De novo sequencing of tree peony (Paeonia suffruticosa) transcriptome to identify critical genes involved in flowering and floral organ development

Abstract

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. Because of the absence of genomic information, 454-based transcriptome sequence technology for de novo transcriptomics was used to identify the critical flowering genes using re-blooming, non-re-blooming, and wild species of tree peonies.

Results: A total of 29,275 unigenes were obtained from the bud transcriptome, with an N50 of 776 bp. The average length of unigenes was 677.18 bp, and the longest sequence was 5815 bp. Functional annotation showed that 22,823, 17,321, 13,312, 20,041, and 9940 unigenes were annotated by NCBI-NR, Swiss-Prot, COG, GO, and KEGG, respectively. Within the differentially expressed genes (DEGs) 64 flowering-related genes were identified and some important flowering genes were also characterized by bioinformatics methods, reverse transcript polymerase chain reaction (RT-PCR), and rapid-amplification of cDNA ends (RACE). Then, the putative genetic network of flowering induction pathways and a floral organ development model were put forward, according to the comparisons of DEGs in any two samples and expression levels of the important flowering genes in differentiated buds, buds from different developmental stages, and with GA or vernalization treated. In tree peony, five pathways (long day, vernalization, autonomous, age, and gibberellin) regulated flowering, and the floral organ development followed an ABCE model. 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. These results could provide valuable insights into the molecular mechanisms of flowering time regulation and floral organ development.

Keywords: Floral model; Flowering induction pathway; MADS-box gene; Re-blooming; Transcriptome; Tree peony.

Fig. 1

Statistics of homology search of unigenes against NR database. a E-value distribution of the top BLASTx hits with a cut-off e-value of 1e-05. b Species distribution of the ten top BLASTx hits

Fig. 2

COG functional classification of the tree peony bud transcriptome

Fig. 3

GO classification of the tree peony bud transcriptome

Fig. 4

KEGG classification of the tree peony bud transcriptome

Fig. 5

The expression level validation of 12 DEGs in the buds of four cultivars and one wild species by qRT-PCR. ZLL T, PD T, HCH T, HN T, and LHY T represent the five samples used for transcriptome sequencing

Fig. 6

The expression levels of 12 important flowering genes in different primordium buds of ‘Ziluo Lan’. UN, Br P, Se P, Pe P, St P, and Pi P represent buds at the following stages: undifferentiated, bract primordium, sepal primordium, petal primordium, stamen primordium, and pistil primordium, respectively

Fig. 7

The expression levels of 12 important flowering genes in eight flowering process buds of ‘Ziluo Lan’. A-H represent stages of bud sprouting, leaflet emerging, flower bud emerging phase, flower bud clearly exposed with leaf appearance, small bell-like flower bud, big bell-like flower-bud, bell-like flower-bud extending, and color exposed, respectively

Fig. 8

The expression levels of flowering genes PsCRY1, PsCRY2, PsLHY, PsGI, and PsSOC1 in buds at the first three developmental stage of ‘Ziluo Lan’. A, B, and C represent stages of bud sprouting, leaflet emerging, flower bud emerging phase, respectively, and Sp and Au represent spring and autumn, respectively

Fig. 9

The expression levels of flowering genes PsAP1, PsFT2, PsLFY, PsSOC1, and PsVRN3 in buds with different treatments. CK represents ‘Ziluo Lan’ with no treatment, while VRN represents ‘Ziluo Lan’ with vernalization treatment

Fig. 10

The expression levels of GA synthesis genes PsGAI and PsGID1, and flowering time genes PsLFY, PsSOC1, and PsSVP in GA₃ treated buds of ‘Ziluo Lan’. CK and CK 1 W represent buds after 0 h and 1 week without GA₃ treatment, respectively, while GA 4 h, GA 8 h, GA 12 h, GA 24 h, and GA 1 W represents buds after 4 h, 8 h, 12 h, 24 h with GA₃ treatment, respectively

Fig. 11

The expression levels of eight floral homeotic genes in five flower parts, and different differentiated primordium stages buds. UN, Br P, Se P, Pe P, St P, and Pi P represent buds at the following stages: undifferentiated, bract primordium, sepal primordium, petal primordium, stamen primordium, and pistil primordium, respectively

Fig. 12

The putative schematic network of flowering induction pathways and floral organ development in tree peony. Arrows indicate positive regulation and bars indicate negative regulation

Fig. 13

The morphology of the seven samples used for transcriptome sequencing. HCH, LHY, PD, HN, ZLL, ZLL-D, and ZLL-E represent the peony names, ‘Huchuan Han’, ‘Luoyang Hong’, P. delavayi’, ‘High Noon’, ‘Ziluo Lan’, clearly exposed buds of ‘Ziluolan’, and small bell-like flower bud of ‘Ziluolan’, respectively