Art16_Ercisli.indd


Journal of Applied Botany and Food Quality 85, 229 - 233 (2012)

1Ministry of Food, Agriculture and Livestock Atatürk Orman Çiftliği Ankara, Turkey
2Department of Horticulture, Ondokuz Mayis University, Samsun-Turkey

3Department of Horticulture, Ataturk University, Erzurum-Turkey
4Ankara University Biotechnology Institute,  Ankara-Turkey

Simple sequence repeat (SSR) analysis for assessment of genetic variability 
in wild cherry germplasm

Z. Turkoglu1,  S. Bilgener2,  S. Ercisli3*,  N. Yildirim4

(Received October 22, 2012)

* Corresponding author

Summary

Conservation of genetic resources is vital for future breeding 
programs and food security for humans. Before conservation of 
genetic resources, it requires objective characterization and a proper 
assignation of individual genotypes to species. The aim of this 
study was to characterize of 58 Prunus accessions which belong to 
Prunus avium, Prunus cerasus and Prunus mahaleb by using 12 SSR 
markers. All twelve SSR markers produced successful amplifi cations 
and revealed DNA polymorphisms. The number of allele per loci 
varied  from 6 (UDP96-019) to 12 (PS12A02) with an average of 
9 per allele. The average of observed and expected heterozygosity 
was found to be 0.609 and 0.720. The allele size varied from 95 to 
276 bp. The number of genotypes per allele were 7 (UCD-CH13) and 
24 (UDP96-005). Genetic distance analysis based on SSRs divided 
the cherry accessions in three main groups based mainly on their 
species characteristics. P. cerasus genotypes had higher similarity 
ratio within species than P. mahaleb and P. avium. 

Introduction

Sweet cherry, sour cherry and mahaleb belongs to Rosaceae family,  
Prunoideae subfamily, Prunus genus and Cerasus subgenus. 
Cerasus is further divided into four groups including Eucerasus, 
Microcerasus, Pseudocerasus and Mahaleb. Sweet cherries (Prunus 
avium L.) and sour cherries (Prunus cerasus L.) are placed in 
Eucerasus group while mahleb (Prunus mahaleb L.) is placed in 
Mahaleb group (ERCISLI, 2004).
Asia minor in Turkey is one of the origins and domestication centers 
for P. avium, P. cerasus and P. mahaleb (OZCAGIRAN et al., 2005). 
P. avium is originated in the area between the Black and Caspian 
seas of Asia minor. Great morphological variation exists among 
P. avium, P.mahaleb and P. cerasus accessions naturally grown as 
wild in Turkey (ERCISLI, 2004). This continuum of morphological 
characteristics makes species assignation diffi cult when considering 
only phenotypic traits. In Turkey Prunus mahaleb, wild Prunus 
avium and Prunus cerasus seedlings commonly have been used as 
rootstocks for both sweet and sour cherry cultivars (ERCISLI et al., 
2011).
Conservation of genetic resources is vital for future breeding pro-
grams and food security for humans. Before conservation of genetic 
resources,  objective characterization and a proper assignation of 
individual genotypes to species is required (KARP et al., 1997). 
Among the different marker systems (Morphological, Biochemical 
and Molecular), molecular markers supply more reliable tools to 
analyze genetic diversity in plant species (KARP et al., 1997). They 
could be helpful by giving an accurate and unambiguous assignation 
of each genotype to a particular species (SZIKRISZT et al., 2011). 
Simple sequence repeats (SSRs or microsatellites) have become the 
genetic markers of choice in many plant species because they are 
PCR-based, highly reproducible, polymorphic, generally codominant 
and abundant in plant genomes (POWELL et al., 1996). SSR loci can 

be identifi ed and their alleles recognized in different genotypes of the 
same species because of  their codominant and usually single-locus 
nature, and often in those of other close relatives. In other words 
a specifi c set of SSRs can be used in different sets of genotypes, 
making them particularly useful for fi ngerprinting. In general, SSRs 
are more transferable between species of the same genus, or between 
closely related genera, than between distant genera of the same 
family (PEAKALL et al., 1998; ZHANG et al., 2005; HENDRE et al., 
2008; LURO et al., 2008). 
Previously SSR (Simple Sequence Repeats) markers have been 
successfully used on genotypes belong to different Prunus genus in 
diversity studies (CHENG and HUANG, 2009; GUARINO et al., 2009; 
LACIS et al., 2009; GULEN et al., 2010; NAS et al., 2011). However, 
the majority of these studies have dealt with cultivars and little 
emphasis has been paid to wild relatives. This study has therefore 
sought to document indigenous knowledge related to uses of wild 
grown genotypes which belong to P. avium, P. cerasus and P. ma-
haleb. 
 

Materials and methods

Plant material
For SSR and genetic relationship studies, 37 Prunus avium, 8 Prunus 
cerasus and 7 Prunus mahaleb rootstock candidates together with 
well-known standard rootstocks of each species, F12/1 (Prunus 
avium L.), Montmorency (Prunus cerasus) and SL 64 (Prunus 
mahaleb L.) were used. These genotypes have been previously 
selected from wild cherry populations as rootstock candidates in 
Ordu region in Turkey. All genotypes are maintained in a germplasm 
collection at the Black Sea Agricultural Research Center in Samsun, 
Turkey. 
      

DNA extraction
Genomic DNA was extracted from young leaf tissue using the 
Wizard® Genomic DNA Purifi cation Kit (Promega, Madison, 
WI) according to the instructions provided by the manufacturer. 
Subsequently, an RNAse treatment was performed on the eluted 
DNA samples. Purity and concentration of the DNA were checked 
both on 1% (w/v) agarose gels and by NanoDrop® ND-1000 
Spectrophotometer. 

SSR analysis
From an initial screening, 12 SSRs were selected to check for 
polymorphism by capillary electrophoresis in 55 genotypes of three 
different Prunus species (Tab. 1). Polymerase Chain Reaction 
(PCR) was conducted in a volume of 10 µL and contained 15 ng 
genomic DNA, 5 pmol of each primer, 0.5 mM dNTP, 0.5 unit 
GoTaq DNA polymerase (Promega), 1.5 mM MgCl2

 
and 2 µL 

5X buffer. The forward primers were “labelled” with WellRED 
fl uorescent dyes D2 (black), D3 (green) and D4 (blue) (Pro ligo, 
Paris, France). Reactions without DNA were included as negative 



230 Z. Turkoglu,  S. Bilgener,  S. Ercisli,  N. Yildirim Molecular characterization of wild cherry germplasm by SSR

controls. PCR amplifi cation was performed using the Biometra® 
PCR System. The amplifi cation condi tions consisted of an initial 
denaturation step of 3 min at 94°C, followed by 35 cycles of 1 min 
at 94°C, 1 min at 52-56°C and 2 mins at 72°C with a fi nal extension 
at 72°C for 10 mins. The PCR products were fi rst separated on a 3% 
(w/v) agarose gel run at 80 V for 2 hrs. The gel was then stained with 
ethidium bromide at a concentration of 10 mg/mL. A DNA ladder 
(100 bp) (Promega) was used for the approximate quantifi cation of 
the bands. The amplifi cation products were visualized under UV 
light, and their sizes were estimated relative to the DNA ladder. For 
further determination of polymorphisms, the PCR prod ucts were run 
on CEQTM 8800 XL Capillary Genetic Analysis System (Beckman 
Coulter, Fullerton, CA). The analyses were repeated at least twice to 
ensure reproducibility of the results. Allele sizes were determined 
for each SSR locus using the Beckman CEQTM frag ment analysis 
software. In each run, SL64, F12/1 and Montmorency were included 
as reference rootstocks.

Genetic analysis
The genetic analysis program “IDENTITY” 1.0 (WAGNER and 
SEFC, 1999) was used according to PAETKAU et al. (1995) for the 
calculation of number of alleles, allele frequency, expected and 
observed heterozygosity, estimated frequency of null alleles, and 
probability of identity per locus. Genetic dissimilarity was de-
termined by the program “MICROSAT” (version 1.5) (MINCH et al., 
1995) using proportion of shared alleles, which was calculated 
by using “ps (option 1 - (ps))”, as described by BOWCOCK et al. 
(1994). The results were then converted to a similarity matrix, and 
a dendrogram was constructed with the UPGMA method (SNEATH 
and SOKAL, 1973) using the software NTSYS-pc (Numerical Taxo-
nomy and Multiware Analy sis System,version 2.0) (ROHLF, 1988).

Results and discussion

The total 12 SSRs studied amplifi ed 108 alleles, an average of 
9 alleles per locus in 58 Prunus accessions belongs to Prunus 
avium, Prunus cerasus and Prunus mahaleb. The highest number 
of allele per primer was observed in PS12A02 primer as 12 alleles 
and followed by Pchgms1, UDP96-001 and UDP96-005 primers 
(11 alleles). The primer UDP96-019 gave the lowest number of 
alleles (6 alleles) (Tab. 1). 
We observed an average SSR observed heterozygosity (Ho) 
of 0.609 and the observed heterozygosity were found between  
0.345 (Pchgms1) and 0.890 (UCD-CH31). The average expected 
heterozygosity (He) was 0.720 indicating higher value than observed 
one (Tab. 1).
The probability of genetic identity (PI) was the lowest in PS12A02 
locus (PI:0.089) indicating that this loci was the most informative 
while the locus UDP96-019 (PI: 0.373) was the less informative.
The allele sizes of 12 SSR locus varied from 95 to 276 bp. The 
number of genotypes per allele were between 7 (UCD-CH13)  and 
24 (UDP96-005) (Tab. 2). 
The genetic similarity measured within species ranged between 
0.17-0.79 within P. avium, 0.96-1.00 within P. cerasus, 0.58-0.83 
within P. mahaleb genotypes. The average similarity ratios within 
species in a descending order were P. cerasus (0.98)> P. mahaleb 
(0.72)> P. avium (0.51), respectively. The similarity ratio were 0.25-
0.58 between P. avium accessions and F12/1; P. cerasus accessions 
and Montmorency was 0.63 and P. mahaleb accessions and SL64 
were 0.46-0.63, respectively. The average similarity ratio between 
P. avium-P. cerasus; P. avium-P. mahaleb and P. cerasus-P. mahaleb 
were 0.33; 0.007 and 0.08, respectively, indicating P. avium is more 
close to P. cerasus. 
A tree constructed from the SSR data divided the accessions into 

3 main clusters according to their taxonomic classifi cation. The fi rst 
cluster included P. avium accessions, the second cluster included 
P. cerasus accessions and the last cluster included P. mahaleb ac-
cessions (Fig. 1). P. cerasus accessions seem to be identical or very 
closely related. In contrast to P. cerasus, P. mahaleb and in particu-
lar P. avium seem to be more differentiated. The reference root-
stocks were also clustered with their associated botanical species 
(Fig. 1). 
The results obtained in the present study show that microsatellites 
could be effectively used for fi ngerprinting purposes in Prunus. In 
the present study, 12 loci in wild Prunus genotypes were assayed. 
The num ber of alleles per locus ranged from 6 to 12 with an average 
of 9 putative alleles per locus. Previously, KACAR et al. (2005) 
obtained a total of 37 alleles among 10 sweet cherry cultivars by 
9 SSR primers. CLARKE and TOBUTT (2003) used 14 sweet cherry 
cultivars for SSR analysis and determined 2 to 7 alleles per SSR 
primer. In addition, VAUGHAN and RUSSELL (2004) used 16 wild 
cherry accessions for molecular analysis by using 10 SSR primers 
and they detected 2 to 6 alleles. In fact all tested microsatellite 
primer pairs worked well and produced variable levels of ampli-
fi cations. The PS12A02 locus was the most polymorphic among the 
twelve loci with the highest effective number of alleles (12 alleles) 
with the one of the lowest PI value (0.089). The UDP96-019 was 
the less informative with the lowest allele number (6). The results 
showed high amplifi cation of cherry groups with plum, apricot and 
peach indicating a congeneric relationship within Prunus species. 
ERCISLI et al. (2011) successfully used SSR markers identifi ed 
in other Prunus species to study genetic diversity in wild sweet 
cherries. Our results demonstrated the cross-species transferability 
of SSR primers developed in cultivated species to wild species in 
Prunus for the discrimination of genotypes. Previously, PS12A02 
loci were found to be the most informative in other studies (DOW-
NEY and IEZZONI, 2000; GULEN et al., 2010; ERCISLI et al., 2011). 
WÜNSCH et al. (2004) reported 7 and 11 alleles in UDP96-005 and 
Pchgms1 locus in sweet cherries. In our study, UDP96-019 and 
UCD-CH13 gave the lowest number of alleles (6 and 7). STRUSS 
et al. (2003) and ZHANG et al. (2008) obtained the lowest allele in 
UCD-CH31 loci and WÜNSCH and HORMAZA (2002) reported the 
lowest loci in UDP96-019 loci. 
The overall genetic diversity within the tested species was relatively 
low as evident from the polymorphic ratio of 21% found by SSR 

Tab. 1: List of genetic parameters obtained with SSR used in this study

Locus N He Ho PI R

CPSCT010 10 0.648 0.581 0.190 0.040

Pchgms1 11 0.723 0.345 0.142 0.219

PS12A02 12 0.806 0.636 0.089 0.094

UCD-CH13 7 0.697 0.836 0.237 -0.081

UCD-CH17 8 0.861 0.418 0.068 0.238

UCD-CH21 8 0.697 0.381 0.175 0.186

UCD-CH31 8 0.773 0.890 0.146 -0.006

UDAp-401 8 0.729 0.636 0.177 0.053

UDAp-404 8 0.606 0.818 0.373 -0.131

UDP96-001 11 0.807 0.545 0.084 0.144

UDP96-005 11 0.850 0.818 0.072 0.017

UDP96-019 6 0.440 0.400 0.370 0.028

Total 133    

Average 13.3 0.81 0.57

N: number of alleles; Ho: observed heterozygosity; He: expected hetero-
zygosity; PI:probability of genetic identity; r: null allele frequencies



Molecular characterization of wild cherry germplasm by SSR 231
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232 Z. Turkoglu,  S. Bilgener,  S. Ercisli,  N. Yildirim Molecular characterization of wild cherry germplasm by SSR
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Molecular characterization of wild cherry germplasm by SSR 233

Fig. 1: Dendrogram of  58 Prunus accessions based on UPGMA analysis using the genetic similarity matrix generated by the Nei and Li similarity coeffi cient
after amplifi cation with 12 pairs of microsatellite primers.

primers (STRUSS et al., 2003) and 19% reported by ZHOU et al. 
(2002) and in cherries. We found high diversity ratio within P. avium 
compared to other species. A higher level of polymorphism was 
expected in sweet cherry due to its predominant self-incompatibility 
(HEGEDUS et al., 2012). 
The observed and expected heterozygosities averaged over the 12 
SSR loci were respectively 0.61 and 0.72 indicating higher mean 
values than those reported for SSRs in Prunus species (ARANZANA 
et al., 2003; BOUHADIDA et al., 2009). High allele number and high 
heterozygosity obtained in the present study refl ect the ability of 
SSR markers to provide unique genetic profi le for individual plant 
accessions, except in P.cerasus accessions. VAUGHAN and RUSSELL 
(2004) reported He and Ho values as 0.61 and 0.60 in 16 wild sweet 
cherry accessions by using 14 SSR locus. 
 

Conclusion

In conclusion, the gene pool of the Prunus species surveyed in 
Black Sea and Northeast Anatolia has signifi cant amounts of genetic 
variation. In regard to germplasm management, our results show 
that the germplasm collection is highly variable and most variation 
is common to all genetic groups identifi ed. Prunus germplasm from 
the region would have economically important adaptive traits that 
can potentially be incorporated into Prunus breeding programs. 
Hence, it is expected that the results of this study will assist current 
Prunus rootstock breeding efforts in Turkey as well as maintain the 
genetic integrity of the genetic resources. The SSR-based phylogeny 
was also generally consistent with Prunus taxonomy based on 
molecular evidence, suggesting the applicability of SSR analysis for 
genotyping and phylogenetic studies in Prunus genus.

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Address of the corresponding author:
E-mail: sercisli@gmail.com