Chloroplast Microsatellite Diversity Among and Within Prunus mahaleb L . and P . avium L . Species

Genetic diversity of 58 Mahaleb cherry (Prunus mahaleb) genotypes and six sweet cherry (P. avium) accessions was studied using 25 cpSSR primer pairs. Thirteen out of them demonstrated two to four alleles with an average of 2.46 alleles per primer pair. The mean of PIC value for the primers was 0.32. The average values of expected heterozygosity (He) and Shannon’s information index (I) for all loci were 0.35 and 0.55, respectively. The dendrogram based on cpSSR markers has been illustrated by MEGA4 software with Maximum Composite likelihood model and the Neighbor-joining method, which clustered the genotypes into four groups. Sum of first three principal components analysis (PCAs) could be represented most of (63.07%) the total variation in the original dimensions and confirmed the results of cluster analysis. Based on the AMOVA results, the allele numbers among groups and species studied in this research were more than those observed within them. In this study cpSSR markers provided a good tool for assessment of genetic diversity among and within mahaleb cherry and sweet cherry genotypes.


Intruduction
Prunus is a large genus of trees and shrubs, which includes plums, cherries, peaches, apricots, and almonds.Botanical classification of species within this genus, partly due to ease of interspecific hybridization is sometimes debatable (Turkoglu et al., 2010).These species belong to the Rosaceae, sub family Prunoidae.There are many different types of rootstocks being used for Prunus species.Each one has a particular set of advantages and limitations for adaptation to different geographic regions.One of the Prunus species used as a common rootstock for sweet cherry that has been known as a cultivar with strong roots, is Mahaleb cherry (P.mahaleb L.), (2n = 2x = 14).This species is native to Mediterranean, Southeast Europe and West Asia, however, it is sometimes found in Central Europe.Another Prunus species is Sweet cherry (P.avium L.), (2n = 2x = 16).This species is typical outcrossing with a mono-factorial and multi-allelic gametophytic incompatibility system (Crane and Lawrence 1929;Lacis et al., 2009;Tehrani and Brown 1992).Sweet cherry is one of spring-summer fruit species which is consumed as a fresh fruit (Jakobek et al., 2009).The available genetic diversity of species such as Mahaleb cherry and Sweet cherry can improve breeding of these species.
The genomic studies concerning the fruit species have increased enormously to characterize fruit germplasm resources and analysis of their genetic diversity including P. mahaleb and P. avium species based on morphological characteristics and molecular markers (Ganji Moghadam and Khalighi 2006;Lacis et al., 2009;Pedersen 2006;Rakonjac et al., 1996;Wunsch andHormaza 2002 and2004).For crop improvement studies, researchers usually request plentiful genetic diversity among materials.
For breeding and commercialization of rootstocks, a precise determination and discrimination method for these materials is desired.Morphological traits are strongly affected by the environment and developmental stage of plants (Casas et al., 1999).Therefore, it is very difficult to follow their morphological traits of rootstocks after grafting.Molecular markers are useful complements to morphological and phenotypic characters because they are plentiful, independent of tissue or environmental effects, and allow reliable identification and discrimination of genotypes in the early stages of development.The superiority of molecular markers over morphological characterization in fruit species is well recognized and widely accepted (Duminil and Di Michele, 2009;Ercisli et al., 2007;Zamani et al., 2007).
In this study, we used cpSSR markers for the first time to identify a set of polymorphic microsatellite loci and analysis of genetic diversity among and within P. mahaleb and P. avium species.It is expected that the information of this research will be useful for selection and more efficient utilization of this germplasm in breeding programs in the future.

Plant Materials and DNA Extraction
Young leaf samples of 64 genotypes from two Prunus species, which include 58 Mahaleb cherry (P.mahaleb L.) and six sweet cherry (P.avium L.), were used as starting material to carry out a chloroplast microsatellite marker analysis.The genotypes used in this study were obtained from the germplasm collection maintained at the Khorasan Agricultural and Natural Resources Research Centre, Mashhad, and Isfahan University of Technology, Isfahan, Iran.Based on morphological characteristics the plant materials were classified to dwarf and vigorous groups (Table1).The plant materials were treated with liquid nitrogen and stored at −80•C until being used.Genomic DNA was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method of Doyle and Doyle (1987).The quality and concentration of the DNA samples were detected on 0.8% agarose gel by electrophoresis and spectrophotometeric measurement according to Sambrook and Russell (2001).

cpSSR Analysis
Twenty five cpSSR primers were selected for cpSSR analysis (Table 2), which purchased from Macrogen Co. (South Korea).PCR reactions were conducted in a 25µl volume consisting of 2.5µl of 10x PCR Buffer, 1.5mM MgCl 2 , 1U of Taq DNA polymerase, 200 µM of dNTPs, 0.3 µM of each primer and approximately 50 ng of template DNA.Amplification were carried out in a Peltier Thermal Cycler PTC-0200 (Biorad Co.) with the following PCR program: 5 min of initial denaturing at 94°C, 30 cycles of three steps: 1min of denaturing at 94ºC, 1min at appropriate primer annealing temperatures (Table 2), 1min of elongation at 72ºC, followed by a final extension of 10 min at 72ºC.The PCR products were mixed with 10µl of formamide loading buffer (95% formamide, 20mM EDTA, pH8.0, 0.25% Xylene cyanol and 0.25% Bromophenol blue) and analyzed on 6% denatured polyacrylamide gels in 1x TBE buffer and then silver stained according to the reported procedure (Bassam et al., 1991;Liu et al., 2007).

Data Analysis
All clearly detectable and reproducible amplified fragments were scored according to their different allele sizes band and the matrix of cpSSRs data was assembled.The diversity level of gene loci was evaluated with the polymorphic information content (PIC).The PIC value was calculated according to the formula: PICi = 1 −ΣP 2 ij , where Pij is the frequency of the jth allele for the ith marker (Smith et al., 1997).
Distance matrix between genotypes was calculated and a dendrogram was constructed by the genetic distance matrix to display relationships among genotypes using MEGA4 software (Tamura et al., 2007) with Maximum Composite likelihood model and the Neighbor-joining method.Bootstrap analysis with 1000 replicates was also performed to obtain the confidence of branches of the cluster tree.
The chloroplast haplotypes of each individual were generated by combination of alleles detected from the polymorphic primers, because of the non-recombination nature of the chloroplast genome.cpDNA haplotypes were treated as alleles at a single locus.Multilocus haplotypes were generated by combining information from all polymorphic loci.Diversity values based on haplotype frequencies were calculated using Arlequin 3.1 software (Excoffier et al., 2005).
For each cpSSR marker, the presence or absence of each single fragment was coded as 1 or 0, respectively to generate a binary data matrix.Genetic relationships among genotypes were further analyzed by the principal component analysis (PCA) of a similarity matrix according to the extracted Eigen vectors in NTSYS-pc version 2.02i (Rohlf 2000).
Population genetic analysis was performed using the model for co-dominant markers with haploid individuals using POPGENE version 1.32 (Yeh et al., 1999) to calculate observed number of alleles (Na), effective number of alleles per locus (Ne), Nei's gene diversity (H) (Levene 1949;Nei 1973) and Shannon's information index (I) (Lewontin 1972).
Analysis of molecular variance (AMOVA) was performed to estimate variance components for cpSSR data, partitioning the variation into within and among populations, using Arlequin 3.1 software (Excoffier et al., 2005) with 1000 bootstrap replicates.

Allelic Variation of cpSSR Loci
Twenty five cpSSR primer pairs were used to amplify DNA fragments from 64 P. mahaleb and P. avium genotypes and 16 out of them were amplified fragments in all genotypes.Eleven out of 16 primer pairs were showed polymorphic bands with a number of alleles ranging from two to four.A total of 25 alleles were identified with an average of 2.27 alleles per locus, while effective number of alleles (Ne) varied from 1.2 to 1.91 with a mean value of 1.56 (Table 3).The genotypes studied revealed significant levels of cpDNA genetic diversity, with percentage of polymorphic bands (PPB) of 68.75%.The mean of polymorphism information content (PIC) was 0.32 which ranged from 0.17 to 0.48 (Table 3).At each single primer pair, the average values of Nei's gene diversity (H) and Shannon's information index (I) were 0.35 (range: 0.17-0.48)and 0.55 (range: 0.31-0.67),respectively (Table 3).
The combination of the alleles at each of the eleven polymorphic loci constituted 43 haplotypes (Table 4).Ten haplotypes (H2, H3, H4, H5, H6, H7, H10, H12, H16 and H36) being found in more than one genotype and the rest of them were in a single genotype.The most of haplotype frequency (0.0862) was seen in five genotypes (T11, T27, T96, T106 and T143) and the lowest one (0.0172) was observed in 29 genotypes (Table 4).

Genetic Relationships among Genotypes
The distance matrix of the 64 genotypes based on cpSSR analysis constructed by MEGA4 software, showed that genetic relationships of 64 genotypes were different and the range of distance varied from 0.0 to 0.427 with an average of 0.22 (data not shown).The lowest genetic distance was showed between "T143" and "T96" genotypes (0.0), whereas the most genetic distance was between "T263" and "Azadi6" genotypes (0.427).

Cluster Analysis
The dendrogram constructed from the distance matrix based on Maximum Composite likelihood model and the Neighbor-joining method was grouped the 64 genotype into two main clusters and four groups (Fig. 1).Cluster I is the biggest cluster, comprised of 58 Mahaleb cherry genotypes which divided in the three groups and Cluster II consisted of six sweet cherry accessions that clustered in one group, separately.
The first three principal axes of PCA analysis explained 53.05%, 5.84% and 4.18% of the total variation, respectively.Sum of first three PCAs could be represented most of (63.07%) the total variation in the original dimensions and confirmed the results of cluster analysis.

Population Genetic Structure of Genotypes
Analysis of molecular variance (AMOVA) was performed to differentiation of dwarf and vigorous genotypes and to estimate the percentage of intra and intergroup genetic variation (Table 5, Analysis 1).Although significant variation was observed among the groups (Fst = 0.06; P =0.0049), 93.88% of the total variance occurred within groups and 6.12% attributed to among groups.The results showed that the haplotype diversity among vigorous genotypes (0.37) is more than dwarf genotypes (0.24).
Another AMOVA analysis was conducted to estimate the percentage of intra and inter-species genetic variation, which revealed a significant variation among the studied species (Fst = 0.56; P < 0.001).This analysis results showed that 55.72% and 44.28% variation accounted for among and within species, respectively (Table 5, Analysis 2).Haplotye diversity within P. mahaleb (0.27) was significantly more than P. avium species (0.16).

Chloroplast Microsatellite Diversity
Assessment of genetic diversity is an essential component which improved breeding of species.Results obtained in genetic diversity studies of P.mahaleb and P.avium based on morphological characteristics and molecular markers indicated that abundant genetic diversity exists in these species (Ganji Moghadam and Khalighi 2006;Lacis et al., 2009;Pedersen 2006;Rakonjac et al., 1996Wunsch and Hormaza 2002and 2004).Using cpSSR as a basis molecular marker in this study is the first attempt to determine genetic variation among and within P.mahaleb and P.avium genotypes.The PPB (68.75%) on the species level was near with that of detected using nuclear SSR markers on the Latvian and Swedish sweet cherry (Prunus avium L.) (Lacis et al., 2009).The number of alleles per locus in this study (Table 3) was similar with that of pines, Clintonia Raf and almond (Echt et al., 1998;Wang et al., 2011;Zeinalabedini et al., 2010;Zhang et al., 2004;) while lower than that of some species (Jiang et al., 2004;Sánchez-Pérez et al., 2005;Setsuko et al., 2007) using nuclear SSR.These show that a lower level of polymorphic for a single locus is detected using cpSSR than using nuclear SSR.
The mean of PIC in this study was in the range of 0.25 to 0.5 (0.5 > PIC > 0.25).This indicates that the cpSSR markers could develop medium loci polymorphism which is useful for genetic variation of genotypes studied (Vaiman et al., 1994;Xie et al., 2010).
Average heterozygosity or gene diversity (He) is more appropriate than the proportion of polymorphic loci in assessment of genetic variation ( Nei 1987).The mean of heterozygosity calculated for each primer pair in this study was similar with that sweet cherries in other study (Schueler et al., 2003;Wunsch and Hormaza 2004), peach (Sosinski et al., 2000;Testolin et al., 2000) and apricot (Hormaza 2002 ).The mean of Shannon's information index in this research was 0.55; in agreement with the results of Jin et al., (2008), Xie et al., (2010) and Xu-Xiao et al., (2008).Shannon's information index (Lewontin 1972) was calculated to provide a relative estimate of the degree of variation within genotypes.

Cluster Analysis and Population Genetic Structure
The clustering results based on polymorphic cpSSR loci fit well to the genetic distance matrix.It was noticed according to dendrogram that P. mahaleb L. and P. avium L. species were separated which shows the ability of cpSSR markers to separate these two species.Results of clustering showed that "T204" genotype from P. mahaleb L. as having the closest genetic relationship with sweet cherry accessions supporting the hypothesis which was based mainly on morphological characteristics.Also, this suggested that "T204" probably arose by hybridization with P. mahaleb L. and P. avium L. Some aspects of interrelation among materials studied that were not recognizable by cluster, revealed by the principal components analysis (PCA).Sum of first three PCAs in this study were 63.07%, which this result demonstrates proper distribution of cpSSR markers through entire genome and confirmed the results of cluster analysis; in agreement with the results of Wang et al., (2011).
The large amount of variation attributed to differences within groups (93.88%) and Fst value (0.06) showed by AMOVA analysis of two groups of the genotypes which shows a moderate differentiation.In another result of this analysis the differences among species showed the most of variation (55.72%) and the rest was attributed to differences within species and Fst value was 0.56.Fst value above 0.25 indicated high genetic variation (Wright 1978), and gene flow was limited among the species.Based on these results, the allele numbers among groups and species studied in this research were more than those observed within them, which shows strong differentiation.These results are in agreement with the results of Wang et al., (2008) and Zhao et al., (2010).

Conclusion
In conclusion, chloroplast microsatellite primers used in this study were able to separate genotypes and species of P. mahaleb L. and P. avium L. according to determine genetic diversity among them.Therefore, cpSSR markers provided a good tool for assessment of genetic diversity among and within species.Consequently, an advantage of microsatellites in the study of conservation genetics is the fact that primers developed for one species are frequently applicable to related taxa.
Figure 1.Dendrogram of 64 genotypes, based on cpSSR markers data, by MEGA4 software with Maximum Composite likelihood model and the Neighbor-joining method

Table 2 .
cpSSR primers sequence used in this study

Table 3 .
Number of the allele, major allele frequency, Gene Diversity, PIC, Nei's gene diversity and Shannon's information index (I) for each cpSSR primers Locus