISSR amplification reactions were carried out in 25 µL volume containing 5 µL master mix (PCR buffer, NTP, Taq DNA polymerase), 1 µL 2.0 µL gDNA (around 10 ng/µL) and 17 µL of ddH PCR cycles with their respective Amplification products were analyzed electrophoresis on 0.8% agarose gels buffered with 0.5X EDTA), stained with ethidium bromide and pictured under ultraviolet light (Figs. 2 and 3).
The pictures were used for the evaluation of the results PCR.In the reading of the bands formed at the end of the amplification, only the bright bands were taken into consideration.
th their respective Amplification products were analyzed by electrophoresis on 0.8% agarose gels buffered with 0.5X EDTA), stained with ethidium bromide and pictured under ultraviolet light (Figs. 2 and 3).
The pictures were used for the evaluation of the results PCR.In the reading of the bands formed at the end of the amplification, only the bright The presence (1) and absence (0) of bands were specified to construct the data matrix.PAUP [Phylogenetic Analysis Parsimony (and Other Methods)] Version 4.0b10 (Swofford, 2002)  In order to visualize gDNA samples, 0.8% standard agarose gel electrophoresis procedure was performed.For PCR amplification, five ISSR primers were used (Table 1).ISSR amplification reactions were carried out in master mix (PCR buffer, ISSR primers, th their respective by electrophoresis on 0.8% agarose gels buffered with 0.5X EDTA), stained with ethidium bromide The pictures were used for the evaluation of the results PCR.In the reading of the bands formed at the end of the amplification, only the bright The presence (1) and absence (0) of bands were specified to construct the data matrix.PAUP [Phylogenetic Analysis Parsimony (and Other Methods)] Version 4.0b10 (Swofford, 2002) was used to perform phylogenetic analyses 555

Results and Discussion
In the ISSR detected, among which polymorphism rate was 60 program was used to calculate the phylogenetic trees and genetic distances between populations PAUP data, the closest genetic distance was 0.03704 between 'Kaburga Apple furthest genetic distance was 0 Apple' and 'Sisli Uruset The phylogenetic tree was obtained using the UPGMA algorithm, and the tree was consisted of two clades (Fig. 4

Results and Discussion
In the ISSR-PCR analysis, a detected, among which 21 bands were polymorphic lymorphism rate was 60 program was used to calculate the phylogenetic trees and genetic distances between populations PAUP data, the closest genetic distance was 0.03704 Kaburga Apple' and furthest genetic distance was 0 Sisli Uruset' (Table 3) The phylogenetic tree was obtained using the UPGMA algorithm, and the tree was consisted of two clades (Fig. 4 Sevindik PCR analysis, a total of 35 bands were were polymorphic lymorphism rate was 60%.PAUP 4.0b10 analysis program was used to calculate the phylogenetic trees and genetic distances between populations.PAUP data, the closest genetic distance was 0.03704 and 'Japon' genotypes furthest genetic distance was 0.48148 between (Table 3).The phylogenetic tree was obtained using the UPGMA algorithm, and the tree was consisted of two clades (Fig. 4 .According to the PAUP data, the closest genetic distance was 0.03704 genotypes, while the 48148 between 'Karanfil The phylogenetic tree was obtained using the UPGMA algorithm, and the tree was consisted of two clades (Fig. 4) Group A consisted Kaba Apple' and 'Limon Sarı Safran' and 'Yaz Apple and' Japon Apple Şah Apple' and Paşa Apple' and 'Karpuz Karanfil Apple' (Fig. 4) .( 2016) used ISSR markers to detect Turkish apple genotypes and their relationships with some foreign sequence distances among some apple genotypes for ISSR According to the PAUP data, the closest genetic distance was 0.03704 while the Karanfil The phylogenetic tree was obtained using the UPGMA algorithm, and the tree was consisted of two clades (Fig. 4).
. ( 2016) used ISSR markers to detect Turkish apple genotypes and their relationships with some foreign genera and species.In their s 'Yaz' group the present study, the apple genotypes named 'Şah' Apple genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, apple genotypes named 'Kaburga four genotypes took place in Clade 1 according to the UPGMA dendrogram obtained via ISSR data in the pr study (Fig. 4).Osmanoğlu 'Uruset apples in another group.In the present study, on the other hand, detected in Clade 1.The results were found partially similar.Daler among six apple varieties cultivated in our country using RAPD markers.10 RAPD primers produced 47 polymorphic bands.analysis through using RAPD markers.Fig. 3.
Not Sci Biol, 2018, 10(4):554-558 genera and species.In their s ', 'Karanfil', 'Paşa group, while 'Kaba the present study, the apple genotypes named ' and 'Kaba' were detected in Clade 1 Apple' was found in Clade 2. Osmanoğlu genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, apple genotypes named Kaburga' and 'Sobe four genotypes took place in Clade 1 according to the UPGMA dendrogram obtained via ISSR data in the pr study (Fig. 4).Osmanoğlu Uruset' apples in a group apples in another group.In the present study, on the other hand, 'Paşa' and detected in Clade 1.The results were found partially similar.Daler et al. (2017) have studied the genetic relationships among six apple varieties cultivated in our country using RAPD markers.10 RAPD primers produced 47 polymorphic bands.analysis of apple genotypes collected from Van p through using RAPD markers.Fig. 3. ISSR-PCR gel photo amplified with 558 genera and species.In their study, apple genotypes named Paşa' and 'Şah Kaba' apple was found in a different group.In the present study, the apple genotypes named were detected in Clade 1 was found in Clade 2. Osmanoğlu genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, apple genotypes named 'Kırmızı Safran obe' were found in the same group.These four genotypes took place in Clade 1 according to the UPGMA dendrogram obtained via ISSR data in the pr study (Fig. 4).Osmanoğlu ( 2008) detected apples in a group, while found apples in another group.In the present study, on the other and 'Kaba', 'Uruset detected in Clade 1.The results were found partially similar.
. ( 2017) have studied the genetic relationships among six apple varieties cultivated in our country using RAPD markers.10 RAPD primers produced 47 polymorphic bands.Kaya et al. (2015) carried out molecular pple genotypes collected from Van p through using RAPD markers.PCR gel photo amplified with tudy, apple genotypes named Şah' were found in the same apple was found in a different group.In the present study, the apple genotypes named were detected in Clade 1, while was found in Clade 2. Osmanoğlu ( 2008) revealed genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, Kırmızı Safran', were found in the same group.These four genotypes took place in Clade 1 according to the UPGMA dendrogram obtained via ISSR data in the pr (2008) detected while found 'Yaz apples in another group.In the present study, on the other Uruset' and 'Yaz detected in Clade 1.The results were found partially similar.
. ( 2017) have studied the genetic relationships among six apple varieties cultivated in our country using RAPD markers.10 RAPD primers produced 47 .( 2015 PCR gel photo amplified with UBC tudy, apple genotypes named were found in the same apple was found in a different group.In the present study, the apple genotypes named 'Paşa', 'Yaz while 'Karanfil ( 2008) revealed genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, , 'Sarı Safran were found in the same group.These four genotypes took place in Clade 1 according to the UPGMA dendrogram obtained via ISSR data in the present ( 2008) detected 'Paşa' and Yaz' and 'Kaba apples in another group.In the present study, on the other Yaz' apples were detected in Clade 1.The results were found partially similar.
. ( 2017) have studied the genetic relationships among six apple varieties cultivated in our country using RAPD markers.10 RAPD primers produced 47 .( 2015) carried out molecular pple genotypes collected from Van province,

UBC-830
tudy, apple genotypes named were found in the same apple was found in a different group.In Yaz', Karanfil (2008) revealed genetic relationships of apple genotypes collected from Ardahan/Posof region using RAPD markers.In their study, Sarı Safran', were found in the same group.These four genotypes took place in Clade 1 according to the esent and ba' apples in another group.In the present study, on the other apples were detected in Clade 1.The results were found partially similar.

Conclusions
The study reports the genetic relationships between 14 apple genotypes distributed in Ardahan province by using five ISSR primers.Genetic distance and phylogenetic relationship among the target populations were detected based on ISSR data, and the rel the phylogenetic tree constructed.The obtained results will be useful to serve plant breeding programs by means of revealing inter great importance in breeding studies also aimed to offer information about the relationships between different populations cultivated in the region.Undoubtedly, more data including more taxa (e.g.morphological and/or DNA sequence data) will improve this conclusion and yi Fig. 4. The UPGMA tree generated using ISSR data of apple genotypes Creating a dendrogram, the similarity index between genotypes was revealed.Molecular techniques including SSR (Hokanson et al., 1998;Kenis and Keulemans, 2005), AFLP (Kenis and Keulemans, 2005), nrDNA ITS, cpDNA (Robinson et al et al., 1995), promoter region of et al., 2016), taxonomic status, genetic diversity and phylogenetic analyses among apple species and genotypes.study reports the genetic relationships between 14 apple genotypes distributed in Ardahan province by using five ISSR primers.Genetic distance and phylogenetic relationship among the target populations were detected based on ISSR data, and the rel the phylogenetic tree constructed.The obtained results will be useful to serve plant breeding programs by means of revealing inter-species genetic relationships which have a great importance in breeding studies also aimed to offer information about the relationships between different populations cultivated in the region.Undoubtedly, more data including more taxa (e.g.morphological and/or DNA sequence data) will improve this conclusion and yield more reliable results.Molecular techniques including 1998; Kenis and Keulemans, 2005), AFLP (Kenis and Keulemans, 2005), nrDNA ITS, cpDNA et al., 2001), cpDNA ., 1995), promoter region of have been used to determine taxonomic status, genetic diversity and phylogenetic analyses among apple species and genotypes.study reports the genetic relationships between 14 apple genotypes distributed in Ardahan province by using five ISSR primers.Genetic distance and phylogenetic relationship among the target populations were detected based on ISSR data, and the relationships were visualized on the phylogenetic tree constructed.The obtained results will be useful to serve plant breeding programs by means of species genetic relationships which have a great importance in breeding studies.Furthermore, it was also aimed to offer information about the relationships between different populations cultivated in the region.Undoubtedly, more data including morphological and/or DNA sequence data) will improve eld more reliable results.dendrogram, the similarity index between Molecular techniques including 1998; Kenis and Keulemans, 2005), AFLP (Kenis and Keulemans, 2005), nrDNA ITS, cpDNA cpDNA atpB ., 1995), promoter region of atpB gene have been used to determine taxonomic status, genetic diversity and phylogenetic analyses among apple species and genotypes.study reports the genetic relationships between 14 apple genotypes distributed in Ardahan province by using five ISSR primers.Genetic distance and phylogenetic relationship among the target populations were detected ationships were visualized on the phylogenetic tree constructed.The obtained results will be useful to serve plant breeding programs by means of species genetic relationships which have a Furthermore, it was also aimed to offer information about the relationships between different populations cultivated in the region.Undoubtedly, more data including more taxa (e.g.morphological and/or DNA sequence data) will improve eld more reliable results.dendrogram, the similarity index between Molecular techniques including 1998; Kenis and Keulemans, 2005), AFLP (Kenis and Keulemans, 2005), nrDNA ITS, cpDNA B-rbcL B gene have been used to determine taxonomic status, genetic diversity and phylogenetic study reports the genetic relationships between 14 apple genotypes distributed in Ardahan province by using five ISSR primers.Genetic distance and phylogenetic relationship among the target populations were detected ationships were visualized on the phylogenetic tree constructed.The obtained results will be useful to serve plant breeding programs by means of species genetic relationships which have a Furthermore, it was also aimed to offer information about the relationships between different populations cultivated in the region.Undoubtedly, more data including more taxa (e.g.

Fig. 1 .
Fig. 1.Location of Ardahan Province, Turkey was used to perform phylogenetic analyses 555

Fig. 2 .
Fig. 2. ISSR-PCR gel ) Clade 1 was divided into five groups , 'Kırmızı Safran , group B consists of group C consist of 'Kaburga Apple group D consist of 'Uruset Apple and group E consist of Clade 2 consisted of only et al. (2016) used ISSR markers to detect Turkish apple genotypes and their relationships with some foreignTable 3. Pairwise sequence distances among some a photo amplified with UBC photo amplified with UBC-826

Fig. 4 .
Fig. 4. The UPGMA tree generated using ISSR data of apple genotypes

Fig. 4 .
Fig. 4. The UPGMA tree generated using ISSR data of apple genotypes Fig. 4. The UPGMA tree generated using ISSR data of apple genotypes

Table 1 .
Primers used in the ISSR

Table 1 .
Primers used in the ISSR

Table 2 .
Cycles and conditions of ISSR PreFig. 1. Location of Ardahan Province

Table 1 .
Primers used in the ISSR

Table 2 .
Cycles and conditions of ISSR

Table 1 .
Primers used in the ISSR-PCR reactions and their Tm degrees

Table 2 .
Cycles and conditions of ISSR-PCR reactions

Table 3 .
Pairwise ) carried out molecular pple genotypes collected from Van p