| Herbaceous peony(Paeonia lactiflora)is a traditional famous flower in China and a new cut flower in the international market.There are tremendous herbaceous peonies in China,but the resource information is not completely clear.The main reason is that the variety identification mainly depends on the name and morphology,currently.Besides,insufficient research on the evaluation of resources cannot well support the utilization of existing variety resources.In this study,based on the previous work of our group,we collected the variety information in the four main planting areas of Chinese ornamental herbaceous peony.Then,we selected high polymorphism SSR primers to construct the molecular ID card of varieties,and established the database of Chinese ornamental herbaceous peony and its retrieval system.Furthermore,we analyzed the phenotypic diversity of ornamental herbaceous peonies,and genetic diversity,population structure and core collections based on the amplified information.Then,we took the stem straightness as the object,from the perspective of cut flower development,and systematically studied the morphological characteristics,stem strength,content of cell wall substance,anatomical characteristics and hormone content of varieties with different stem characteristics,and conducted transcriptome sequencing.We focused on the differences of the substrate content of cellulose synthesis and its key enzyme activities,cellulose structure and arrangement,the content of auxin synthesis precursor substances and auxin,and explored the mechanisms that led to the different stem straightness of herbaceous peony varieties,which could serve the development and cultivation of cut peonies.The main results are as follows:1.A total number of 163 varieties with 47 phenotypic traits and photos were obtained based on the investigation of Heze,Shandong province,Luoyang,Henan province,Yangzhou,Jiangsu province and Lintao,Gansu province.Combining initial variety information database information obtained by our research group before,a total of 420 varieties of morphology record and image information were acquired.These information of phenotypes,images and molecular ID cards of 420 ornamental herbaceous peonies constituted the database and its information management systems with the use of Spring Boot technology based on the Java Web framework.It realized the convenient query and management of the graphic and text information of ornamental herbaceous peonies.2.960 pairs of SSR primers were picked and designed for assessment of primer specificity and polymorphism.Successful amplification was achieved with 89.05% of total SSR primers,55.83% of which were polymorphic.46 pairs of SSR primers with relative high polymorphism were selected to further amplify in 24 varieties and seven species of the genus Paeonia.The results showed that the average PIC value was 0.77,and the frequency of invalid alleles for 22 SSR locus was less than 0.08(or did not exist),and for four SSR locus,it ranged from 0.20 – 0.40.These SSR primers could distinguish 31 germplasms,and principal coordinates analysis(PCo A)and cluster dendrogram analyses produced results consistent with known genealogical relationships.Then,using the EST-SSR primers developed above,393 varieties and seven wild species were distinguished.The discrimination rate of 25 pairs of primers was greater than 2%,combing three pairs of them(T852 + S024 + S025)could distinguish 221 kinds of accessions,adding three more pairs of SSR primers(TA564 + TA704 + T125)could distinguish 87.34% of total accessions,and10 primers could distinguish all of the samples.Based on the amplification information of the 10 pairs of primers,the molecular ID codes of 420 kinds of herbaceous peonies were constructed and the QR codes were generated.3.The phenotypic diversity analysis of ornamental herbaceous peonies showed that the diversity index of 24 qualitative traits and the seven quantitative traits were 1.90 – 2.08 and0.29 – 1.90,respectively,and most of the traits had high diversity.Herbaceous peony cultivars in China existed some problems,such as insufficient numbers of cultivars with straight stems,light flower fragrance,having side buds and concentrated flowering period.Varieties from various regions had significant differences in coefficient of variation of flower height,and varieties from Lintao had relatively low coefficient of variation,varieties in Lintao and Heze had some advantages of cut flowers in stem,bud and flower diameters.However,only seven herbaceous peonies basically met the shape requirements of the first grade cut flowers.Thus,parent from other variety groups should be introduced in future herbaceous peony development.Moreover,based on the principal component analysis,it was believed that factors related to petal characteristics had a greater impact on the phenotypic differences of herbaceous peony.The clustering results were closely related to flower traits,including the degree of stamen petalization and flower height,stem traits including stem straightness and length also showed some aggregation characteristics,while there was no obvious relationship with the cultivation regions.4.Analysis of genetic diversity based on allelic information showed that there was no obvious genetic differentiation in four cultivation regions of herbaceous peonies,and they had strong gene exchanges,showing frequent exchanges among different regions.STRUCTURE analysis divided herbaceous peony resources into two subgroups,and the varieties from four regions did not form subregions.From the perspective of average genetic distance and allele coverage,a core collection of herbaceous peonies containing 39 varieties was obtained.Although it covered all flower colors,no cultivars with the flower type of single-petal or golden stamen were included.Therefore,it is of necessity to construct the core collection of herbaceous peonies in the future by multiple methods.5.The results of comparative study of upright ‘Da Fu Gui’ and bending ‘Chui Tou Hong’ showed that there were significant differences in lignin,hemicellulose,soluble sugar and cellulose crystallinity contents,sclerenchyma and xylem areas and thickening in cell walls in pith at stage S3,when ‘Chui Tou Hong’ bending began.And the time when the cell wall hardens was also related to the straightness.6.Illumina sequencing of ‘Da Fu Gui’ and ‘Chui Tou Hong’ was performed and 44182 significantly differentially expressed genes(DEGs)were found,and these DEGs were mainly enriched in 36 pathways,such as phenylpropanoid biosynthesis,flavonoid biosynthesis,stilbenoid,diarylheptanoid and gingerol biosynthesis,biosynthesis of secondary metabolites,alpha-linolenic acid metabolism,and plant hormone signal transduction.Among the DEGs,hub genes involved in lignin,cellulose,and xylan biosynthesis and transcription factors that regulated these processes were identified by weighted correlation network analysis(WGCNA),including 4CL1,Ces A4,Ces A7,LAC3,MYB44,IAA31 and other 40 genes.In view of the lack of stable reference genes in P.lactiflora,reference genes selection was conducted,and the combination of F-box and ACT were the most suitable and stable in the stems of herbaceous peony in different development stages,and the q PCR results of the above genes were analyzed by using them.The expression trends obtained by q PCR were basically consistent with the gene expression trends plotted by transcriptome RPKM values,indicating the reliability of transcriptome sequencing and analysis in this study.In conclusion,this study established a database of Chinese ornamental herbaceous peonies and its information management system,and made it clear that the number of cultivated herbaceous peonies was 420 in China.A set of highly polymorphic EST-SSR primers was developed,and 10 pairs of primers were used to construct molecular ID cards of 420 varieties(including 27 varieties constructed by the research group previously).The phenotypic differentiation,genetic diversity and the relationship between the current cultivated herbaceous peonies and the cultivation regions were clarified through resource evaluation,which provided a basis for production and utilization.And the core collection construction method of ornamental herbaceous peony was explored from the perspective of genetic distance and allele coverage.In addition,it was revealed that the difference in the content of compounds that provided rigidity to the cell wall,the crystallinity of cellulose,and the content of hormones such as auxin,abscisic acid and ACC,and the maturity of the stem were important factors that affected the straightness of the herbaceous peony stem. |
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