| Double flowers,which are characterized by the excessive development of petals,are important in ornamental flowers because they are considered to be more attractive ingardens than their normal counterparts.Sagittaria sagittifolia is an aquatic herb in the Alismaceae.It also known as ‘Zizania latifolia’,‘Zizania latifolia’,commonly known as ‘scissors grass’,‘swallowtail grass’,the ancient name ‘Yugu’,‘Hefu’,‘white chestnut’.It is native to China and is distributed in temperate and tropical zones of Asia,Europe and Africa.Europe is mostly used for ornamental,while China,Japan,India and North Korea are used for vegetables.S.sagittifolia with double flowers is an aquatic plant with a beautiful flower type and a flowering period that lasts for half a year,and it is suitable for landscape viewing.The species is highly popular as an aquatic flowering plant for balconies,and it has substantial potential for market popularization and application.However,little is currently known about the molecular regulatory mechanism in its flower development stage.The research on S.sagittifolia is mainly focused on germplasm resources,population hybridization,pollination habits and reproduction,but the mechanism related to the morphogenesis of flower organs,especially the formation of double flower,is not clear.For this reason,the formation mechanism of double valve character of S.sagittaria was studied in this paper and the main results are as follows:1.Based on the observation of single and double flowers of S.sagittifolia under optical microscope and floral physiology,the flower buds of S.sagittifolia were divided into three key stages of flower bud differentiation:(1)sepal differentiation;(2)petal differentiation;and(3)stamen(pistil)differentiation.Paraffin sections showed that there were normal carpels and stamens in the single flowers,while the petals were produced instead of carpels and stamens in double flowers during the early stage of flower bud differentiation.2.This study is the first to use SMRT sequencing technology to analyze the full-length transcriptome of S.sagittifolia.Using Pacific Bio Sciences Iso-Seq-SEQ combined with second generation transcriptome sequencing,the transcriptome sequencing of 34 samples was completed,and high-quality sequencing data were obtained.After sequencing,656,275 circular consensus(CCS)readings were obtained including 53,422 complete open reading frames(ORF)were obtained.The average consensus of isoforms read lengths was 2,208 bp.A total of 502,228 differential genes were identified.The number of DEGs in female flowers was higher than that in male flowers,and the number of most DEGs was down-regulated in double flowers.Most DEGs were enriched in phenylalanine metabolism,starch and sucrose metabolism,plant hormone signal transduction,phenylalanine biosynthesis and other metabolic pathways.A total of 5,980 transcription factors that belonged to 67 families were identified.Weighted gene co-expression network analysis(WGCNA)of transcription factors and differential genes showed that transcription factors related to plant hormone signal regulation played an important role in double flower formation.3.The genes IAA8、ARF6、YUC、LOX、IPT、ARR、GA20、KAO1、AOC4、MYC etc.,which are closely related to the signal transmission and response of endogenous hormones such as IAA,ZR,GA and JA,were obtained.The results show that,the contents of JA,GA and ZR in single flowers were always higher than those in double flowers,the contents of IAA,JA and ZR in female flowers were higher than those in male flowers,while the content of GA3 in male flowers was slightly higher than that in female flowers.During flower development,the expression of endogenous hormone IAA and GA biosynthesis and signal pathway related DEGs was up-regulated,while the expression of JA and ZR biosynthesis and signal pathway related DEGs was down-regulated.In double-petal flowers,the expression of most plant hormone biosynthesis and signal pathway-related DEGs was down-regulated,while most of DEGs expression in female flowers was up-regulated compared with male flowers.The changes of DEGs expression related to plant hormone biosynthesis and signal pathway are generally consistent with the change trend of endogenous hormone content.The expression of IAA8、ARF6、YUC LOX 和 OPR11、AOC4、MYC related to IAA and JA signal transduction and response is closely related to the phenotypic formation of S.sagittifolia double flower.4.48 MADS-box genes related to flower development were identified,which contained intact conserved domains of MADS-box,K-box and SRF-TF,and the amino acid lengths were located between 140～330 aa.All proteins were basic proteins with isoelectric points greater than 7.The prediction of subcellular localization showed that except for F01＿transcript＿156288 located on the cell membrane,all the others were located in the nucleus.Evolutionary analysis showed that the MADS-box gene was divided into 11 subfamilies,which were clustered with AGL6,SEP,AG,SOC1,AP3/PI,SVP,AP1 subfamilies in Arabidopsis thaliana and water lilies respectively.The analysis of expression patterns of MADS-box gene family members showed that the expression of most genes in double flowers was down-regulated,and the expression level in female flowers was higher than that in male flowers.Among them,class B genes AP3 and PI were highly expressed in double flowers and male flowers,and class C gene AG was highly expressed in single flowers,but low or no expression in double flowers.The expressions of AP3,PI,AP2 and WUS were all up-regulated in double flowers,while the expressions of AG and SEP genes were down-regulated and significantly different,indicating that these genes are involved in flower development and are closely related to the regulation of double-petal traits.5.103 AP2/ERF family transcription factors were obtained,and all AP2 transcription factors were located in the nucleus,which had high homology with Arabidopsis AP2 gene.Among them,the expression of TOE3-homologous AP2 transcription factor was significantly up-regulated in double flowers,while the expression of AP2-homologous transcription factor was higher in single flowers.The full-length c DNA sequence of Ss AP2 gene of S.sagittifolia was screened and cloned,which has the highest homology with TOE3 gene in the eu AP2 family of angiosperms.Compared with the wild type,the flowering time of Ss AP2 transgenic S.sagittifolia was delayed by about 15 days,and the number of petals increased in varying degrees.Among them,the male flower of transgenic S.sagittifolia has three petals of normal outer whorl,and the stamens were petalized completely,while part of the carpel of female flower petals into petals in varying degrees.The petalized petals were obviously smaller than the normal petals,forming a semi-double flower phenotype.There was a negative correlation between the expression of Ss AP2 and AG in transgenic S.sagittifolia,and the expression of Ss AP2 gene was closely related to the character of double flower.6.The micro RNA library of single and double S.sagittifolia was constructed and sequenced for the first time.21 known mi RNA and 96 new predicted mi RNAs were identified.61 mi RNA were differentially expressed,of which 29 were up-regulated and 32 down-regulated.A total of 3750 mi RNA target genes were predicted,and most of the candidate target genes of differentially expressed mi RNAs were mainly concentrated in plant hormone metabolism and signal transduction pathways.In double S.sagittifolia,the relative expression of mi R156 j was higher,while the relative expression of mi R164 a,mi R167 a and mi R172 e decreased significantly;in single stamens and carpels,the expression of mi R156 j and mi R167 a was significantly up-regulated,while in single sepals and petals,the expression of mi R172 e was significantly up-regulated;the expression of mi R164 was not significantly different in different organs of flowers.The expression of the above key candidate mi RNAs was negatively correlated with the expression of the corresponding target genes,indicating that mi R156 j,mi R164 a,mi R167 a and mi R172 e may be involved in the regulation of double flower development and floral organ morphogenesisIn conclusion,this study excavated the key candidate genes related to the double valve trait,screened and identified the differential genes related to plant hormone biosynthesis and signal transduction affecting the double valve trait,and the transgene further revealed that the Ss AP2 gene is involved in regulating the formation of the double valve trait.At the same time,the expression patterns of four mi RNAs and their target genes during flower bud differentiation were identified and analyzed,which laid a foundation for further analysis of the molecular regulatory network for the formation of double characters,and provided a theoretical basis for the morphological formation of double floral organs and breeding of new varieties. |
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