Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis

Abstract

Background: To understand the gene expression networks controlling flower color formation in alfalfa, flowers anthocyanins were identified using two materials with contrasting flower colors, namely Defu and Zhongtian No. 3, and transcriptome analyses of PacBio full-length sequencing combined with RNA sequencing were performed, across four flower developmental stages.

Results: Malvidin and petunidin glycoside derivatives were the major anthocyanins in the flowers of Defu, which were lacking in the flowers of Zhongtian No. 3. The two transcriptomic datasets provided a comprehensive and systems-level view on the dynamic gene expression networks underpinning alfalfa flower color formation. By weighted gene coexpression network analyses, we identified candidate genes and hub genes from the modules closely related to floral developmental stages. PAL, 4CL, CHS, CHR, F3'H, DFR, and UFGT were enriched in the important modules. Additionally, PAL6, PAL9, 4CL18, CHS2, 4 and 8 were identified as hub genes. Thus, a hypothesis explaining the lack of purple color in the flower of Zhongtian No. 3 was proposed.

Conclusions: These analyses identified a large number of potential key regulators controlling flower color pigmentation, thereby providing new insights into the molecular networks underlying alfalfa flower development.

Keywords: Alfalfa; Cream color; Floral pigmentation; Hub gene; PacBio Iso-Seq; Transcriptome.

Fig. 1

Phenotypes and anthocyanins compounds of the alfalfa materials. a Phenotypes of the different flower development stages from Defu and Zhongtian No. 3. b Anthocyanin compound contents in the peels of the two cultivars in S4. C, Defu; M, Zhongtian No. 3. Error bars indicate SEs

Fig. 2

Alternative splicing events from the Iso-Seq. IR, intron retention. A3SS, alternative 3ˊ splice sites. ES, exon skipping/inclusion. A5SS, alternative 5ˊ splice sites. MXE, mutually exclusive exons

Fig. 3

Comparison of isoforms from the PacBio Iso-Seq data and contigs from the RNA-Seq data

Fig. 4

Global gene expression statistics in different floral development stages. a Numbers of detected transcripts in each sample. b Principal components analysis (PCA) of the RNA-Seq data

Fig. 5

Number of DEGs between the different floral development stages. a DEGs of alfalfa cultivar C. b DEGs of alfalfa cultivar M. C, Defu; M, Zhongtian No. 3

Fig. 6

Comparison of the DEGs between the two cultivars. C, Defu; M, Zhongtian No. 3

Fig. 7

Expression heatmap of the DEGs of flavonoid biosynthesis. The expression of DEGs is displayed as log₁₀ (FPKM+ 1). PAL, phenylalanine ammonia-lyase; 4CL, 4-coumarate: coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FLS, flavonol synthesis; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase

Fig. 8

Gene co-expression modules detected by WGCNA. The clustering dendrogram of the genes across all the samples exhibits dissimilarity based on topological overlap, together with the original module colors (dynamic tree cut) and assigned merged module colors (merged dynamic)

Fig. 9

Module-trait associations using WGCNA. Each row corresponds to a module eigengene and each column to a stage. Each cell contains the corresponding correlation and P-value. The table is color-coded by correlation, according to the color legend

Fig. 10

Expression profiles of 12 candidate genes and RT-qPCR validation. EF1a is used as the internal control. The error bars represent the SEs of the RT-qPCR data (n = 3). “r” represents the Pearson correlation coefficient. Pearson’s correlations between the RNA-Seq data and RT-qPCR data are calculated using the log₂ fold change and the relative expression level. aPAL6; bPAL9; cCHS2; dCHS4; eCHR1; fCHR2; gCHR3; hF3’H4; iDFR1; jDFR2; kUFGT22; lUFGT23

Fig. 11

A referred model for the process of anthocyanin synthesis in the purple flowers of C and cream flowers of M. The crucial isoform IDs are indicated at the side of each gene. Upstream of M, PAL and 4CL are suppressed, and an increasing branch of isoflavone biosynthesis regulated by CHS and CHR is dominant. Furthermore, the up-regulation of F3H/FLS, F3’H, and F3’5’H causes an increase in other flavonoid compounds, such as myricetin and kaempferol, further reducing the anthocyanin synthesis. Finally, the low expression level of DFR accompanied with the low abundance of UFGT might disrupt the anthocyanin synthesis, leading to the formation of the cream color