Erogul D; Cakir B

Research Article

Austin J Biotechnol Bioeng. 2015;2(3): 1044.

Molecular Characterization of Sweet Cherry Genotypes and Rootstocks by using Prunus SSR Sequences

Erogul D* and Cakir B

Department of Horticulture, University of Ege, Turkey

*Corresponding author: Erogul D, Department of Horticulture, University of Ege, Turkey

Received: April 09, 2015; Accepted: May 29, 2015; Published: June 02, 2015

Abstract

Sweet cherry nurseries graft rootstock onto Prunus avium, Prunus cerasus, Prunus mahaleb and hybrids of other Prunus species. In this study, we used eight Simple Sequence Repeat (SSR) primer pairs to describe the genetic variability of the commercial rootstocks CAB6P, MaxMa 60, MaxMa 14, PHL-C, SL 64 and an unknown rootstock widely used in the production of sweet cherries in Turkey and twelve sweet cherry genotypes. These SSR primers are widely used for molecular characterization of Prunus species. Amplification of SSR loci was obtained for all microsatellite primer pairs and the microsatellites generated multiple alleles (42 in total) in the cherry rootstocks. All tested loci were polymorphic, with four to seven (average = 5.3) alleles per locus. The allele size varied from 95 to 206 bp. UCDCH-17 and UCDCH-21 had the lowest number of alleles per locus (four), while PS12A02 had the most (seven). The observed mean heterozygosity value for different loci was 0.79, while the expected heterozygosity was 0.58.

Keywords: Sweet cherry; Microsatellites; Prunus; Rootstock; SSR transferability

Introduction

Cherry (Prunus avium L.) is economically very important temperate fruit in Turkey. Cherries are thought to have originated around the Caspian and Black Seas and currently they are found across mainland Europe and in western Asian areas [1]. Cherry is mainly produced in the Central Anatolia, Marmara region [2] and Aegean region due to the early harvest time and sunshine.

Use of rootstocks is very important in fruit-tree nurseries and the numerous benefits of rootstocks necessitate their use for sweet cherry production. Many different types of rootstocks are used for Prunus species worldwide [3,4] each of which has a specific set of advantages and limitations in adapting to different geographic regions. Rootstocks can influence the performance of sweet cherry varieties and are responsible for water and nutrient uptake, resistance to soilborne pathogens, and tolerance to environmental stressors. Scion– rootstock interactions also affect water relations, plant size, pathogen resistance, environmental stress tolerance, nutrient uptake, plant size, flowering, vigor, survival of fruit buds, fruit set, fruit quality, yield, and leaf gas exchange [5-10].

Over the past decade, intensive evaluation of sweet cherry rootstocks has taken place; sweet cherry rootstocks that can impart a wide range of tree vigor and that are appropriate for different pedo-climatic conditions and cultivation systems are available [11]. Sweet cherries are traditionally cultivated by grafting on generative rootstocks or seedlings of Prunus avium, Prunus mahaleb and sometimes other Prunus species [12,13]. Strong interest in sweet cherry production has led to increased interest in breeding existing rootstocks and in selecting new, more appropriate rootstocks.

Microsatellites are present in many organisms and are found in the genomes of plants and organelles [14,15]. Generally, Simple Sequence Repeats (SSRs) are considered the marker of choice for genetic fingerprinting of fruit trees because of their high levels of polymorphism, high degree of reliability and reproducibility and codominant mode of inheritance [16,17]. These markers have been used widely in plants for cultivar identification [18,19]. Microsatellites are accepted as good genetic markers in sweet cherry [20,21]. Primers that frame SSR markers have been cloned, sequenced and used in peach [18,21-27], cherry [20,21,24,28,29-34], apricot [35-37], almond [38] and other Prunus rootstocks [39-41] for molecular characterization and identification of different varieties. In the present study we verified the high cross-species transferability of four SSR markers obtained from peach, plum and apricot to other Prunus species such as sweet cherry and sweet cherry rootstocks and we discriminated sweet cherry genotypes and sweet cherry rootstocks by using eight SSR markers.

Materials and Methods

Plant materials

Twelve sweet cherry genotypes (Kp1, Kp2, Kp3, Kp4, Kp5, Kp6, Kp7, Kp8, Kp9, Kp10, Kp11, Kp12) and ‘0900 Ziraat’ as a reference which is the most important sweet cherry variety grown in Turkey and six sweet cherry rootstocks (CAB6P, SL 64, MaxMa 14, MaxMa 60, PHL-C and one unknown rootstock) (Table 1) were used for SSR analysis. The commercial rootstocks and ‘0900 Ziraat’ were obtained from a commercial nursery located in Kemalpasa, Izmir (Figure 1). All the Prunus genotypes and unknown rootstock were collected from Kemalpasa, a significant growing area for early sweet cherry production. The unknown rootstock has no compatibility problems and a positive effect on yield. Its relationship to other commercial rootstocks and sweet cherry varieties has also been determined.

Table 1: Rootstocks and their genetic origin.




  
    Rootstocks 
    Genetic Origin 
  
  
    CAB 6P
    Prunus cerasus L.
  
  
    SL 64
    Prunus mahaleb L.
  
  
    Maxma 14
    Prunus mahaleb L. X Prunus avium L.
  
  
    Maxma 60
    Prunus mahaleb L. x Prunus avium L.
  
  
    PHL-C
    Prunus avium L. x Prunus cerasus L.
  
  
    Unknown
    -



Table 1:  Rootstocks and their genetic origin.

Figure 1: Kemalpaşa, Izmir, Turkey.

    
    
    Figure 1:  Kemalpaşa, Izmir, Turkey.

DNA extraction

Genomic DNA was extracted from young leaf tissue using a DNA easy plant mini kit (Qiagen, Germany) according to the manufacturer’s instructions. And RNase treatment was performed on the eluted DNA samples, and the purity and concentration of the DNA were checked on 1% (w/v) agarose gels and a NanoDrop ND- 1000 spectrophotometer.

SSR marker analysis

Eight previously published primer pairs were used for SSR analysis (Table 2). These primers have been widely used in molecular characterization of Prunus species. Polymerase Chain Reaction (PCR) amplification was performed in a 10-μl reaction volume containing 15 ng of DNA, 5 pmol of each primer, 0.5 mm dNTP, and 0.5 units GoTaq DNA polymerase (Promega, Madison, WI) that included 1.5 mM MgCl2. Forward primers of each primer pair were labeled with WellRED fluorescent dyes D2 (black), D3 (green), and D4 (blue) (Proligo, Paris, France). PCR conditions consisted of an initial cycle of 3 min at 94ºC, followed by 35 cycles of 1 min at 94ºC, 1 min at 55–60ºC, and 2 min at 72ºC, with a final extension at 72ºC for 10 min. PCR products were diluted with sample loading solution in proportion to the quantity of fluorescent dye used in labeling, applied to sequencing reaction according to the manufacturer’s instructions (Genome Lab DNA Standard Kit-400) and followed by electrophoresis with a CEQ 8800XL capillary DNA analysis system (Beckman Coulter, Fullerton, CA). Allele sizes were determined for each SSR locus using Beckman’s CEQ fragment analysis software. The analyses were repeated a minimum of two times to ensure reproducibility of the results.

Table 2: Simple sequence repeat primer pairs used in this study to characterize sweet cherry genotypes and rootstocks.




  
    SSR locus
    Sequence  (5’-3’)
    Species    origin
    References
  
  
    CPSCT010
    TTG GGT AAA TAC TTT    ATC ATT TCC TCC CTG AAT AAG GGT TGT GC
    Plum
    Mnejja et al. [46]
  
  
    UCD-CH13
    ACC  CGC  TTA     CTC  AGC   TGA     ACT  TAG  CAC     TAA  GCC  TTT     GCT  GC
    Sweet cherry
    Struss et al. [17]
  
  
    UCD-CH17
    TGG ACT TCA CTC ATT    TCA GAG A ACT GCA GAG AAT TTC CAC AAC CA
    Sweet cherry
    Struss et al. [17]
  
  
    UCD-CH21
    TTG TTG ACC ATC GAA    TAT GAA G GAA GGT ACA TGG CGT GCC
    Sweet cherry
    Struss et al. [17]
  
  
    UCD-CH31
    TCC GCT TCT CTG TGA    GTG TG CGA TAG TTT CCT TCC CAG ACC
    Sweet cherry
    Struss et al. [17]
  
  
    UDAp-401
    AAA CCC TAG CCG CCA    TAA CT GCT AAA GGC CTT CCG ATA CC
    Apricot
    Messina et al. [49]
  
  
    UDP96-005
    GTA ACG CTC GCT ACC    ACA AA CCT GCA TAT CAC CAC CCA G
    Peach
    Cipriani et al. [18]
  
  
    PS12A02
    GCCACCAATGGTTCTTCC    AGCACCAGATGCACCTGA
    Cherry
    Downey and Iezzoni [21]



Table 2:  Simple sequence repeat primer pairs used in this study to characterize sweet cherry genotypes and rootstocks.

The genetic analysis program ‘IDENTITY’ 1.0 [42] was used in order to calculate the number of alleles, expected and observed heterozygosity (He and Ho, respectively), estimated frequency of null alleles, and the Probability of Genetic Identity per locus (PI) [43]. The results were then converted to a similarity matrix, and a dendrogram was constructed using the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) [44] with the Numerical Taxonomy and Multiware Analysis System software, version 2.0 [45].

Results and Discussions

SSR analysis

Each of the eight tested primer pairs yielded PCR amplification products in the sweet cherry genotypes and sweet cherry rootstocks and all tested loci were polymorphic (Table 3). All microsatellites generated multiple alleles, with a total of 42 alleles and four to seven alleles per locus (average = 5.3). Allele size range varied from 95 to 206 bp. The lowest number of alleles (four) was found in the microsatellite markers UCDCH-17 and UCDCH-21 and the most (seven) were found in PS12A02 (Table 3). The observed mean Ho value for different loci for all of the rootstocks was 0.79 while the He was 0.58. The Ho was higher than expected for all microsatellites except PS12A02 because of the presence of null alleles, which are alleles that fail to amplify during PCR. The Ho identified in the primer pairs ranged from 0.30 (PS12A02) to 0.95 (UCDCH17 and UDP96005). The He value ranged from 0.53 (PS12A02) to 0.63 (CPSCT10). The marker PS12A02 had the highest information value as reflected in the lowest PI (0.27) value, while the least informative locus was UCDCH17 (PI = 0.52) (Table 3). Null allele frequency was the highest in locus PS12A02 (0.14) and lowest in UCD-CH17 (–0.26). Null alleles are generally referred to as alleles that fail to amplify during the PCR.

Table 3: List of genetic parameters obtained with simple sequence repeat used in this study.




  
    Locus
    N
    Allelic size    range (bp)
    He
    Ho
    PI
    r
  
  
    CPSCT010
    5
    170-184
    0.63
    0.90
    0.35
    -0.16
  
  
    UCD-CH13
    5
    127-145
    0.62
    0.85
    0.31
    -0.14
  
  
    UCD-CH17
    4
    180-206
    0.55
    0.95
    0.52
    -0.26
  
  
    UCD-CH21
    4
    95-119
    0.62
    0.90
    0.36
    -0.17
  
  
    UCD-CH31
    6
    124-182
    0.60
    0.60
    0.31
    0
  
  
    UDAp-401
    6
    142-188
    0.61
    0.90
    0.37
    -0.18
  
  
    UDP96-005
    5
    103-135
    0.56
    0.95
    0.46
    -0.24
  
  
    PS12A02
    7
    143-185
    0.53
    0.30
    0.27
    0.14
  
  
    Total
    42
     
     
     
     
     
  
  
    Average
    5.3
     
    0.58
    0.79
     
     
  
  
    N:    Number of Alleles; Ho: Observed Heterozygosity; He: Expected Heterozygosity;    PI: Probability of Identity; r: Null Allele Frequencies.



Table 3:  List of genetic parameters obtained with simple sequence repeat used
in this study.

Genetic similarity measured within the rootstocks and sweet cherries ranged between 0.25-1.00. The dendrogram generated from the UPGMA cluster analysis based on the Jaccard coefficient of genetic similarity classified all thirteen sweet cherry (twelve sweet cherry genotypes and a reference sweet cherry variety, ‘0900 Ziraat’) and six sweet cherry rootstocks (five commercial sweet cherry rootstocks and one unknown rootstock) into two main groups, which are depicted in Figure 2. The unknown rootstock formed a separate group and displayed a distinct branching pattern (Figure 2).

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Citation: Erogul D and Cakir B. Molecular Characterization of Sweet Cherry Genotypes and Rootstocks by using Prunus SSR Sequences. Austin J Biotechnol Bioeng. 2015;2(3): 1044. ISSN: 2378-3036

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Rootstocks	Genetic Origin
CAB 6P	Prunus cerasus L.
SL 64	Prunus mahaleb L.
Maxma 14	Prunus mahaleb L. X Prunus avium L.
Maxma 60	Prunus mahaleb L. x Prunus avium L.
PHL-C	Prunus avium L. x Prunus cerasus L.
Unknown	-