- De novo sequencing of tree peony ( Paeonia suffruticosa ) transcriptome to. - Background: Tree peony (Paeonia suffruticosa Andrews) is a globally famous ornamental flower, with large and colorful flowers and abundant flower types. - However, a relatively short and uniform flowering period hinders the applications and production of ornamental tree peony. - Unfortunately, the molecular mechanism of regulating flowering time and floral organ development in tree peony has yet to be elucidated. - Moreover, it was also found that the genes PsAP1, PsCOL1, PsCRY1, PsCRY2, PsFT, PsLFY, PsLHY, PsGI, PsSOC1, and PsVIN3 probably regulated re-blooming of tree peony.. - Conclusion: This study provides a comprehensive report on the flowering-related genes in tree peony for the first time and investigated the expression levels of the critical flowering related genes in buds of different cultivars, developmental stages, differentiated primordium, and flower parts. - Tree peony (Paeonia suffruticosa Andrews) belongs to section Moutan DC of the genus Paeonia and family Paeoniaceae and is the first candidate for China’s national flower. - Tree peony is valued all over the world due to its large and colorful flowers [1, 2]. - suffruticosa were the result of tree peony breeding breakthroughs since 1997 (Martin, 1997. - Now, tree peony cultivars can be geographically classified into seven worldwide groups: (1) Chinese Zhongyuan cultivars, (2) Chinese Xibei cultivars, (3) Chinese Xinan cultivars, (4) Chinese Jiangnan cultivars, (5) European cul- tivars, (6) American cultivars, and (7) Japanese cultivars [1]. - The dif- ferent flowering time and long flowering period are very important for applications and potted production of tree peony. - Thus, understanding of the molecular mechanism of flowering time in tree peony could provide a theoretical basis for flowering regulation and breeding.. - In the past decade, forcing culture technology and re-blooming in autumn was first investi- gated to achieve tree peony flowering at the proper time. - on flowering quality, endogenous hormones, and hor- mone- and flowering-associated gene expression in a forcing culture of tree peony were also deciphered [13]. - Endo-dormancy-imposed growth arrest is one of the key characteristics preventing tree peony from flowering well. - used a subtractive cDNA library and transcriptome sequencing, respectively, to identify key genes associ- ated with the release of dormant buds in tree peony;. - PsFT, PsVIN3, PsCO, and PsGA20ox were identified to play import- ant roles in the regulation of re-blooming in tree peony by transcriptome sequencing [16]. - However, the detailed mechanism of the flowering induction pathway is unclear in tree peony, which affects the improvement of the quality of the for- cing culture of tree peony.. - Then, the critical flowering-related genes were also se- lected to do expression analysis in different tree peony cultivars, and buds at different developmental stages or under different treatments. - Our re- sults provide valuable insights into the molecular mechanisms of flowering time regulation and floral organ development of tree peony.. - 454 GS-FLX sequencing and a de novo assembled tree peony transcriptome. - Functional annotation of tree peony transcriptome We performed BLASTx (version 2.2.21) analysis against several protein databases: NCBI non-redundant (NR) protein, Swiss-Prot, Clusters of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) using a cut off E-value of e-5 to annotate tree peony transcriptome. - In addition of the unigenes were annotated in the Pfam database. - The detailed results for annotation of the tree peony unigenes are summarized in Table 1.. - A summary of the findings is presented in Fig. - It was suggested that the circadian rhythm was probably important for tree peony flowering.. - Table 1 The annotations of tree peony bud unigenes against the public databases. - The possible reason was that HN is a tree peony hybrid (P. - 2 COG functional classification of the tree peony bud transcriptome Fig. - b Species distribution of the ten top BLASTx hits. - Unlike in other model plants, the genetic network of flowering for tree peony is unclear. - 3 GO classification of the tree peony bud transcriptome. - Relative expression analysis of DEGs related to flowering in the buds of four tree peony cultivars and one wild species. - Combining the flowering characters of five tree peony cultivars, AP1, COL1, CRY1, FT, GI, LFY, LYH, SOC1, SPL9, SVP , and VIN3 were shown to be associated with tree peony autumn flowering or re- blooming. - It was deduced that tree peony flowering was. - The expression patterns of the above genes were also detected in the buds from stage A (bud swelling) to stage H (color exposing) to detect the function of regulating flowering again. - Generally, stages A to E are very important for tree peony flowering, especially for flowering of the forcing culture tree peony. - 4 KEGG classification of the tree peony bud transcriptome. - these genes are suspected to repress tree peony flowering. - PsSPL9 had higher ex- pression in the bud from stages A to G and may also take part in flowering regulation and bud develop- ment in tree peony (Fig. - Above all, long day, GA, age, and vernalization pathways were shown to be important for the flowering induction pathway in tree peony. - Tree peony is long day plants, and the differentially expressed unigenes (Phy A, Phy B, FKF1, CRY, GI, LHY , FT, TOC1, etc.) were mainly involved in the circadian rhythm pathway (Additional file 3: Figure S2). - This result indicated that the long day pathway is very important for regulating tree peony autumn flowering or re-blooming. - Most of the genes had high expression in the spring buds (Fig. - According to the gene function in the model plants, those genes were key genes regulating plant flowering, and higher expression of these genes may be induced by tree peony flowering. - The expression levels of the flowering time genes were consistent with their functions. - Expres- sion results of those genes further validated that tree peony flowering could be induced by the GA pathway in the short day pathway rather than the long day pathway.. - Petal develop- ment was very complex most likely due to extremely abundant flower types of tree peony. - Tree peony has large and colorful flowers that are valued globally. - However, the short and relatively uniform flowering period is an important hindrance for tree peony production. - In this paper, we employed RNA-seq technology on buds of different tree peony cultivars to identify putative genes in- volved in flowering, floral organ development, and re-. - Flowering habit and period of different tree peony cultivars. - Now, more than 2000 tree peony cultivars have been cultivated by long-term artificial selection and cross breeding [1, 2, 23]. - Almost all of the cultivars flower once in the spring. - The large genome of tree peony (about 12.5 Gb) coupled with a large amount of repetitive DNA has prevented genome sequencing projects in this species.. - Thus, four tree peony cultivars with dif- ferent flowering habit and period and one wild species were used to do RNA-seq to decipher the mechanism of. - The highest matched species of the annotated sequences was V. - Different developmental buds of the same cultivar also had a large number of DEGs. - Although the analysis of DEGs in re- blooming and non-re-blooming cultivars, or early and late flowering period cultivars has identified some im- portant flowering time genes and re-blooming genes using Illumina HiSeq™ 2000 and Illumina HiSeq™ 2500 platforms the flowering induction pathway in tree peony remains unknown. - Some flower- ing genes, such as FRI, CRY1, PHYA, TFL, and FVE were identified for the first time in tree peony. - Finally, 67 flowering time genes involved in the flowering induction pathway, floral integrators, repres- sors, promoters, and organ development were obtained (Table 2), representing the most comprehensive report of flowering genes in tree peony.. - Expression levels of the flowering-related genes were compared in the buds of four tree peony cultivars, one wild species, and two developmental buds of ‘Ziluo Lan’ to determine the putative schematic network of flowering in tree peony. - expression levels of those genes (Additional file 5: Table S2), and their functions in model plants [9], the completed schematic network of flowering induction pathways of tree peony was proposed. - it was deduced that the GA pathway and vernalization pathway were also important pathways in tree peony [13, 16]. - 12 The putative schematic network of flowering induction pathways and floral organ development in tree peony. - transition, suggesting that the age pathway was also important in tree peony [12]. - Above all, five main pathways, autonomous, long day, vernalization, age, and gibberellin regulated flowering in tree peony.. - the expression patterns of the above genes further confirmed the reliability of the flower- ing induction pathways of tree peony. - More flower- ing genes should be identified to improve the genetic network of flowering regulation in tree peony in the future.. - DEGs and the specification of flower organ development Flower type is an important ornamental character, and there are ten flower types in tree peony. - All of the floral homeotic genes in tree peony were characterized in our study, providing valuable gene resources for flower type investigation.. - The bract covering the outside of the sepal was very large and unique in tree peony and the expression levels of the eight genes were investigated in the bract. - Based on these data, the genetic network of floral organ development in tree peony was postulated (Fig. - The floral organ development model of tree peony is an ABCE model, consistent with previous studies [32]. - Alternative splicing and variation of floral organ genes is also involved in abun- dant flower type forming in tree peony (data unpub- lished). - This postulated genetic network could provide a theoretical basis for tree peony flower type breeding.. - The important re-blooming genes of tree peony. - Four genes, PsCO, PsFT , PsGA20ox, and PsVIN3 probably play important roles in the regulation of the re-blooming process in tree peonies [16]. - This work presents de novo transcriptome sequencing analysis of tree peony flower development using the 454 GS-FLX platform. - A total of 67 flowering-related genes were identified in tree peony, and the genetic regulation network of the flowering induction pathways and floral organ de- velopment were postulated. - Moreover, the genes that regulated re-blooming in tree peony were proposed.. - Our work provides a theoretical basis for tree peony forcing culture and breeding for flowering period and flower type.. - All of the cultivars and one wild species were introduced from Luoyang Tree Peony Gene Bank, China (there are no Genbank numbers, and only cultivar names in the Tree Peony Gene Bank), and grown in the Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences. - ‘Luoyang Hong’ was always used for tree peony. - 13 The morphology of the seven samples used for transcriptome sequencing. - Based on “the significance of digital gene ex- pression profiles”, differentially expressed genes (DEGs) between samples and their corresponding P-value were determined using methods described by Audic and Clav- erie [38]. - Relative expression levels of the candidate genes were calculated by normalizing to the reference gene ACTIN [1]. - The unigenes involved in the plant circadian rhythm in bud of tree peony. - The differentially expressed genes based on comparisons of any two samples in bud transcriptome sequencing in tree peony. - The expression level analysis of the flowering-related genes in the seven samples by the Reads per Kilobase per Million mapped reads (RPKM) method. - Primer name Sequences of the primers. - Molecular characterization and expression patterns of PsSVP genes reveal distinct roles in flower bud abortion and flowering in tree peony (Paeonia Suffruticosa). - Molecular cloning and potential function prediction of homologous SOC1 genes in tree peony. - Forcing of tree peony for december shipping using spring- and winter-blooming cultivars. - Effect of exogenous GA 3 on flowering quality, endogenous hormones, and hormone-, and flowering-associated gene expression in forcing-cultured tree peony (Paeonia suffruticosa). - Transcriptome analysis of tree peony during chilling requirement fulfillment: assembling, annotation and markers discovering. - Screening of genes related to early and late flowering in tree peony based on bulked segregant RNA sequencing and verification by quantitative real-time PCR. - Molecular cloning and expression analysis of cryptochrome gene PsCRY2 in tree peony.. - Analysis of the formation of flower shapes in wild species and cultivars of tree peony using the MADS-box subfamily gene. - Tree peony of China
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