SSR Markers Development And Flowering Genes Discovery Of Cymbidium Kanran Based On RNA-seq

Orchidaceae is one of the most evolutionary monocotyledonous,widely distributed in various habitats and showing highly specific features of morphology,structure and physiology,which make it the ideal material for the study of development of flowers.Cymbidium kanran(Orchidaceae)is an important member of Chinese orchids with polymorphic lips,four seasons flowering and long flowering period,etc.,known as the "king of the orchids".However,a very long time is required for orchids from the seeds to flowering,which restricted the related research of orchids flower development and resource development.Although the system of vitro flowering has been established in many orchids,but the flower bud induction rate and subsequent reproductive growth situation has yet to be improved.Besides,the induction in vitro flowering increased operation cost and technical difficulty,so the study on flowering molecular mechanism of C.kanran is great significance to its development.In this study,to reveal the molecular mechanism of flowering,the transcriptome of C.kanran were identified by de novo and the reference sequence was obtained.Digital gene expression profiling(DGE)was used to select the differentially expressed genes from C.kanran buds at different stages,then the quantitative real-time reverse-transcription polymerase chain reaction(RT-qPCR)was used to verify the differentially expressed genes.The results laid the foundation for the molecular regulation mechanism of C.kanran,and also provided the theoretical basis for the flowering control of orchids.The main results were summarized as follows:1.By using the Solexa/Illumina second generation RNA-seq platform,the transcriptome of the mixture of C.kanran root,stem,leaf and flower were sequenced.The valid sequence of 108,927,860 nt was obtained,and 68,699 Unigene with an average length of 867 nt was yielded by de novo assembly.2.A total of 11,646 loci were obtained from 68,699 Unigene in the library of C.kanran,accounting for 16.95%.141 pairs of SSR primers were randomly selected,of which 79 pairs could amplify specific bands with an amplification rate of 56.03%,15 pairs of primers with polymorphic bands could be amplified,up to 10.64%.3.The 23,720 differentially expressed genes were obtained by comparison of three transcriptome databases by using DGE in the buds during three different periods of C.kanran,The nine differentially expressed genes related to flowering were choosed,which are: CkFPA,CkGA2 OX,CkGID1,CkCO,CkPHYB,CkELF3,CkFRI,CkFLC and CkAP1.4.By using RT-qPCR technolology,the expression pattern of the differentially expressed genes was analyzed in C.kanran.The five genes of CkFPA,CkGA2 OX,CkCO,CkFLC and CkAP1 were found that may be closely related to flowering in C.kanran,which has presented high concentration and similarity pattern during flowering.Overall,this study showed that C.kanran flowering is controlled by autonomous pathway,gibberellin regulatory pathway,photoperiod pathway and vernalization pathway,and the up-regulation of expression of CkFPA,CkGA2 OX,CkCO and CkFLC combined the peak expression of CkAP1 during induction,thus induced C.kanran flowering.In this study,the high quality and comprehensive transcriptome database were constructed and the key candidate genes involved in C.kanran flowering were screened.This research laid a foundation for further revealing the mechanism of flowering time,and provided genetic resources for molecular breeding in C.kanran,which has important theoretical and practical significance.