Genetic Diversity In Cymbidium Based On RAPD Markers And PCR-RFLP Analyses Of Organellar DNAs

Cymbidium is perennial herbs. With bright-colored flowers, regular and satisfactory shape, rich varieties, Cymbidium is highly commercial value and great market potentiality. Due to the higher variabilities and distinction obscurity among species, the phylogenetic relationships between species are controversial. It also restricted their genetic improvement. To investigate the genetic diversity among Cymbidium are essential to the development of new varieties. The objective of this study was to be evaluated the genetic diversity among the species in Cymbidium by using RAPD, PCR-RFLP of cpDNA and mtDNA markers. The main results were as following:(1) The genetic diversity among the species in Cymbidium was investigated by RAPD markers of the 50 arbitrary 10-mer primers used for PCR amplification, the amplified products of 36 primers (72.0%) showed polymorphism.The RAPD-based genetic similarity (GS) among 20 Cymbidium accessions ranged from 0.503 to 0.765, with the mean of 0.598. Based on the GS matrix, a dendrogram showing the genetic relationships between accessions was constructed using UPGMA. The results showed that four groups were evident for 20 accessions. Pearl Dawson 'Procyon' was the most different, so it was shown a separate group.(2) The genetic diversity among 20 accessions of Cymbidium, was detected by using 7 cpDNA PCR-RFLP markers. Of the 7 cpDNA PCR-RFLP markers, 6 markers(87.5%) could produce one or polymorphic distinct bands were detected by direct electrophoresis in 2% agrose gels. 6 markers and 7 marker/enzyme combinations gave products which generated polymorphic bands upon digestion with Hinf Ⅰ, EcoR Ⅰ, Hind Ⅲ, BamH Ⅰ, EcoR321, BsuR`(HaeⅢ), EcoR881. A total of 53 bands were detected in 19 cpDNA PCR-RFLP marker/enzyme combinations, among which 37 bands (69.8%) were polymorphic. The cpDNA PCR-RFLP-based GS among 20 Cymbidium accessions ranged from 0.571 to 0.949, with the mean of 0.766. Based on the GS matrix, a dendrogram revealing the genetic relationships between accessions was constructed using UPGMA. The results showed that three groups could be distinguished. Great Flower 'Venus' was the most different, so it was shown a separate group. It indicated the cpDNA genome is conserved in Cymbidium, and extend of the genetic variations is very limited.(3) By using 8 mtDNA PCR-RFLP markers, the genetic diversity among 20 accessions of Cymbidium was investigated. Of the 8 cpDNA PCR-RFLP markers, 3 markers(37.5%)could produce one distinct band was detected, and no polymorphism was detected by direct electrophoresis in 2% agrose gels. 3 markers and 7 marker/enzyme combinations gave products which revealed polymorphisms upon digestion with Hinf Ⅰ, EcoR Ⅰ, Hind Ⅲ, BamH Ⅰ, EcoR881, BsuR Ⅰ (HaeⅢ) and Hapll. A total of 33 bands were detected in 10 mtDNA PCR-RFLP marker/enzyme combinations, among which 21 bands (63.6%) were polymorphic. The mtDNA PCR-RFLP-based GS among 20 Cymbidium accessions ranged from 0.634 to 1.000, with the mean of 0.829. Based on the GS matrix, a dendrogram showing the genetic relationships between accessions was constructed using UPGMA. The results demonstrated that three groups could be distinguished, whereas all of them could not distinguished by mtDNA PCR-RFLP markers. Pearl Dawson 'Procyon' was the most different, so it was shown a separate group. It indicated the mtDNA genome is highly conserved in Cymbidium, and extend of the genetic variations is very limited.(4) Pearl Dawson 'Procyon' was a separate group based on GS in RAPD and mtDNA PCR-RFLP. Miliaria Great Flower 'Ballerina', Lucky Gloria 'Aguri', Enzan Stream 'Orpheus '和 03-024 were a group based on GS in cpDNA PCR-RFLP and mtDNA PCR-RFLP. It suggested that the GS of Cymbidium was closely correlated with color and size of flower.