- In the three comparisons (the floral initiation stage of spring flowering in once-bloomers (OB-T1) vs the floral initiation stage of spring flowering in rebloomers (RB-T1). - As a result, the following four genes, PHYTOCHROME A (PHYA), GIGANTEA (GI), SHORT VEGETATIVE PERIOD (SVP) and AUXIN RESPONSE FACTOR (ARF), were considered to be involved in the second floral initiation of the rebloomers.. - In the long breeding his- tory, some reblooming bearded irises occurred, which could bloom for the second time in autumn of the same. - The more ornamental stages, the unique three falls and standards, the various flower colors and the ease of propagation, make the rebloomers increasingly popular in the market. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The floral transition has been widely studied in the an- nual plant Arabidopsis, which is controlled by six main pathways [6, 7]. - As for the reblooming perennials, temperature influenced the remontancy in the remon- tant octoploid strawberry (Fragaria × ananassa) [8], which could attribute to the temperature tolerance in the flower initiation of remontant strawberry [9]. - In the study of remontant Hydrangea macrophylla, floral in- duction occurred under short-day (SD) and extended- day (ED) conditions but was more rapid under SD than ED [10]. - In Fragaria vesca, a 2-bp deletion in the coding region of the TFL1 homologue in- troduced a frame shift and reversed the photoperiodic requirement for flowering, thus generating continuous flowering behavior [11, 12]. - The putative regulatory genes related to reblooming may be applied in the future molecular breeding of rebloomers, which could accelerate the breeding process. - In the F 1 population, the flowering rate in spring was 65.23%, while the reblooming rate in autumn was 27.94%, suggesting that there existed once-bloomers and rebloo- mers in the same hybrid population (Additional file 1:. - In the reblooming season (au- tumn), the phenotypic values of PH (plant height) and DF (diameter of flower) were smaller than those of their parents, the decrease of which may be owing to the comparatively cold climate in autumn. - In the GO analysis, 29,310 unigenes were assigned to 42 GO terms under three categories (Fig. - The proportions of “catalytic activity” and “binding” were the largest in the “molecular function” category. - In the comparison of OB-T1 vs RB- T1, a total of 1363 genes were differentially expressed (786 up-regulated and 577 down-regulated). - Fur- thermore, in the Venn diagram, a total of 27 DEGs existed in all the three comparisons (Fig. - In the KEGG pathway analysis, we mainly focused on the “plant hormone signal transduction” under the “en- vironmental information processing” category and the. - In the comparison of OB-T1 vs RB-T1, two DEGs were annotated into the “plant hormone signal transduction” pathway, and five DEGs were annotated into the “circadian rhythm-plant” pathway (Additional file 1: Table S7, Additional file 2: Fig. - In the com- parison of RB-T1 vs RB-T5, the numbers of DEGs anno- tated into “plant hormone signal transduction” pathway and “circadian rhythm-plant” pathway were 43 and 25, respectively (Additional file 1: Table S8, Additional file 2: Fig. - Similarly, in the comparison of OB-T1 vs RB- T5, the numbers of DEGs annotated into “plant hor- mone signal transduction” pathway and “circadian rhythm-plant” pathway were 38 and 20, respectively (Additional file 1: Table S9, Additional file 2: Fig. - In addition, these two pathways were listed in the top 20 enriched KEGG pathways (Fig. - In the GO analysis of DEGs from the three compari- sons (OB-T1 vs RB-T1, RB-T1 vs RB-T5, OB-T1 vs RB- T5), “metabolic process” was the most enriched GO terms in the category of “biological process”. - “cell” was the most annotated in the “cellular component” cat- egory. - “catalytic activity” was most abundant in the “mo- lecular function” category (Additional file 1: Table S10, Additional file 2: Fig. - The redundant GO terms were removed and the GO differences between the three comparisons were visualized in the Reduce and Visualize GO (REVIGO) analysis. - The GO terms in the “biological process” part, such as photosynthesis, light harvesting in photosystem I (GO:0009768), auxin biosynthesis (GO:. - Similarly, photosystem I antenna complex (GO:0009782), photosystem I (GO:0009522) and light- harvesting complex (GO:0030076) in the “cellular com- ponent” part (Fig. - (C) Species distribution of the top BLAST hits. - 0009881) in the “molecular function” part (Fig. - Therefore, the photoperiod related genes, as one of the output pathways of the circadian clock, may partici- pate in the formation of reblooming.. - In the comparison of the two floral initiation stages in rebloomers (RB-T1 vs RB-T5), SUPPRESSOR OF PHYTO- CHROME A1 (SPA1), PHYTOCHROME A (PHYA), CRYP TOCHROME (CRY) and GIGANTEA (GI) were signifi- cantly up-regulated in RB-T5 compared to RB-T1 (Fig. - Moreover, in the comparison of the two floral initi- ation stages in once-bloomers and rebloomers (OB-T1 vs RB-T5), CCA1, GI, SPA1, PHYA and CRY were signifi- cantly up-regulated. - 7a-b), three DEGs were categorized into the “ starch and sucrose me- tabolism ” pathway in the KEGG database (Fig. - Besides, one AUXIN RESPONSE FACTOR (ARF) was detected to differentially express in all the compari- sons of two adjacent periods in rebloomers, but was ab- sent in the counterparts of once-bloomers (Fig. - SHORT VEGETATIVE PERIOD (SVP), an important flowering suppressor, was identified as a DEG in the comparison. - In rebloomers, the expres- sion level of SVP in the second floral initiation period (RB-T5) was much lower than that of the first one (RB- T1), thus fascinating the second floral initiation. - How- ever, TFL1, another flowering repressor controlling the remontant flowering in rose and strawberry showed a higher expression level in the second floral ini- tiation period, the reason of which need further investi- gation. - 8 were analyzed in the STRING database (Additional file 2: Fig. - In the hub gene analysis by Cytoscape, GI was identified as a hub gene that interacted with all the top 10 interaction nodes (Additional file 2: Fig. - Therefore, GI, PHYA and FT may be key regulators in the development of flowers and played an essential role in the formation of rebloomers.. - The construction of a hybrid population is an essential step for effectively detecting key genes of reblooming Transcriptome profiling is an effective way to detect DEGs from various sequencing groups, thereby provid- ing clues to the detection of key genes involved in the target characters. - 5 GO pathway analysis for DEGs in the three comparisons (OB-T1 vs RB-T1, RB-T1 vs RB-T5, OB-T1 vs RB-T5) by using REVIGO. - Bubble color represents the log10 (P-value) for false discovery rates, as shown in the scale on the right. - germanica is an essential ornamental perennial in the spring garden, and the reblooming ones can provide di- versity to the autumn landscape. - The anno- tation rate could be improved if the genomic informa- tion is available in the future.. - The analysis of the most enriched GO terms in the three categories showed that most terms were related to light sensing and harvesting, cold accli- mation and growth speed, such as photosynthesis, light harvesting in photosystem I (GO:0009768), auxin bio- synthesis (GO:0009851) and cold acclimation (GO:. - In the comparison of the floral initiation stages of spring flowering in once-bloomers and rebloomers (OB- T1 vs RB-T1), the numbers of DEGs annotated into. - It could be concluded that the gene expression levels of once-bloomers and rebloomers were similar in the spring flower initiation stage. - S4B-C), indicating the different gene expression pat- terns in the first and second floral initiation stages.. - This periodicity is mirrored by daily periodicity in the behavior and physiology of or- ganisms [15]. - In the hub gene analysis, IgPHYA, IgGI and IgFT are among the top 10 nodes with most interactions to other genes, indicating their important roles in the regulation of reblooming (Additional files 2: Fig. - In the circa- dian rhythm regulatory network, the photoreceptors PHYA (a far-red-light receptor) and CRY2 (a blue-light receptor) could stabilize the expression of CONSTANS (CO) at the end of a long day [34, 35]. - In the putative pathway, the expres- sion level of PHYA and GI in T5 was much higher than that in T1, which resulted in the different expression of its downstream gene CO in OB-T1 vs RB-T5, and the rapid increase of FT and AP1 on the stage of second flower initiation (Fig. - In the flower development stages, although the expression of FRI was enhanced in the second floral initiation (T5), its down- stream gene FLC was not screened as a DEG (Fig. - However, TFL1 was highly expressed in the second floral initiation period of reblooming iris, the reason of which need further investigation.. - All the upstream signals integrated and resulted in the lower ex- pression of IgSVP (a flowering repressor) in the second floral initiation stage, thereby causing the reblooming characteristics. - R20130005), introduced from the Hall’s Flower Garden (West Alexandria, Ohio, USA), have been conserved in the scientific base of Beijing Botanical Garden, Beijing, China for many years. - The experimental materials were all cultivated in the Changping District of Beijing, China (40°09′15″N E). - The individuals in the F 1 generation were distributed in a randomized complete block design. - The significant differences among various characters were tested with one-way ANOVA using Duncan’s new multiple range in the SPSS 22.0 software. - De novo assembly of the I. - In the GO enrichment analysis, the DEGs were imple- mented using topGO R packages based on the Kolmogo- rov–Smirnov test. - The putative DEGs in the reblooming network were ana- lyzed in the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) online database, in order to construct the PPI network (https://string-db.org/) [48].. - Then the credible PPI interactions were further analyzed in the Cytoscape software to visualize the hub genes [49].. - The expression levels and annotations of the DEGs. - germanica in the six sampled stages. - KEGG pathways significantly enriched in DEGs in the comparisons of OB- T1 vs RB-T1 (A), RB-T1 vs RB-T5 (B) and OB-T1 vs RB-T5 (C). - GO terms significantly enriched in DEGs in the comparisons of OB-T1 vs RB- T1 (A), RB-T1 vs RB-T5 (B) and OB-T1 vs RB-T5 (C). - (B) The graphical depiction of the top 10 interaction nodes in the hub module.. - CJG helped in the molecular biology analysis of this experiment. - The funders did not have any role in the design of the study, collection, analysis, or interpretation of data or the writing of the manuscript.. - 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