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Whole-transcriptome profiles of Chrysanthemum seticuspe improve genome annotation and shed new light on mRNA–miRNA–lncRNA networks in ray florets and disc florets

Abstract

Background

Chrysanthemum seticuspe has emerged as a model plant species of cultivated chrysanthemums, especially for studies involving diploid and self-compatible pure lines (Gojo-0). Its genome was sequenced and assembled into chromosomes. However, the genome annotation of C. seticuspe still needs to be improved to elucidate the complex regulatory networks in this species.

Results

In addition to the 74,259 mRNAs annotated in the C. seticuspe genome, we identified 18,265 novel mRNAs, 51,425 novel lncRNAs, 501 novel miRNAs and 22,065 novel siRNAs. Two C-class genes and YABBY family genes were highly expressed in disc florets, while B-class genes were highly expressed in ray florets. A WGCNA was performed to identify the hub lncRNAs and mRNAs in ray floret- and disc floret-specific modules, and CDM19, BBX22, HTH, HSP70 and several lncRNAs were identified. ceRNA and lncNAT networks related to flower development were also constructed, and we found a latent functional lncNAT–mRNA combination, LXLOC_026470 and MIF2.

Conclusions

The annotations of mRNAs, lncRNAs and small RNAs in the C. seticuspe genome have been improved. The expression profiles of flower development-related genes, ceRNA networks and lncNAT networks were identified, laying a foundation for elucidating the regulatory mechanisms underlying disc floret and ray floret formation.

Peer Review reports

Background

Comprising approximately 25,000 species, Asteraceae is the largest family of flowering plants, with members occurring across all continents except Antarctica [1]. Plants of Asteraceae are characterized by complex inflorescences (capitulum), which include both disc florets in the center and ray florets outside. There are only pistil and zygomorphic petals on ray florets, while there are developed pistil, stamen and actinomorphic petals on disc florets [2]. Chrysanthemums (Chrysanthemum morifolium Ramat.) are among the most important cut flowers in the world, are traditional flowers in China, and have high economic value [3]. Due to both polyploidy and hybridization within the genus Chrysanthemum, it is difficult to understand the evolution of these species and their underlying molecular basis [4]. Chrysanthemum seticuspe, a model plant species of cultivated chrysanthemum plants, has been used for studies involving diploid and self-compatible pure lines (Gojo-0) [5]. The whole genome of Gojo-0 has been sequenced and assembled at the chromosome level [6]. However, organ-specific whole transcriptomes, including mRNAs, lncRNAs and miRNAs, have not been sequenced. To better elucidate the regulatory mechanism of ray florets and disc florets, these molecular networks should be studied.

The development of floral organs is determined by ABCE genes, which regulate floral morphogenesis at various levels together with elaborate networks [7]. The expression levels of ABCE genes determine the specifying organ identity, including that of petals, pistils, sepals and stamens. Suppression of the C-class gene CAG results in the conversion of the pistils and stamens into corolla-like tissues in florets of C. morifolium [8]. Furthermore, transcription factors other than ABCE genes were also reported to be related to the morphogenesis of florets in the capitulum. Dorsal identity is closely related to the TCP family transcription factor CYCLOIDEA (CYC) [9]. Overexpression of GhCYC5 increases the flower density in the capitulum of Gerbera hybrida [10]. Moreover, knockdown of CYC2g in Chrysanthemum lavandulifolium promotes the gradual transition from ray florets into disc florets [11]. CUP SHAPED COTYLEDON (CUC) transcription factors were found to limit cell growth and create a creased shape in the boundaries [12]. Additionally, the YABBY gene family has been found to specify abaxial cell fate, and mutants of this family showed that they can act in both distinct and redundant manners [13]. However, studies of flower development genes in the genera Chrysanthemum and Gerbera are still not sufficient to elucidate the formation mechanism of ray florets and disc florets.

Noncoding RNAs (ncRNAs) have been found to be involved in various biological processes in both animals and plants [14]. Including small RNAs and long noncoding RNAs (lncRNAs), ncRNAs participate in almost all biological processes by interacting with coding genes at both the transcriptional and posttranscriptional levels in plants [15]. MicroRNAs (miRNAs) can trigger posttranscriptional repression by targeting mRNA through high complementarity [16]. In tomato, sly-miR160a regulates blade outgrowth, leaf and leaflet initiation and floral organ development by targeting SlARF10A and adjusting auxin-mediated development [17]. lncRNAs are RNAs with a length of more than 200 nt and no protein-coding capability; lncRNAs include long intergenic noncoding RNAs (lincRNAs) and long noncoding natural antisense transcripts (lncNATs), which are denoted according to their position relative to genes [18]. lncRNAs regulate biological mechanisms of gene expression, including chromatin remodelling, modulation of alternative splicing, fine tuning of miRNA activity, and the control of mRNA translation or accumulation [19]. A lncNAT (TWISTED LEAF) has been shown to influence leaf blade flattening in rice through its regulation of the expression of OsMYB60 [20]. Another lncRNA (MISSEN) in rice binds to a helicase family protein (HeFP), which ultimately damages the protein complex of HeFP and tubulin during endosperm nuclear division, resulting in abnormal cytoskeletal polymerization [21]. LncRNAs can also act as competing endogenous RNAs (ceRNAs) to modulate gene expression by mimicking miRNAs. Both lncRNA23468 and NBS-LRR genes are targeted by miR482b in tomato, and the downregulation of lncRNA23468 leads to the downregulation of NBS-LRR genes [22]. Although lncRNAs play an important role in plant development, their biological mechanisms in flower morphogenesis remain unclear, which prompted us to explore their expression profiles in flowers.

Whole-transcriptome sequencing is useful for constructing mRNA–miRNA–lncRNA regulatory networks and has been used in Populus and Liriodendron chinense [23, 24]. In this study, we sequenced whole transcriptomes of mRNAs, lncRNAs and small RNAs in five different organs (leaves, stems, roots, disc florets and ray florets) of C. seticuspe, which are shown in Fig. 1. These transcriptomes improved the annotation of the genome and showed overall gene expression profiles. To identify networks related to flower development genes in C. seticuspe, latent mRNA–lncRNA-miRNA regulatory networks were also discovered with weighted gene correlation network analysis (WGCNA), positional relationships in the genome and prediction of targeting miRNAs. In summary, our study will contribute to our understanding of the mechanism of lncRNAs in capitulum flower morphogenesis.

Fig. 1
figure 1

Morphology of five organs sampled from the seedlings of Gojo-0. (a-b) The adaxial surface and abaxial surface of a leaf. Bar, 1 cm. (c) A flower bud at the sampling stage. This stage is the final stage of corolla primordia differentiation according to Wen’s study [25]. Bar, 1 mm. (d) Root. Bar, 5 cm. (e-f) Single ray floret and disc floret of a head-like inflorescence. Bar, 1 mm. (g) Intact seedling. Bar, 5 cm

Results

Transcriptome sequencing

The Q20 values, percentage of clean data and total mapping percentage of the lncRNA, mRNA and small RNA library sequences of each sample are shown in Table 1 and Table 2. The mean Q20 value of the lncRNA and mRNA libraries was more than 97% and that of the small RNA library was more than 98%. The percentage of clean reads in the lncRNA and mRNA libraries was more than 91%, and the percentage of clean tags in the small RNA library was more than 92%. The total mapping percentage of the lncRNA and mRNA libraries was more than 77% and that of the small RNA library was more than 89%. After filtration, a total of 1,844,502,354 clean reads were generated for the lncRNA and mRNA libraries, and a total of 418,586,690 clean tags were generated for the small RNA libraries.

Table 1 Results of the lncRNA–mRNA library sequencing of five organs
Table 2 Results of the small RNA library sequencing of five organs

Following assembly, 501 miRNAs and 22,065 siRNAs were identified in the small RNA library. Except for 74,259 mRNAs identified in C. seticuspe, there were 18,265 novel mRNAs and 51,425 novel lncRNAs annotated in the lncRNA and mRNA libraries. The lncRNA and mRNA distribution within chromosomes was analysed, the results of which revealed a more well-distributed location of lncRNAs (Fig. 2). Although the most frequent number of exons for lncRNAs and mRNAs was 1, the average exon number of mRNAs was greater than that of lncRNAs (Fig. S1). The most frequent sequence length distributions of lncRNAs and mRNAs were 0–500 bp and 500–1000 bp, respectively, which indicated that the average length of mRNAs was larger than that of lncRNAs (Fig. S2). The average fragments per kilobase per million mapped reads (FPKM) values of the lncRNAs and mRNAs were 15.7 and 8.9, respectively.

Fig. 2
figure 2

Distribution of mRNAs and lncRNAs on each chromosome in physical bins of 10 Mb (visualized with circlize version 0.4.15 [26])

Expression quantification and comparison of differentially expressed genes (DEGs)

The expression levels of mRNAs, lncRNAs and small RNAs were quantified in different samples. Three biological replicates showed relatively high Pearson correlation coefficients for all three RNAs (Fig. 3 a-c). The clustering analysis of expression profiles also showed a high degree of consistency across biological replicates (Fig. 3 d-f). With respect to mRNAs, the expression profile of disc florets was strongly related to that of ray florets, while the roots were weakly related to the other four organs. Regarding lncRNAs, the expression profile of the leaves was weakly related to the other four organs, which indicated the unique expression patterns of lncRNAs in the leaves. With respect to miRNAs, the expression profile of the leaves was strongly related to that of the roots and stems but was weakly related to that of the ray florets and disc florets. DEGs were identified in different organ comparisons with the following threshold criteria: p value ≤0.01 and |log2| ≥ 1. The DEGs of mRNAs and lncRNAs were counted in all comparisons (Fig. S3 and S4). The most significant comparison for both mRNAs and lncRNAs was ‘disc floret vs. root’, in which there were 23,184 and 17,567 DEGs, respectively. The least significant comparison for both mRNAs and lncRNAs was ‘disc floret vs. ray floret’, in which there were 7061 and 5211 DEGs, respectively.

Fig. 3
figure 3

Relevance and expression profiles of mRNAs, lncRNAs and small RNAs among different samples. Calculations and visualizations of Pearson correlation coefficients of mRNAs (a), lncRNAs (b) and small RNAs (c). The expression profiles of mRNAs (d), lncRNAs (e) and small RNAs (f) and the transcripts per million (TPM) and FPKM values were log2 transformed. The heatmap plots were visualized with the R package Pheatmap (version 1.0.12) [27]. Clustering was performed by the hclust method in R

Expression patterns of flower development-related genes

To investigate the developmental differences between ray florets and disc florets, we identified flower development-related genes with the BLASTX algorithm by inputting related genes from Arabidopsis thaliana. The expression levels of flower development-related genes are shown in Fig. 4. ABCE flower development-related genes displayed higher expression levels in ray florets and disc florets than in the other three organs. Three transcripts (AP3.1/CsG_LG4.g53779.1, AP3.2/CsG_LG8.g58037.i1 and PI.1/CsG_LG9.g33362.i1) of B-class genes had higher transcription in ray florets than in disc florets, similar to the expression of an E-class gene (SEP3.3/CsG_LG7.g49236.1) (Fig. 4 a). However, two of the C class genes (AG.1/CsG_LG9.g43875.i1 and AG.2/CsG_LG7.g04334.i1) showed higher expression levels in the disc florets than in the ray florets. The expression levels of three transcripts of the CUC family showed relatively low expression in all five organs. There were higher expression levels of YABBY1.1, YABBY1.4 and YABBY5 in the disc florets than in the ray florets, suggesting that these genes have a possible regulatory function in the development of flower symmetry (Fig. 4 b). TCP2.1, TCP4.2, TCP5.1 and TCP19 showed relatively high expression levels in the ray florets; however, TCP2.2 displayed higher expression levels in both the ray florets and the roots (Fig. 4 c). In addition, there was a relatively high expression level of TCP7 in the roots.

Fig. 4
figure 4

Expression patterns of flower development-related (a) ABCE genes, (b) CUC and YABBY family genes and (c) TCP family genes. Bar, log2-transformed FPKM values

Network analysis based on the WGCNA results

To elucidate the latent regulatory network of C. seticuspe, we performed WGCNA to identify the hub genes related to the development of ray florets and disc florets. After clustering analysis was performed, 12 major modules were identified, which were related to different organs (r > 0.7 and P < 0.001) (Fig. 5 a and b). In the brown module, there were 8840 genes, which were found to be highly associated with disc florets. The blue module (14,787 genes) was associated with ray florets, the yellow module (7238 genes) was associated with leaves, the green module (6284 genes) was associated with stems, and the turquoise module (20,044 genes) was associated with roots.

Fig. 5
figure 5

Weighted gene correlation network analysis. a Results of a hierarchical cluster analysis performed by WGCNA. Each leaf of the tree represents a single gene, and the tree branches cut to a height of 0.4 produced different modules with specific colours. b Relationships between modules and organs. Each column and each row correspond to different organs and modules, respectively. In every box, the upper values are the correlation coefficients, and the lower values are the p values

The regulatory networks of ray florets and disc florets were constructed with the genes associated with high KME values in the modules, and the hub mRNAs and lncRNAs are shown in the center of the networks with orange colour; these hubs were selected based on a combination of their FPKM values and gene function (Fig. S5 a and b). In the ray floret-specific module, 3 hub mRNAs and 8 hub lncRNAs were identified. Two of these three genes encoded transcription factors, namely, a MADS-Box family gene (CMD19/CsG_LG8.g58037.i1) and a B-BOX gene (BBX22/CsG_LG6.g06424.i1). The other gene (HTH/CsG_LG5.g56450.i1) encodes a glucose-methanol-choline (GMC) oxidoreductase involved in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids [28]. The hub lncRNAs were LXLOC_086819, LXLOC_034411, LXLOC_026470, LXLOC_015065, LXLOC_042903, LXLOC_041244, LXLOC_053379 and LXLOC_037271. In the disc floret-specific module, one hub mRNA and three hub lncRNAs were identified. The mRNA was related to a heat shock protein family gene (HSP70/MXLOC_096069). The three hub lncRNAs were LXLOC_096252, LXLOC042445 and LXLOC_106636.

Analysis of ceRNAs and lncNATs of flower development-related genes

The predicted miRNAs targeting lncRNAs and mRNAs are listed in Table S1. The number of miRNAs targeting lncRNAs was 251, and that of miRNAs targeting mRNAs was 206, of which there were 139 miRNAs targeting both lncRNAs and mRNAs (Fig. S6). To identify the ceRNAs related to flower development, we predicted the miRNAs targeting both flower development-related genes and lncRNAs, the results of which are shown in Fig. 6. Five transcripts of AP2 (AP2.1/CsG_LG3.g24432.i1, AP2.2/CsG_LG4.Cse_sc005454.1_g020.1, AP2.3/CsG_LG2.g46212.i1, AP2.4/MTCONS_00030951 and AP2.5/MTCONS_00057150) and 8 lncRNAs were predicted to be targeted by miR172s (Fig. 6 a). In the TCP gene family, TCP9 (TCP9.2/MTCONS_00032755) and two transcripts of TCP2 genes (TCP2.1/CsG_LG9.g53441.1 and TCP2.2/CsG_LG7.g04758.1) were predicted to be targeted by miR5138 and miR319, respectively (Fig. 6 b and c). Two CUC2 genes (CUC2.1/CsG_LG5.g54126.1 and CUC2.2/CsG_LG7.g63595.i1) were predicted to be targeted by miR164b together with 6 lncRNAs (Fig. 6 d).

Fig. 6
figure 6

Prediction of ceRNA networks of (a) miR172-AP2, (b) miR5138-TCP9, (c) miR319-TCP2 and (d) miR164-CUC2. The rectangles represent mRNAs, the triangles represent miRNAs, and the diamonds represent lncRNAs

The lncNATs of flower development-related genes were also found to improve the regulatory network (Fig. 7). In the ray floret-specific module, one of the hub lncRNAs (LXLOC_026470) was located in the opposite chain of MIF2 (CsG_LG3.g28582.1). There was also a lncRNA of the hub mRNA CDM19 in the ray floret-specific module. There was a lncNAT of CUC2.2 (CsG_LG7.g63595.i1), which is targeted by miR164 as mentioned above, indicating that there is a complex gene regulatory network involving CUC2. There were also two lncNATs (LXLOC_031428 and LXLOC_023025) of AGL70 (CsG_LG3.g30433) and AP1.3 (CsG_LG2.g44914.i1).

Fig. 7
figure 7

LncNATs of flower development-related genes. The rectangles represent mRNAs, and the diamonds represent lncRNAs

qRT–PCR verification of transcriptomic data

qRT–PCR was performed to verify the FPKM values of flower development-related genes and hub genes in flower-specific modules. As shown in Fig. 8, the FPKM values show a high degree of consistency with the qRT–PCR results, indicating the reliability of the data. Notably, the expression level of MIF2, whose lncNAT (LXLOC_026470) is in the ray floret-specific module, was also quantified. The relative expression level of MIF2 in the disc florets was significantly greater than that in the ray florets, as shown in Fig. 8 n-o. In contrast, the relative expression level of LXLOC_026470 in the ray florets was significantly greater than that in the disc florets. This opposite trend for the relative expression levels suggests that MIF2 and its lncNAT-LXLOC_026470 might interact to control the development process of ray florets and disc florets.

Fig. 8
figure 8

Relative expression levels and FPKM values of flower development-related genes and hub genes in modules; correlation coefficients are shown at the top right. a CsG_LG8.g33949.1-AP1.1, (b) CsG_LG4.g53779.1-AP3.1, (c) CsG_LG9.g33362.i1-PI.1, (d) CsG_LG9.g43875.i1-AG.1, (e) CsG_LG7.g04334.i1-AG.2, (f) CsG_LG6.g50199.i1-SEP2.3, (g) CsG_LG7.g49236.1-SEP3.3, (h) CsG_LG7.g11579.i1-YABBY1.1, (i) CsG_LG5.g64343.i1-YABBY5, (j) MTCONS_00042147-CRC, (k) CsG_LG8.g58037.i1-CDM19, (l) CsG_LG6.g06424.i1-BBX22, (m) CsG_LG5.g56450.i1-HTH, (n) CsG_LG3.g28582.1-MIF2, (o) LXLOC_026470, (p) LXLOC_037271, (q) LXLOC_053379, and (r) LXLOC_086819. Blue bars represent the mean FPKM values, and red dots represent the mean qRT–PCR values. Error bars indicate the SD for three biological replicates based on qRT–PCR, and shared letters indicate no statistically significant difference between the means (P > 0.05) as determined by ANOVA. The correlation coefficient was calculated by R with mean FPKM values and relative transcript levels generated by qRT–PCR

Discussion

Differences in the expression of flower development-related genes between ray florets and disc florets

Petal type is of great importance for both the ornamental and evolutionary value of chrysanthemums. The expression levels of two B-class genes, AP3 and PI homologues (AP3.1, AP3.2 and PI.1) of ray florets, were also higher than those of disc florets in both C. lavandulifolium and C. morifolium [29], the results of which are consistent with the expression patterns in this study (Fig. 4 a). There was a protein–protein interaction between ClAP3 and ClPI in C. lavandulifolium, illustrating that the formation of heterodimers is needed for proper functioning. In C. morifolium, both AP3 and PI have relatively higher expression levels in the petals of ray florets, petals of disc florets and stamens of disc florets [2]. Therefore, B-class genes might play major roles in both petal and stamen development of both types of florets with conserved functions in the Chrysanthemum genus. Three of the C-class genes (AG.1, AG.2 and AG.3) were more highly transcribed in the disc florets than in the ray florets. The knockdown of two C-class genes (CAG1 and CAG2) in C. morifolium resulted in the transformation of reproductive organs into petaloid organs in both disc florets and ray florets, showing that these genes play important roles in the development of pistils and stamens [30]. C-class genes function synergistically with B-class genes to regulate the development of stamens [31]. These findings indicate that B-class and C-class genes might work together to control the development of petals and stamens in C. seticuspe. The E-class genes SEPALLATA are essential for floral identity; the expression levels of these genes determine the development of ectopic flowers instead of vegetative organs [32]. Most E-class genes were transcribed strongly in floral organs in this study (Fig. 4 a), indicating that these genes play essential roles in flower development. However, one transcript of SEP3 (SEP3.3) had a higher expression level in ray florets than in disc florets. SEP3 is able to function as a component of various protein complexes [33]. Therefore, the SEP3.3 protein might interact with other proteins to mediate the development of ray florets and disc florets in C. seticuspe.

YABBY genes encode proteins that include an N-terminal zinc finger domain and a C-terminal helix-loop-helix motif; these genes are involved in the abaxial domain determination of flower development [34]. In this study, the expression levels of YABBY family genes were higher in floral organs than in vegetative organs. Overexpression of YABBY1 in C. morifolium changed petal curvature and inflorescence morphology, of which the petals of ray florets in CmYAB1-overexpressing plants tend to be flat [35]. Therefore, the different flower symmetries of disc florets and ray florets in C. seticuspe might be related to the differential expression of YABBY family genes, which regulate zygomorphic and actinomorphic petal types in ray florets and disc florets, respectively.

Potential regulatory function of hub genes in ray floret- and disc floret-specific modules

Several hub mRNAs and lncRNAs were identified through WGCNA in both ray floret- and disc floret-specific modules (Fig. S5 a and b). CDM19 is also an AP3 homologue and shows a higher expression level in ray florets than in disc florets (Fig. 8 k). The ectopic expression of CDM19 in C. morifolium in Arabidopsis leads to altered carpel development and multi-carpel siliques [36]. Therefore, CDM19 might control carpel development in Chrysanthemum. BBX22, a B-box zinc transcription factor, occupied a central position in the ray floret-specific module and had a higher expression level in ray florets than in disc florets (Fig. 8 l). In C. morifolium, BBX20 was identified in the ray petal-specific module, and the expression level of BBX20 was higher in ray florets than in disc florets in six different cultivars [2]. This suggests the conservation of B-box proteins in the regulation of flower morphogenesis in Chrysanthemum. HTH, encoding a GWC oxidoreductase involved in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids, participated in floral organ fusion and cuticle membrane structure formation in Arabidopsis thaliana [37]. The expression level of HTH was higher in ray florets, and HTH might control the flower development of ray florets by the biosynthesis pathway of long-chain α-,ω-dicarboxylic fatty acids in C. seticuspe. HSP70 is a type of heat shock protein and has been found to participate in cabbage flower development [38], indicating that this protein has a possible role in the regulation of disc floret development. In the ray floret- and disc floret-specific modules, there were not only coding genes but also many lncRNAs, which indicated that they might function together to regulate flower development in C. seticuspe.

ceRNA and lncNAT regulatory networks

LncRNAs can act as ceRNAs and be targeted by miRNAs, which increases the expression levels of mRNAs targeted by miRNAs [39]. In this study, AP2, CUC2 and TCP family genes were found to be targeted by miRNAs together with lncRNAs. AP2 has been implicated as a target of miR172 family members [40], which have been found to regulate the floral transition and flower development in Arabidopsis [41, 42] and the fruit development process in tomato [43]. miR164 can target CUC2, the interaction of which influences leaf margin serration in Arabidopsis [44]. In strawberry, the balance between miR164 and CUC2 was shown to control the serration of leaf margins and flower margins [45]. The balance between miR319 and TCP family genes controls the development of the cotyledon boundary and leaf serration, and miR319 and TCP genes form complex regulatory networks to determine leaf development when combined with miR164 and CUC family genes [46, 47]. In the present study, these miRNAs were predicted to target not only related genes but also lncRNAs. LncRNA-TCONS_00021861 was shown to be targeted by miR528-3p together with YUCCA7 in rice, and overexpression of TCONS_00021861 led to the sequestration of miR528-3p to upregulate YUCCA7, followed by activation of the indole-acetic acid (IAA) biosynthetic pathway [48]. These findings indicate that there is a potential regulatory function of mRNA–miRNA–lncRNA regulatory networks in flower development in C. seticuspe.

LncNATs have emerged as pivotal regulators of plant biological processes. In C. morifolium, a lncNAT of TCP1-lncTCP1 can enhance the expression levels of TCP1 to improve cold tolerance via the histone modification protein DgATX [49]. The lncNAT of FLC-COOLAIR recruits Polycomb repressive complex 2 (PRC2) and deposits H3K27me3 in the chromatin region to facilitate silencing of FLC mRNA transcription, which has been shown to affect seed dormancy and flowering time in Arabidopsis [50]. In Chinese cabbage, the lncNAT of MAPK15 showed involvement in resistance to downy mildew, and there was enhanced expression of MAPK15 in MSTRG.19915-silenced seedlings [51]. In rice, the lncNAT of MYB60-TWISTED LEAF might recruit H3K27me3 to silence the expression of MYB60, controlling twisted leaf blades [20]. In this study, we identified several flower development-related genes and their lncNATs, of which the expression levels of MIF2 and its lncNAT-LXLOC_026470 tended to be the opposite in ray florets and disc florets. The positional relationship of MIF2 and LXLOC_026470 on chromosome 3 is shown in Fig. S7. The MIF gene family encodes small zinc finger proteins, and overexpression of two homologues of MIF2 (MIF1 and MIF3) has been shown to influence the determinate leaf growth of Arabidopsis [52]. Together with TOPLESS and HISTONE DEACETYLASE 19, the MIF2 homologue in tomato-SlIMA recruits SlKNU to form a transcriptional repressor complex, which represses the expression of SlWUS, participates in the floral meristem termination process and ultimately determines carpel number [53]. Therefore, LXLOC_026470 may recruit histone proteins to silence the expression of MIF2 to influence the development of ray florets and disc florets in C. seticuspe.

Conclusion

Through whole-transcriptome sequencing of C. seticuspe, annotation of the genome has been improved with information concerning mRNAs, lncRNAs and small RNAs. mRNA–miRNA–lncRNA networks were constructed to increase our understanding of ray floret and disc floret development in chrysanthemums. We identified a lncNAT-mRNA combination, LXLOC_026470 and MIF2, that might be related to flower development of the capitulum via histone modification.

Methods

Plant materials

Seedlings of Gojo-0 were obtained from the Graduate School of Integrated Sciences for Life, Hiroshima University, 1–4-3, Kagamiyama, Higashi-Hiroshima, Japan [6]. The seedlings were planted in the greenhouse of Nanjing Agricultural University, and the growing environment was the same as that described by Nakano et al. [54]. The leaves, stems and roots were sampled from plants at the vegetative stage, while the ray florets and disc florets were sampled from plants at the flowering stage.

Small RNA library construction, sequencing and data processing

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the provided protocol. Extracted RNA was used to construct a small RNA library for each sample. After electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel, the bands of the 18–30 nt small RNA region were recovered. After being adenylated with 3′ adapters annealed to unique molecular identifiers (UMIs) and ligated with 5′ adapters, the small RNAs were transcribed into cDNA via SuperScript II Reverse Transcriptase (Invitrogen, USA). cDNA fragments were enriched using several rounds of PCR amplification. The PCR products were selected by agarose gel electrophoresis with target fragments of 110 ~ 130 bp and then purified by a QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The small RNA libraries were subsequently sequenced on the MGISEQ-2000RS platform (BGI-Shenzhen, China).

The raw data of small RNA libraries were considered raw tags. The raw tags were filtered by removing unqualified tags (tags of law quality, tags with 5′ primer contaminants, tags without 3′ primers, tags without insertions, tags with poly(A) nucleotides and tags shorter than 18 nt). After filtering, the clean tags were mapped to the reference genome sequence and sequences in other sRNA databases, including miRbase [27], siRNA sequences [28] and snoRNA sequences [29], with Bowtie2 [30]. The quantification of small RNA expression was performed by counting the absolute numbers of molecules with unique molecular identifiers [31].

mRNA and lncRNA library construction, sequencing and data processing

The RNA used in the lncRNA and mRNA libraries was the same as the RNA used in the small RNA libraries. However, 1 μg of total RNA was purified with a Ribo-Zero™ Magnetic Kit (Plant Leaf, Epicentre) to deplete the rRNA. The purified RNA was fragmented by adding First Strand Master Mix (Invitrogen, USA). Subsequently, RNA was transcribed into first-strand cDNA using random primers, followed by second-strand cDNA synthesis. The synthesized cDNA was then subjected to end-repair and 3′ adenylation. The ends of these 3′-adenylated cDNA fragments were subsequently ligated with adapters. The cDNA fragments were enriched using several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix. Then, the PCR products were purified with AMPure XP Beads. The resulting mRNA and lncRNA libraries were sequenced on the BGISEQ-500 platform (BGI-Shenzhen, China).

The raw data of the lncRNA and mRNA libraries were assessed with SOAPnuke [32] by (1) removing reads containing sequencing adapters; (2) removing reads whose low-quality base percentage (base quality less than or equal to 5) was more than 20%; and (3) removing reads whose unknown base (‘N’ base) percentage was more than 5%. Subsequently, the clean reads of the lncRNA and mRNA libraries were aligned using HISAT [33] and assembled using StringTie [34] and Cufflinks [35]. The coding availability was assessed by CPC [36], txCdsPredict (https://github.com/ENCODE-DCC/kentUtils/tree/master/src/hg/txCds/txCdsPredict), CNCI [37] and the Pfam database [38]. Only when at least three of the four judgement methods were consistent could we confirm that a transcript was an mRNA or lncRNA. The quantification of transcripts was transformed into FPKM values for lncRNA and mRNA libraries using RSEM [39]. A heatmap was constructed by Pheatmap according to the gene expression patterns in different samples.

Prediction of miRNA targeting

The prediction results are shown in Table S1. Software including psRobot [55] and TargetFinder [56] was used to predict the target genes of the miRNAs. Functional annotations of targeted mRNAs were performed with the NCBI-NR database and Gene Ontology (GO) [57].

WGCNA and network visualization

The transcriptome profiles of biological replicates were also calculated to obtain a comprehensive regulatory network. The FPKM values were log2 normalized and then input into the WGCNA package version 1.70–3 [58] in R. The outliers were removed to standardize the whole dataset. A soft threshold power of 18 was used to construct the adjacency matrix. The dynamic tree was cut with a minimum module size of 30 and a merging threshold of 0.25. Each organ was defined as a trait, and the correlations between modules and traits were analysed using Pearson correlation coefficients (PCCs) [59]. The genes with a high KME were identified as hub genes [60]. The whole-gene coexpression, ceRNA and lncRNA network was visualized by Cytoscape v3.9.1 software (https://cytoscape.org/).

qRT–PCR

RNA was extracted from the five organs as mentioned above. The primer pairs designed and used for qRT–PCR are listed in Table S2. cDNA was synthesized using a PrimeScript RT Reagent Kit (TaKaRa, Japan), and qRT–PCR was performed with a SYBR Premix Ex Taq II Kit (TaKaRa, Japan) according to the manufacturer’s protocol on a 480 Real-Time PCR System (Roche, Switzerland). Three biological replicates were included, of which three technical replicates were used for each biological replicate. The reference gene CsEF1α was used to normalize the expression levels using the 2−ΔΔCT method [61].

Availability of data and materials

The relevant data in this study are included in this article and the supplementary files. The transcriptome data have been uploaded to the National Center for Biotechnology Information under accession number PRJNA82048835 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA820488?reviewer=t9sj9hjp5oetuede4ka11lsjfd).

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Acknowledgements

We would like to thank Prof. Makoto Kusaba, Hiroshima University, for sharing the Gojo-0 materials.

Funding

This research was funded by the Natural Science Fund of Jiangsu Province (BK20190076), the National Natural Science Foundation of China (32172609, 31870694, 31872149), the Foundation of Central Laboratory of Xinyang Agriculture and Forestry University (FCL202002), and a project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institution.

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Contributions

A.S. conceived the project. D.S., J.Z., J.H., Z.G., S.L., J.L.Z., P.L. and L.Z. cultivated plants, collected samples and performed experiments. D.S. and J.H. visualized the data. D.S. performed the bioinformatic analysis and drafted the manuscript. Z.W., L.W., F.C. and A.S. revised the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Aiping Song.

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Our research did not involve any human or animal subjects, material, or data. The plant materials were provided by Prof. Makoto Kusaba, Hiroshima University, Japan. The plant materials involved in this study were stored in the life science building of Nanjing Agricultural University. We declare that the plant material in the experiment was collected and studied in accordance with relevant institutional, national, and international guidelines and legislation.

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Sun, D., Zhang, J., He, J. et al. Whole-transcriptome profiles of Chrysanthemum seticuspe improve genome annotation and shed new light on mRNA–miRNA–lncRNA networks in ray florets and disc florets. BMC Plant Biol 22, 515 (2022). https://doi.org/10.1186/s12870-022-03889-y

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