Expression Pattern Analysis Of Flower Development Genes In The Capitulum Of Chrysanthemum Lavandulifolium And Chrysanthemum × Morifolium

Chrysanthemum flowers have a typical,complex capitulum.The capitulum consist of four main parts,from outside to inside,bracts,receptacle,ray florets and disc florets.The major characteristic of the capitulum is the differentiation of two types of florets,which form various flower shapes via their differentiation and development,even under the same genetic background.Florets vary in location,symmetry,gender,structure,organ fusion,pigmentation and function.So far,the genetic regulation model of floral organ development in higher plants still cannot explain the developmental mechanism of the capitulum and two types of florets,which restricts novel flower shapes in breeding chrysanthemums.Chrysanthemum × morifolium has a complex genetic background,leading to difficulties in molecular biology research.Chrysanthemum lavandulifotium,one of the original species of C.morifolium,is diploid.It has a simple and stable capitulum and it is an ideal material for studying the molecular mechanism of floret differentiation.Whole-transcriptome profiles from two stages of ray florets and disc florets of C.lavandulifolium were investigated using next-generation sequencing(NGS).To study the main transcription factors of floral organs,the gene expression patterns of floral organs were examined.Genetic transformation system of C.lavandulifolium was built to analyze the gene function.Furthermore,interactions among CYC2-like proteins and floral homeotic proteins were examined using a yeast two-hybridization system.The main results are described as follows:1.Nine candidate reference genes were selected and evaluated for their expression stabilities across the samples during the process of flower development,and their stability was validated by four different algorithms(Bestkeeper,NormFinder,GeNorm and Ref-finder).SAND(SAND family protein)was found to be the most stably expressed gene in all samples or in different tissues during the process of C.lavandulifolium development.Both SAND and PGK(Phosphoglyceratekinase)were the most stable in Chinese large-flowered chrysanthemum cultivars,and PGK was the most stable in potted chrysanthemums.There were differences in best reference genes among varieties as the genetic background of varieties were complex.It is advisable to select different reference genes for specific varieties.In addition,the author analyzed the relative expression level of the DFR(dihydroflavonol 4-reductase)and CYE(lycopene e-cyclase)genes in different samples to verify the reference genes that the author selected.These studies provide a guide for selecting reference genes for analyzing the expression pattern of floral development genes in chrysanthemums.2.Whole-transcriptome profiles from two types of florets in C.lavandulifolium were investigated using next-generation sequencing.A total of 47893 unigenes with a mean length of 1772 bp were obtained.All unigenes were annotated based on BLASTx and BLASTn(E-value<1× 10-5)searches of five public databases:Nt,Nr,KEGG,Swissprot and eggNOG.A total of 41611 unigenes(86.88%)were annotated by at least one public database.The numbers of unigenes expressed higher in disc florets and expressed higher in ray florets were 16 and 411,respectively.It was obvious that the number of unigenes with increased expression was much larger than that with decreased expression during the development in both ray florets and disc florets.3.According to comparative studies between the two types of florets,the author found that the CYCLOIDEA(CYC2-like)genes and B-class MADS-box genes were expressed to a higher degree in ray florets than in disc florets,and the C-class MADS-box genes and genes of pollen development were expressed to a higher degree in disc florets.The author also found that genes response to hormones showed regular expression changes between two stages of florets.One of the gibberellin biosynthesis-related genes was up-regulated in the development of the two types of florets.Eight auxin biosynthesis genes had differential expressions.Four of them increased,and the other four decreased.PIN is an important type of auxin response transcription factor.The expression of PIN increased with the development of disc florets.Four genes involved in jasmonate increased in disc florets.Genes implicated in ethylene and cytokinin biosynthesis were down-regulated at the second stage of ray floret development.Gene expression in two types of florets in C.morifolium showed similar patterns with that of C.lavandulifolium.In addition,the author also found expression level of some genes in anemone were between those in ray florets and disc florets.4.Full length cDNA of 11 genes that include floral development homoeotic genes and homologous genes of CYC were cloned from C.lavandulifolium.The gene expression patterns of floral organs(bract,receptacle,ovary,pistil,stamen,corolla of disc floret,corolla of ray floret)were examined through qRT-PCR.DenFUL,ClCYC1 and ClCYC3 were highly expressed in bracts.In the receptacle,DenFUL,ClAP2,ClSEP1,CISEP3 were highly expressed.In the corolla of the ray floret,ClAP3,ClPI,CISEPl,CISEP3 as well as four CYC2-like genes were highly expressed.In the corolla of the disc florets,ClAP3,ClPI,ClAG1,CIAG2,ClSEP1,ClSEP3 as well as DenFUL,were highly expressed.In the stamen,the CIAP2,CICYC2 and CICYC4 genes were highly expressed.In the pistil,the DenFUL,CISEP3,and C-class genes as well as three CYC2-like genes were highly expressed.In the ovule,the ClAP2 and ClSEP1 genes,as well as three CYC2-like genes,were highly expressed.Accordingly,the author proposes that the distribution pattern of the genes in floral organ development in chrysanthemum is different from model plants.The expression pattern of some genes showed differences between C.lavandulifolium and C.morifolium.For example,CIAP2 was expressed to the highest degree in the ovule in C.lavandulifolium,but in small chrysanthemum cultivar(SCI),the highest expression was in the stamen.Also,C-class genes were highly expressed in ovule and stamen in SCI,but were less expressed in the ovule of C.lavandulifolium.5.Interactions among CYC2-like proteins and floral homeotic proteins were examined using a yeast two-hybridization system,which confirmed that some of these proteins can form heterodimers:DenFUL with CAG2,C1AP2 with C1SEP1,C1AP3 with C1PI,C1CYC1 with C1CYC2,DenFUL with C1CYC2,C1AP2 with C1CYC1.Moreover,there are some proposed heterodimers that showed one way interactions.6.Genetic transformation system of C.lavandulifolium has not been established.Using hypocotyl of C.lavandulifolium as explants,an efficient and stable genetic transformation system mediated by Agrobacterium was established through investigating some factors influencing the transformation efficiency.The highest transformation frequency was obtained,with no pre-cultivate,the suitable infection duration 10 min with bacterial concentration OD600=0.4,appropriate co-culture for 2 days.Several putative transformants were obtained after two rounds of selection procedure.PCR analysis indicated that target gene had been integrated into the genome of C.lavandulifolium and its transgenic frequency was 5%.Also,the author got 6 transgenic lines of CICYC3.The establishment of efficient transformation system can provide an effective technical basis for subsequent gene function verification.Thus,the author proposed that expression of homologous genes of floral organ development were redistributed in the capitulum compared with those in model plants such as Arabidopsis.CYC2-like genes,as well as B-class and C-class MADS-box genes,were involved in the differentiation of the two types of florets.Based on the expression pattern of floral organs and analysis of protein interactions,the author established a gene expression model for floral organ development in the capitulum of C.lavandulifolium and C.morifolium.This study provided a novel insight for understanding the transcriptional regulation and differentiation between two types of florets as well as floral organ development in the chrysanthemum.Moreover,it provided a massive amount of sequence information for future molecular biological research on capitulum development in C.lavandulifolium.