Genetic Analysis of Prunus salicina L. by Random Amplified Polymorphic DNA (RAPD) and Intersimple Sequence Repeat (ISSR)

Background Prunus salicina L. is an important fruit tree species of great economic value which is mainly distributed in the northern hemisphere. Methods 25 samples of Prunus salicina L. were collected from 8 provinces in China, Japan, USA, and New Zealand. The genetic variations of these samples were characterized by the random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) technique, respectively, and in combination. Results Totally, 257 RAPD bands ranging 200∼2300 bp was found, and 81.59% of these bands were polymorphic. ISSR analysis identified 179 bands ranging 300∼2500 bp, and 87.74% of the bands were polymorphic. ISSR results showed that the similarity coefficient index between samples P10 (Maihuangli in Anhui, Chin) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest, while that between samples P10 (Maihuangli in Anhui, Chin) and P15 (Baili in Japan) was highest. Combined analysis of RAPD and ISSR demonstrated that the similarity coefficient index between samples P4 (Qiepili in Ningbo, Zhejiang, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest, while that between samples P19 (Laroda in USA) and P20 (Red heart in USA) was highest. Conclusion RAPD combined with ISSR analysis can be used for genetic characterization of Prunus L. species.


Background
Prunus salicina L., belonging to the family of Rosaceae, are one of the most important economical fruit trees and are widely cultivated all over the world. ey are mainly distributed in the northern hemisphere, especially in the temperate zone [1,2]. China is one of the origin and distribution centers of Prunus L. species. Prunus L. species contain more than 430 species and are first segregated into six genera according to the morphology of fruit: Amygdalus L., Armeniaca Scop., Cerasus Mill., Laurocerasus, Padus Mill., Prunus species, and Tourn. ex Duh. However, phylogenetic analysis showed that Cerasus, Laurocerasus, and Padus were not monophyletic [3,4]. Besides, an increasing number of new cultivars from different countries result an important renewal of plant material worldwide [2]. It is thus necessary to characterize genetic information of Prunus L. species to cultivate new breed with improved quality characteristics.
DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence has been widely used for genetic diversity analysis of species [5]. Several studies have been devoted to the genetic diversity in Prunus L. species [6][7][8].
Recently, a number of molecular marker techniques including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), intersimple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) have been developed and widely used in the identification of various organisms [4,6,[9][10][11]. Among these techniques, RAPD and ISSR methods are two PCR-based methods that require only small amounts of DNA sample without involving radioactive labels and therefore have been widely used for genetic characterization [12]. RAPD is a technique based on the amplification of the genomic DNA with either a single or multiple short oligonucleotide primers of an arbitrary or random sequence [12]. RAPD is simple, cost-efficient, and does not require DNA sequences before application [13]. ISSR is derived from SSR, which is more abundant, informative, highly polymorphic, and efficient [14]. RAPD and ISSR methods have separately been used for genetic characterization in many species, such as Lonicera japonica unb. [15], synthetic hexaploid wheats [16], Atractylodes lancea [17], and Ocimum basilicum L. [18]. However, because of their advantages and disadvantages, more studies applied RAPD combined with ISSR to characterize the genetic variation of species, such as Litchi chinensis Sonn. [19], Allium species [20], date palm [21], and Cymbopogon [22]. However, only limited studies have been conducted to characterize the genetic relationships among different genus or cultivars of Prunus L. species [23][24][25].
In this study, we applied the RAPD and ISSR technique for the genetic characterization of 25 P. salicina from China and other countries. is study may provide valuable insight into the genetic diversity of P. salicina L. and provide information to cultivate new breed with improved traits.

Plant Material Collection and DNA Extraction.
is study included 25 P. salicina L. which were collected from 14 different regions from China (13 samples), Japan (4 samples), USA (7 samples), and New Zealand (1 sample) ( Figure 1 and Table 1). Among them, P1, P2, and P3 are the three lines with different maturity of one cultivar. e flowers of the 25 P. salicina L. are shown in Figure 2. e genomic DNA of 25 P. salicina L. was extracted from fresh leaves using a modified cetyl trimethylammonium bromide (CTAB) method as described previously [15,26]. DNA integrity was checked by 0.8% agarose gel electrophoresis, and DNA purity was determined by the absorbance ratio at 260 nm : 280 nm on spectrophotometry. e final concentration of DNA samples was adjusted to 10 ng/µl for PCR and stored at −20°C until use.

Amplification of DNA by RAPD-PCR.
e random RAPD primers were selected randomly for PCR amplification (Table 2). e PCR system in 10 μL volume contains 1 μL of 2.5 μmol/L primers, 1 μL of DNA template, 5 μL of 2 × PCR Taq Mastermix (TianGen Biotech Co. Ltd., Beijing), and 3 μL of deionized water. e PCR was executed on Applied Biosystems Veriti 96-Well ermal Cycler ( ermo Fisher, USA) in the following procedure: initial denaturation at 95°C for 90 s, followed by 40 cycles of 40 s at 94°C, 60 s at 36°C, 90 s at 72°C, and final extension of 5 min at 72°C.

Agarose Gel Electrophoresis.
e amplified PCR products were separated by electrophoresis on 1.8% agarose gel in 1 × TAE buffer. Gels were visualized by 0.5 g/ml ethidium bromide staining, and the images were documented using the ChemiDoc XR (Bio-Rad, USA). Bands that were unambiguous and reproducible in successive amplifications were selected for scoring.

Data
Analysis. All PCRs were repeated five times for each of five samples. Bands in the gel profiles were scored as 1 for present and 0 for absent. e similarity matrix (SM) and the similarity index (SI) were calculated using SM coefficient in Numerical Taxonomy Multivariate Analysis System (NTSYS pc 2.1) software. e dendrogram based on the unweighted pair group method with arithmetic mean algorithm (UPGMA) was generated using the SAHN module in the NTSYS pc 2.1 software.

Amplification of DNA by RAPD and ISSR.
A total of nineteen RAPD primers and fifteen ISSR primers were used in this study for the evaluation of DNA polymorphism ( Table 2). All RAPD primers and ISSR primers generated evaluable bands. Figure 3 shows the representative reproducible polymorphic amplification bands in these 25 samples generated from ISSR primer UBC807 and RAPD primer S201. For the RAPD primers, a total of 315 bands with an average of 16.58 bands per primer were obtained. Among these bands, 257 (81.59%) bands were polymorphic, and the approximate band size ranged from 200 bp to 2300 bp. e minimum number of bands was 10, which was generated by primer OPA-4 and the maximum was 21, which was produced by primer S43. e total number of polymorphic fragments ranged from 7 (primer OPA-4) to 18 (primer OPA-10). e average polymorphic fragments ratio (PFR) (in %) was 81.60% (min: 65%; max: 94.74%). e other information of the bands generated by RAPD primers, including polymorphism information content (PIC), resolving   Table 3. For the ISSR primers, a total of 204 bands with an average of 13.60 bands per primer were produced; of them, 179 (87.74%) were polymorphic. e approximate range of band size was 300 bp to 2500 bp (Table 4). e minimum number of bands was 8, which was yielded by primer UBC829, and the maximum was 19, which was produced by primer   UBC807. e total number of PFs ranged from 8 (primer UBC829) to 15 (primers UBC807, UBC810, UBC846, and UBC881). e average PFR% was 87.80% (min: 69.23%; max: 100%). e other information of the bands generated by ISSR primers, including PIC, RP, EMR, and MI, are presented in Table 4.

Genetic Distance and Cluster Analysis of RAPD and ISSR
Markers. Based on the RAPD amplification profiles, cluster dendrogram was obtained using UPGMA ( Figure 4). Since P1, P2, and P3 belong to one cultivar, we ignored their coefficients in the following analysis. e dendrogram showed that the similarity coefficients ranged from 0.584 to 0.860. In the RAPD-based dendrogram, the 25 P. salicina samples formed four clusters at a cutoff of 0.692. e similarity coefficient between sample P4 (Qiepili in Ningbo, Zhejiang, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest (0.584), while that between sample P19 (Laroda in USA) and P20 (Red heart in USA) was highest (0.860) (Figure 4). e ISSR analysis showed similar results to the RAPD analysis. e dendrogram showed that the similarity coefficients ranged from 0.558 to 0.892. In the ISSR-based dendrogram, the 25 P. salicina samples were divided into five clusters at a cutoff of 0.692. e similarity coefficient between sample P10 (Maihuangli in Anhui, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest (0.558), while that between sample P10 (Maihuangli in Anhui, Chin) and P15 (Baili in Japan) was highest (0.892) ( Figure 5).

Integrating Analysis of RAPD and ISSR Data.
e dendrogram results of RAPD combined with ISSR showed that the similarity coefficients ranged from 0.597 to 0.865. Total 519 DNA fragments were yielded, of which 435 (84.7%) were polymorphic. e average number of PF per primer was 12.7. e mean PIC, RP, EMR, and MI values observed for all primers were 0.42, 17.77, 10.80, and 4.54, respectively (Table 5).

Typical Band Patterns Amplified by ISSR and RAPD
Markers. Sixteen primers, including 11 ISSR primers and 5 RAPD primers, could be used as the markers of molecular identification for 25 Prunus L. samples ( Table 7). As shown in Table 7, UBC810, UBC834, and UBC836 could be considered as the markers of P1 (Zuili1 in Jiaxing, Zhejiang, China), P2 (Zuili2 in Jiaxing, Zhejiang, China), and P3 (Zuili3 in Jiaxing, Zhejiang, China). S17 could be considered as a marker of P4 (Qiepili in Ningbo, Zhejiang, China). UBC881 might be a marker of P5 (Jintangli in Zhoushan, Zhejiang, China). UBC847 was a marker of P6 (Furongli in Fujian, China). UBC847 and UBC855 could be used to distinguish P7 (Yuhuangli in Hubei, China). UBC848 could be considered as a marker of P8 (Jiuqianli in Guizhou, China). UBC857 might be a potential marker of P9 (Huangguli in Tongxiang, Zhejiang, China). RAPD-1 could be used as a marker of P12 (Niuxinli in Shandong, China). UBC889 could be considered as a marker of P16 (Akihime in Japan). S43 and S1403 might be the markers of P17     (Zhenzhuli in Japan). UBC829 might be a potential marker of P23 (Queen rose in USA). RAPD-5 also might be used a marker of P25 (Misili in New Zealand). e representative banding profiles obtained by ISSR primers UBC834, UBC847, UBC857, and RAPD primer S1403 are shown in Figure 6.

Discussion
Illustration of the genetic relationships or characterization of genetic diversity is important to provide genetic guidance for hybrid breeding. In this study, the genetic diversity and relationship among 25 P. salicina L. varieties were evaluated by RAPD and ISSR, respectively, and integrated. e Dice's similarity coefficient of RAPD ranged from 0.584 to 0.860, and that of ISSR ranged from 0.558 to 0.892. Integrating analysis of RAPD and ISSR indicated the similarity coefficient varied from 0.597 to 0.865. e results indicated high diversity among the 25 varieties. ISSR and RAPD were widely used for genetic diversity evaluations of Prunus L. species. Tian et al. used ISSR and RAPD for genetic diversity evaluations of 48 Prunus mira L. samples, the high levels of polymorphism, and the results imply that Tibet samples preserved higher genetic diversity and most genetic variations occurred [27]. However, the efficiency of RAPD markers and ISSR markers in detecting polymorphism is controversial. Tian et al. demonstrated that ISSR found 77.80% polymorphism, which is higher than that found by RAPD (72.73%). In the study of Kumar et al. the phylogenetic relationships of 36 locally grown P. armeniaca genotypes were analyzed using 20 RAPDs and 11 ISSRs markers. RAPD markers were found more efficient for polymorphism detection, as they detected 97.84% as compared to 96.5% for ISSR markers, and the pattern of (d) Figure 6: e representative results of banding profiles obtained by ISSR primers UBC834 (a), UBC847 (b), UBC857 (c), and RAPD primer S1403 (d). Lanes P1-P25 represent different samples listed in Table 1. Lane "M" represents the DL2000 DNA marker. e typical bands for molecular identification of P. salicina L. are indicated by a red arrow. 8 Genetics Research clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR [28]. In our study, the PFR% of RAPD primers was 81.60%, which is lower than that of ISSR primers (87.80%). Our results support the view that ISSR markers are more efficient than RAPD with regards to detecting polymorphism. e RAPD results showed that the index of similarity coefficient between sample P4 (Qiepili in Ningbo, Zhejiang, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest (0.584), while that between sample P19 (Laroda in USA) and P20 (Red heart in USA) was highest (0.860). However, the ISSR results showed that the index of similarity coefficient between sample P10 (Maihuangli in Anhui, Chin) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest (0.558), while that between sample P10 (Maihuangli in Anhui, China) and P15 (Baili in Japan) was highest (0.892). In addition, the analysis of RAPD combined with ISSR showed that the similarity coefficient between sample P10 (Maihuangli in Anhui, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest (0.597), while that between sample P10 (Maihuangli in Anhui, Chin) and P15 (Baili in Japan) was highest (0.865), which was consistent with the RAPD analysis. ese findings demonstrated that the RAPD technique not only increased the resolution and yield but also was a reliable molecular tool for the genetic characterization of various organisms, which was reported in previous studies [6,15]. Our RAPD and ISSR analysis showed potentiality to distinguish P. salicina L. from related genus or species.

Conclusion
In summary, our study indicates that the RAPD combined with ISSR techniques would be used for the genetic diversity, molecular-assisted breeding, and genetic characterization of P. salicina L. Our results might assist in parental gametophytes selection for hybrid breeding of P. salicina L.

RAPD:
Random amplified polymorphic DNA SSR: Simple sequence repeat ISSR: Intersimple sequence repeat AFLP: Amplified fragment length polymorphism CTAB: Cetyl trimethylammonium bromide SM: Similarity matrix SI: Similarity index NTSYS: Numerical taxonomy multivariate analysis system UPGMA: Unweighted pair group method with arithmetic mean algorithm PIC: Polymorphism information content RP: Resolving power EMR: Effective multiplex ratio MI: Marker index TF: Total number of fragments PF: Polymorphic fragments PFR (%): Polymorphic fragments ratios (%).
Data Availability e data that support the findings of this study are available on request to the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.