Analysis Of MRNAs And Non-coding RNAs Expression Involved In The Female And Male Floral Develoment Of Eucommia Ulmoides

Eucommia ulmoides Oliver.,which belongs to Eucommiaceae,is a typical dioecious plant with crucial economic and ecological value native to China.Its leaf,flower,fruit,bark,and root contain a varity of secondary metabolites and cotton-like rubber called Eu-rubber.The epicarp and seed of female fruit is a crucial raw material for extracting Eu-rubber and the seed oil,while the male flower pollen of E.ulmoides,a rarest medicinal pollen resource,which is a new raw material for tea,wine and other drinks.The quality and quantity of the furits and male flower depend on the development female and male floral bud,which is essential to the products engineering.However,the key genes and non-coding RNAs involved in the development female and male floral bud are still unclear.In this article,the morphological structure of female and male floral buds of E.ulmoides during the whole developmental periods were observed.Based on the external morphology and internal structure,endogenous hormones of female and male flower buds at different developmental periods were detected.At the same time,RNA-seq technology is applied for comparing transcriptome differences of female and male flower buds at diffenrent development stages.Candidate genes involved in floral organ development of E.ulmoides were mined.The lnc RNA,mi RNA and circ RNA of female and male flower buds of E.ulmoides were further identified at the key differentiation stage of floral bud.According to the prediction of targeted genes,the function of lnc RNA,mi RNA and circ RNA omplicated in floral organ development were further predicted.And the regulatory network of non-coding RNA and m RNA was established,in order to provide crucial theoretical basis and practical guidance for studing flower development mechanism and molecular breeding of E.ulmoides.The main research results are as follows:(1)The morphological differences of female and male floral organ was more obvious with the development of female and male floral bud,and the endogenous hormones had a different role on the development of female and male floral organ.Based on the morphological structure of female and male flower bud of E.ulmoides at different development stages,and thedevelopment process of floral bud could be divided into inflorescence primordium formation,bract differentiation,pistil and stamen differentiation,the staminate and pistil morphological formation,dormancy period and the development of male and female gametophytes.The period of bract differentiation was the pivotal stage of pistil and stamen morphological differentiation of female and male flower buds.However,the inflorescence primordia differentiation,pistil and stamen differentiation and meiosis of megasporocyte and microspore mother cell were the important stages of induction,development and maturation of the pistil and stamen.The floral bud development was accompanied by the changes of IAA contents,ABA contents and ZR contents.The changing trend of IAA and ZR were coincident,but the contents of IAA and ZR were remarkable difference between female and male floral buds at different developmental periods.However,the change trend of ABA were opposite when the inflorescence primordia developed to female and stamen primordia,at the same time,the ABA content was remarkable difference between female and male floral buds.The hormone changing trend showed that the induction of the pistil primordia needed high level of IAA content,low level of ABA and ZR content.However,the induction of stamen primordia needed high level of ABA and IAA content and low level of ZR content.(2)These differentially expressed genes(DEGs)of female and male floral bud among three development stages was invovled in the floral organ morphological development and biosynthesis,metabolism and signal transduction of endogenous hormones.A total of 152.19 Gb of clean reads was acquired from eighteen samples of female and male flower buds at the stage of inflorescence primordia differentiation,pistil and stamen differentiation and the previous stgage of meiosis of megasporocyte and microspore mother cell.And 92% clean reads were mapped to E.ulmoides genome.A total of 7945 and 5780 DEGs were screened of female and male floral bud among three differentiation stage.Based on the analysis of the DEGs between the inflorescence primordia differentiation stage and pistil/stamen differentiation stage,a total of 36 and 56 DEGs involed in the induction of pistil and stamen primordium were obtained from female and male floral bud,respectively.A number of DEGs related to photoperiod and plant hormones,flowering inhibitory factor FLC,flowering integron SOC1 and transcription factors including WRKY,MYB,GATA and b HLH were obtained.However,more DEGs involved in auxin and ethylene biosynthesis and metabolism in male floral bud than female.Besides,a total of 98 DEGs was obtained from female foral bud among three stages,including flower organ identity genes such as A(AP1),C(AG2 and AGL1)and D(FBP2 and FBP9)genes,and 71 DEGs involed in the auxin,ABA,cytokinin and other endogenous hormones process,DEGs controling the transition of meristem and the development ovule primordia,which co-controled the pistil development.At the same time,a total of 103 DEGs were obtained from male foral bud among three stages,including flower organ identity genes such as B(DEF),C(AG1,AG2 and AGL1)and E(SEP1 and SEP2)genes,DEGs involed in the differentiation of male germ cells and meristem,and 60 DEGs involed in various endogenous hormones process,which jointly promote the development of stamen.(3)These differentially expressed genes between female andmale floral bud(sex-biased genes)at different floral bud development stages were also all related to the floral organ morphological development and endogenous hormones processes.A total of 3869 sex-biased genes were obtained at three development stages,of which 16 sex-biased genes closely involed in female and male floral organ development were obtained from three develoment stages,including B genes(DEF),MADS4,CLV1 and ERECTAL controling the meriatem and floral organs developmente,DEGs invloed in hormone biosynthesis and signal transduction,and other transcription factors of WRKY,GATA,b HLH and TFIIA.A total of 25 sex-biased genes involved in the intiation of floral organs were obtained at the bract differentiation stage including 15 sex-biased genes involved in the endogenous hormones processes.A total of 34sex-biased genes involved in the floral organ morphological differentiation were obtained at the pistil and stamen differentiation stage including 24 sex-biased genes involved in the endogenous hormones processes.A total of 128 sex-biased genes involved in the development of megasporocyte and microspore mother cell were obtained at the previous stage of meiosis of megasporocyte and microspore mother cell.Furthermore,these sex-biased genes at different development stages were also all consist of these special genes involved in the inflorenscencemeristem,floral organ meristem and germ cell differentiation,which directly regulating the flowring process.(4)These sex-biased lnc RNA and mi RNA were also involved in the floral organ morphological development and endogenous hormones processes,while the sex-biased circ RNA were involved in endogenous hormones processes.A total of 32 937 ? lnc RNA?10014 TUCP,26,732 m RNA,and 1658 circ RNA were identified based on the sequencing of chain specific library,and 187 mi RNA were identified based on the sequencing of s RNA library.A total of 312 DE-lnc RNA,417 DE-m RNA,191 DE-TUCP,31 DE-mi RNA and 31DE-circ RNA were obtained from the female and male floral bud.The DE-lnc RNA,DE-mi RNA and DE-m RNA were all related to the floral organ morphological development and endogenous hormones processes,while the DE-circ RNA were related to latter.Based on the functional analysis of differential transcripts,six DEGs were selected as key candiated genes,including GH3.0,PLC2,MADS4,AGL8,DEF and GATA5,which expressed differently between the female and male floral bud among different development stages.According to the correlation analysis of these key candiated DEGs,DE-lnc RNA,DE-mi RNA and DE-circ RNA,five DE-lnc RNA(including three TUCP)and eight DE-mi RNA as well as five DE-circ RNA were determined to be the key non-coding RNA involved in floral organs development of E.ulmoides.Importanly,the EUC24067-RA homologous to Antirrhinum DEF was determined to be the most important candiated gene.Ce RNA regulatory network of lnc RNA-mi RNA-m RNA was established based on the EUC24067-RA(DEF),which indicated that selected novel?23and LNC?025233 competitively regulate EUC24067-RA,rather than indirectly regulate EUC24067-RA through ce RNA.