| Chinese orchid is a kind of terrestrial orchid with high ornamental value in Cymbidium.The commonly cultivated species are C.goeringii,C.faberi,C.ensifolium,C.sinense,C.kankan,C.longibracteatum,C.tortispalum and C.serratum.Some species of Chinese orchid have been cultivated for thousands of years and formed a large variety group,but the genetic relationship between species is not clear,and the cultivar classification lacks scientific basis.In this study,the species cultivated in in the history of China were studied from the ancient orchid books,and the evolution history of ancient Chinese orchid varieties was analyzed.Through observation and analysis of important ornamental characteristics of Chinese orchid,stable ornamental characteristics with classification value were found,and the classification standard of Chinese orchid varieties was formulated.The whole chloroplast genomes of the 8species were compared and analyzed,and a large number of SNP data were obtained based on transcriptome.Genotyping of SNP loci in eight species was carried out,and phylogenetic trees were constructed to clarify the genetic relationship between eight species.Then SSR molecular marker technology was used to classify and identify 96 accessions.Finally,the classification results obtained by different test methods were comprehensively analyzed,and the classification opinions of Chinese orchid species and varieties were put forward.The main conclusions are as follows :(1)There are 73 ancient books about Chinese orchid,including 4 in the Tang and Song dynasties,12 in the Ming Dynasty,46 in the Qing Dynasty,11 in the National Republic of China,and 56 books spread so far.The earliest classification of Chinese orchid according to the number of flowers in the stalk,one flower is C.goeringii,five to seven flowers is C.faberi.The Zhong Lan Pian which was written in tang Dynasty reflects the cultivation of C.goeringii or C.faberi in Zhejiang Province.The cultivation of C.ensifolium(including C.sinense and C.goeringii)in Fujian was first reflected in Jin Zhang Lan Pu which was written in song Dynasty.The Nan Zhong You Fang Lu which was written in Ming Dynasty described the varieties of C.tortispalum.It can be inferred that the color evolution process of Chinese orchid is as follows: it was yellow and green at first,and gradually appeared red,pink,purple,white and multi-color varieties in Ming Dynasty.In the Qing Dynasty,it returned to yellow or green,and required the pure color of the lip without plaque(suxin).(2)Referring to the classification method of ancient and modern flower types,considering the timing of the emergence of various flower types,the Chinese orchid flower types are divided into conventional and unconventional flower types,there are10 conventional and 8 unconventional flower types.The classification system of Chinese orchid varieties was established.Chinese orchid varieties were classified according to the five criteria of species,petal shape,flower type,color and lip plaque.(3)Nineteen stable and easily observed quantitative traits were selected,and 8species 66 varieties of Chinese orchid were observed.The data were analyzed by coefficient of variation,principal component analysis and cluster analysis.The results showed that the variation of quantitative traits was rich and the floral traits were the most stable.R cluster analysis showed that sepal length and petal length,sepal width and petal width,scape height and sepal aspect ratio were closely linked.Q cluster analysis showed that C.goeringii and C.faberi,C.ensifolium and C.sinense,C.longibracteatum and C.kankan,C.tortispalum and C.serratum were closely related,which was not completely consistent with the classical botany classification.In the classical classification,C.serratum was used as a variety of C.goeringii,and C.longibracteatum was used as a variety of C.tortispalum or C.goeringii.(4)whole chloroplast genome sequencing and mapping of wild materials of C.serratum and C.longibracteatum were performed.The chloroplast genome sequences of another 6 species and 1 outgroup were downloaded from NCBI database,and IR boundary changes,sequence homology analysis and collinearity analysis of 8 species were performed.The results showed that the total length of the chloroplast gene of C.serratum was 149998 bp,including 129 genes.C.longibracteatum is 150070 bp and has 130 genes.The evolutionary tree showed that the Chinese orchid was divided into three branches,C.goeringii and C.faberi were gathered together,with the support rate of 95%.C.longibracteatum and C.tortispalum gathered together,with the support rate of 82%.They were in the same group as C.sinense and C.kankan.C.ensifolium is a single branch.(5)Transcriptome sequencing was performed on the wild materials’ fresh leaves of 8 species of Chinese orchid,and SNP information of mixed populations was analyzed.The population structure diagram showed that it was most suitable to divide the orchid into eight independent species.The phylogenetic tree based on 496480 SNP loci showed that C.longibracteatum and C.tortispalum were closely clustered with the support rate of 100%,while C.sinense and C.ensifolium were closely related to C.kankan.(6)Twenty-eight pairs of SSR primers were used to perform capillary electrophoresis genotyping test on 96 populations of Chinese orchid.The clustering results showed that all the materials were divided into three branches: C.goeringii,C.faberi and C.serratum were in one branch.C.ensifolium,C.sinense,C.kankan,C.longibracteatum and C.tortispalum gather in another,among which C.longibracteatum and C.tortispalum are closely related.The hybrid orchid clustered in one branch.Population structure analysis revealed a variety of gene exchange phenomena.Based on the above results,this study supports eight independent species.The cpDNA,SNP and SSR markers all supported the close genetic relationship between C.longibracteatum and C.tortispalum.SSR markers and cpDNA support the close genetic relationship between C.goeringii and C.serratum.Based on morphological,cpDNA,SNP and SSR data,the authors put forward five criteria for cultivar classification.The classification of orchid varieties includes 8 species,10 petals,18 flower types,8 major color lines,Labial with or without plaque. |
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