Genetic Diversity Analysis And Molecular Identification Of Major Chinese Cymbidium Cultivars

Chinese cymbidium belongs among a terrestial group of Cymbidium inOrchidaceae and generally have a small-sized flower, with a high ornamental andcultural value. However, traditionary Chinese cymbidium mainly includes sevengermline: Series Goeringii, Series Longibractium, Series Faberi, Series Ensifolium,Series Kanran, Series Sinense and Series Lianpan, furthmore, some hybrids may beincluded in them. According to incomplete estimates has been named the Chinesecymbidium species has reached more than3,000, however, the studies about theirgermplasm mainly focused on their classification and genetic breeding, less onaspects of molecular biology research at home and abroad, especially less on geneticdiversity and molecular identification at the DNA level. The current reports abouttheir genetic diversity clearly are unsystematic, and also there were less cultivars andranges examined. Therefore, in this paper139Chinese cymbidium samples werecollected to the genetic diversity research and molecular identification with twomolecular markers including SRAP and ITS+trnH-psbA sequence, the main resultsare as follows:1. The DNA was extracted by the modified method of CTAB, and cancompletely meet the analyses of SRAP and nrDNA ITS and cpDNA psbA-trnHsequencing.2. A optimized reaction system of SRAP-PCR for analyzing the genetic diversityto Chinese cymbidium was build by orthogonal design, bulk volume25μL containing10×PCR buffer2.5μL,50ng template DNA(2μl)、Mg2+2.5mmol·L-1(2.5μl)、dNTPs0.25mmol· L-1(1.56μl)、 primers0.3μmol· L-1(0.75μl)、 TaqDNA polymerase1.0U(0.25μl)、ddH2O15.44μl.3.17pairs SRAP primers amplified a total of489DNA bands, of484polymorphic bands, percentage of polymorphic bands(PPB) ratio is98.89%; Geneticsimilarity coefficient varied from0.59-0.91, observed number of alleles (Na), theeffective number of alleles (Ne), Nei’s genetic diversity diversity index (H), and Shannon ’s information index (I) were respectively2.0000,1.4917,0.2968,0.4548.These data show that the Chinese cymbidium examined have a higher geneticdiversity level. In addition, a molecular identification card of139samples examinedwas built by17pairs primers combinations with48core bands, its confidenceprobability is99.99%.4. rDNA ITS sequences of Chinese cymbidium identified range from672bp to729bp, the average of GC content, variable sites, non-variable sites, and simplicityinformation sites is65.62%,72.2%,54.4%and21.1%respectively. According to theresults of comparison between different cultivars, AG, CT transversion and some basedeletion obviously exist in those sequences examined; However, psbA-trnHsequences length range from804bp to905bp, the average of GC content, variablesites, non-variable sites, and simplicity information sites is35.7%,29.2%,70.3%,and6.1%respectively. The variation present higher level at ITS sequence than atpsbA-trnH.5. The methods sequencing for the genetic diversity analysis and identification tothe Chinese cymbidium is: SRAP> psbA-trnH+ITS> ITS> psbA-trnH. As for thegenetic relationship about seven germlines, generally, series Longibractium is closerto series Lianpan than with other series; Series Ensifolium is closer to series Sinense;Series Kanran posses a more complicated genetic background. In addition, SRAP isan effective technique for Chiese cymbidium cultivars to be identified, for theirpedigree and genetic relationships to be analyzed, however, ITS and psbA-trnHsequence analyses have an extent significance for their species identification, andhave a limited significance for these cultivars identification.