Genetic diversity of Cymbidium kanran detected by Polymerase chain reaction – restriction fragment length polymorphism ( PCR-RFLP ) markers

Fifty-four Cymbidium kanran cultivars from China, Japan and South Korea were examined and analyzed by using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) markers to determine their molecular diversity and relationships. In chloroplast (cp) PCR-RFLP analyses, genetic differences were revealed in 4 of 6 primer sets (66.67%) and 19 of 72 primer-enzyme combinations (26.38%), 116 polymorphic bands were detected. For mitochondrion (mt) PCR-RFLP markers, genetic differences were revealed in 2 of 8 primer sets (25%) and 55 polymorphic bands (53.49%) were detected with 7 restriction primer-enzyme combinations. According to the PCR-RFLP marker profiling data, all the cultivars were classified into four groups. The group 1 has included three subclusters that two of them were generally consistent with geography classification. Subcluster 1 and Subcluster 2 were mainly composed of Chinese cultivars. Comparably, Subcluster 3 was composed of two cultivars which originated from Japan separately. Group 2 comprised Huangchengzhiyue and Xiongnu originated from Japan. Group 3 was included in seven Chinese cultivars and five Japanese cultivars. Group 4 was composed of eight Chinese cultivars and four Japanese cultivars. Therefore, we demonstrated that the PCR-RFLP technique could provide a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in cymbidium kanran.


INTRODUCTION
Orchidaceae, one of the largest families of angiosperms, and one of the most numerous epiphyte groups in many tropical and subtropical areas of the world, are diverse in both their specialized pollination and ecological strategies.They provide a rich source of material for the investigation of evolutionary relationships and developmental biology (Dressler, 2005;Kuehnle, 2007).The orchids represent a group of botanically significant and commercially important flowering plants because of their ornamental value, that is, their variation in shape, form, size, and coloration surpasses the flowers of the other angiosperms (Kuehnle, 2007).A species' breeding system, its population size, and its colonizing ability play an important role in determining the distribution of genetic variation and genetic differentiation within and between populations (Hamrick and Godt, 1989;Sun, 1997).However, in recent decades, the over-collection of wild Cymbidium species has been a direct cause for the rarity and endangered nature of these species and populations, which led to a further loss of their genetic diversity and the alteration of their population genetic structure.
The plant cytoplasmic genome includes chloroplast (cp) DNA and mitochondrion (mt) DNA.Compared with the nuclear genome, the cytoplasmic genomes have simple genetics, little recombination, and a slow rate of structural evolution; thus it is a powerful tool for the study of landplant evolutionary processes, both at the intraspecific level and in molecular systematic (Toshio et al., 2005;Roy et al., 2000;Yoshiya, 2001).Inter-Simple sequence repeats (ISSR) are one of the most powerful genetic markers in biology.They are a source of abundant, nondeleterious mutations that provide variation in the face of stabilizing selection (Qi-Lun and Fang, 2008).To improve the process of orchid breeding, we have identified the 54 C. kanran varieties and evaluated their molecular relationship and difference using the cpPCR-RFLP, and mtPCR-RFLP markers.

Plant materials and DNA extraction
We used 54 C. kanran cultivars collected from China (40) and Japan (14) (Table 1) and extracted genomic DNA from leaves by a modified cetyltrimethylammonium bromide (CTAB) method (Knapp and Chandlee, 1996).

PCR-RFLP marker development
Approximately 1 mg each of extracted DNA was digested using six endonucleases that recognize four to six base pairs.The Primers are listed in Table 2.The 25 μl PCR mixtures contained 2.5 μl of buffer; 2 μl dNTPs; 0.4 μmol/L primers; 1.4 U Taq DNA polymerase and 300 ng of DNA.The cycle sequencing used the following procedure: step1, 94°C for 4 min; step 2, 55°C for 1 min, step 3, 72°C for 3 min, steps 1 to 3 were repeated for a total of 35 cycles; and finally, step 4, 72°C for 2 min.The PCR products were electrophoreses in a 1.8% agarose gels.

Data and cluster analysis
The presence of amplified bands were detected and analyzed with the Quantity One (BioRad, Hercules, CA, USA) software .Bands were scored for their presence (1) or absence (0) for numerical analysis.Genetic distances were calculated using Nei's coefficient of genetic distance (Nei and Li, 1979).The dendrogram of these C. kanran cultivars were constructed based on the similarity matrix data by applying unweighted pair group method with arithmetic averages (UPGMA) cluster analysis using the NTSYS program (Exeter Software, Setauker, NY).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) maker development
In cpDNA PCR-RFLP analysis, four out of six primers could amplify more than one clear band that could be digested by the restriction endonucleases EcoRI, HindIII, BamHI, EcoR321, BsuR1, and EcoR881, Nine out of seventy-two primer-enzyme combinations amplified 518 bands, and 116 bands revealed genetic differences.The product of trnL-trnR digested by EcoRI obtained relatively higher levels of polymorphisms (24 bands) than the others.Few primers could produce polymorphic bands in different C. kanran varieties.
In mtDNA PCR-RFLP analysis, two out of six primers could amplify one clear band, and they were digested by the restriction Endonucleases EcoRI, HindIII, BamHI, EcoR321, BsuR1, and EcoR881.Only one primerenzyme combination (Cox1) amplified 109 bands and 55 (53.49%) polymorphic bands were revealed in 54 C. kanran cultivars.

Cluster analysis
The PCR-RFLP classified all cultivars into four groups (Figure 1); the group 1 has included three subclusters that two of them were generally consistent with geography classification.Subcluster 1 and Subcluster 2 were composed of Chinese cultivars.Comparably, Subcluster 3 was composed of four cultivars which originated from Japan separately.Group 2 comprised Huangchengzhiyue and Xiongnu originated from Japan.Group 3 was mainly composed of Chinese C. kanran cultivars which was included in seven Chinese cultivars and five Japanese cultivars.Group 4 was composed of eight Chinese cultivars, Four Japanese cultivars a.The results from this study demonstrated that most cultivars have genetic polymorphisms that correspond with the ecological diversity in C. Kanran.
In this study, PCR-RFLP markers were be used for the first time to measure genetic variation and determinate of genetic relationships in C. kanran cultivars, which could be produced complete, very reliable, reproducible and highly polymorphic fingerprints among 54 C. kanran cultivars.
According to different genetic and chemical characteristics of orchid, the use of PCR-RFLP fingerprints needed to modify optimal pattern in C. kanran cultivars.Four cpDNA and two mtDNA PCR-RFLP primers were selected and provided robust, complete and polymorphic fingerprints which could be clearly distinguished all the tested C. kanran cultivars.
In fifty-four C. kanran cultivars, we detected 290 cpDNA PCR-RFLP fragments using four primers and 109 mtDNA PCR-RFLP fragments using two primers, indicating that there was abundance information in the genome of C. kanran.
Polymorphisms were useful for genotype identification, genotype relationship and genetic variability.A high level of DNA polymorphism (94%) was detected across special C. kanran cultivars using PCR-RFLP genotyping.Lower polymorphism (78%) using RAPD marker was found in 36 Cymbidium cultivars (Obara-Okeyo and Kako 1997).High polymorphism for ISSR markers was also reported , such as C. goeringii cultivars (Hui-zhong, 2009), which was proved that the genetic variation may be more directly related to the number of polymorphisms detected by the marker system and indicated that PCR-RFLP and ISSR markers were equally effective for assessing molecular diversity and identifying cultivars in C. kanran.

Conclusion
Molecular markers are scattered throughout the genome and their association with various agronomic traits is influenced by the cultivator under selection pressure induced by domestication.In conclusion, we have established a genotyping system using the PCR-RFLP technique in C. Kanran.This genotyping system can be used efficiently for specific genes that confer commercially important traits, and exploration of diversity among these C. kanran cultivars would be of great significance for breeding programmers.1.

ACKNOWLEDGEMENT
This research was funded by the science and technology Foundation of Guizhou Province in China(No.QiankeheJzi [2012]2012).

Figure 1 .
Figure 1.Dendrogram of 54 C. kanran cultivars using PCR-RFLP markers as per unweighted pair group method with arithmetic average (UPGMA) clustering.Cultivar numbers are the same as in Table1.

Table 2 .
The primer sequences and source for cpDNA and mtDNA RFLP markers.