Construction Of Molecular Identification System Of Cymbidium(Orchidaceae:Epidendroideae)

Accurate identification of species is the basis of biological research and application,and there are also extensive and realistic needs in various fields of society.DNA barcoding technology,based on a complete DNA barcodes reference database,provides a new means for accurate species identification.Cymbidium Sw.is a group with important cultural and ornamental value,but due to the comprehensive influence of the environment and growth period,as well as artificial cultivation,hybridization,etc.,its classification and identification are controversial.This study used genome skimmming sequencing technology to obtain 2 Gb sequencing data,assembled and analyzed 237 chloroplast genomes of 50 species of Cymbidium,downloaded 33 chloroplast genome data of 18 species of Cymbidium from NCBI database,and constructed a database containing 50 Cymbidium species of 270 chloroplast genomes.A phylogenetic tree of Cymbidium was constructed based on the maximum likelihood method,and DNA barcoding and ultra-barcoding were used to identify Cymbidium.In addition,this study also used flow cytometry to detect the size of some Cymbidium genomes,and explored the auxiliary role of the genome in the identification of Cymbidium species.The main findings are as follows:1 ? Application of chloroplast genome sequence in the identification of CymbidiumThe chloroplast genomes of Cymbidium have a typical tetrad structure,including a large single copy(LSC),a small single copy(SSC)and two inverted repeats(IR),the length is 148,359(Cymbidium madidum)? 167,829 bp(C.wenshanense),the difference is mainly caused by the expansion of the IR region and the loss of ndh gene.Among the maximum likelihood trees constructed based on the chloroplast genome and its parts sequence data set,the species identification rate of chloroplast genome and LSC are the highest both at 56%,followed by: IR at 52%,SSC at 50%,and coding and hypervariable regions at 46%,mat K+rbc L is 44%.Compared with standard plant DNA barcoding,the species identification rate of the chloroplast genome has increased from 44% to 56%,and the identification rate has increased slightly.Some species of Cymbidium still cannot be identified with chloroplast genome data,the reasons may be:a.The species differentiation time is short,and the chloroplast genome is not enough to distinguish;b.Chloroplast capture occurs after hybridization.In order to further explore the identification of Cymbidium,more sample size and nuclear genome data are needed.2?Explore the application of genome size in the identification of Cymbidium speciesThe genome sizes of 139 individuals of 42 Cymbidium species were newly measured by flow cytometry.The results showed that the genome size of Cymbidium ranges from 2.41(C.chloranthum)to 9.13 Gb(C.teretipetiolatum),and the average genome size is 4.06 Gb.This study also found that C.aloifolium and C.mannii with overlapping distribution,similar morphology and the same ploidy have a large difference in genome size.The genome size of C.aloifolium is 5.37 ? 5.91 Gb,C.mannii is 2.64?3.02 Gb.Therefore,the significant difference in the genome size of C.aloifolium and C.mannii can be used to assist in the identification of the two species.