Transcriptome Analysis of Flower Development and Mining of Genes Related to Flowering Time in Tomato (Solanum lycopersicum)

Abstract

Flowering is a morphogenetic process in which angiosperms shift from vegetative growth to reproductive growth. Flowering time has a strong influence on fruit growth, which is closely related to productivity. Therefore, research on crop flowering time is particularly important. To better understand the flowering period of the tomato, we performed transcriptome sequencing of early flower buds and flowers during the extension period in the later-flowering "Moneymaker" material and the earlier-flowering "20965" homozygous inbred line, and we analyzed the obtained data. At least 43.92 million clean reads were obtained from 12 datasets, and the similarity with the tomato internal reference genome was 92.86-94.57%. Based on gene expression and background annotations, 49 candidate genes related to flowering time and flower development were initially screened, among which the greatest number belong to the photoperiod pathway. According to the expression pattern of candidate genes, the cause of early flowering of "20965" is predicted. The modes of action of the differentially expressed genes were classified, and the results show that they are closely related to hormone regulation and participated in a variety of life activities in crops. The candidate genes we screened and the analysis of their expression patterns provide a basis for future functional verification, helping to explore the molecular mechanism of tomato flowering time more comprehensively.

Keywords: RNA-seq; flowering time; genetic pathways; tomato.

Figure 1

Pearson correlation coefficients of all 12 samples. The Pearson correlation coefficients of all gene expression levels between every two schemes.

Figure 2

Volcano map of DEGs. (A) Volcano map of MM_1-vs-MM_2. (B) Volcano map of 20965_1-vs-20965_2. (C) Volcano map of MM_1-vs-20965_1. (D) Volcano map of MM_2-vs-20965_2. The red dots represent upregulated genes, the blue dots represent downregulated genes, and the gray dots represent non-DEGs. The X-axis represents the fold change of the difference after conversion to log2, while the Y-axis represents the significance value after conversion to -log10.

Figure 3

Venn diagram of DEGs included in the four groups. The four groups are MM_1-vs-MM_2, 20965_1-vs-20965_2, MM_1-vs-20965_1, and MM_2-vs-20965_2. Each circle represents a group of gene sets. The overlapping area of different circles represents the intersection of these gene sets, that is, the co-expressed genes. Non-overlapping parts indicate the uniquely expressed genes. The numbers on the figure represent the number of DEGs in the corresponding area.

Figure 4

GO functional enrichment of differentially expressed genes in tomato flower buds. (A) GO_biological process enrichment bubble chart. (B) GO_cellular component enrichment bubble chart. (C) GO_molecular function enrichment bubble chart. The sizes of the bubbles indicate the number of genes enriched in the GO term. The color of the bubble represents the Qvalue. The rich factor is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene numbers annotated in this pathway term. The greater the rich factor is, the greater the degree of pathway enrichment.

Figure 5

Bubble chart of KEGG pathway enrichment. (A) KEGG pathways enriched with upregulated DEGs from the MM_1-vs-MM_2 comparison. (B) KEGG pathways enriched with upregulated DEGs from the 20965_1-vs-20965_2 comparison. (C) KEGG pathways enriched with upregulated DEGs from the MM_2-vs-20965_2 comparison. The sizes of the bubbles indicate the number of genes enriched in the KEGG pathway. The color of the bubble represents the Qvalue. The rich factor is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene numbers annotated in this pathway term. The greater is the rich factor, the greater is the degree of pathway enrichment.

Figure 6

Analysis of expression patterns in tomato flower samples by WGCNA. (A) Gene clustering tree and module division. (B) Module-sample association. The abscissa represents the samples; the ordinate represents the modules. The upper value in each cell represents the correlation coefficient, and the lower value represents the p value.

Figure 7

The clustering pattern of DEGs generated with the MapMan tool. (A) Overview of DEG regulation in MM1. vs-MM_2. (B) Overview of DEG regulation in 20965_1-vs-20965_2. (C) Cellular response of DEGs in MM_1-vs-MM_2. (D) Cellular response of DEGs in 20965_1-vs-20965_2. (E) Transcription of DEGs in MM_1-vs-MM_2. (F) Transcription of DEGs in 20965_1-vs-20965_2. Each square represents a separate gene. Red represents upregulation and blue represents downregulation. The color brightness represents the degree of difference (see scale).

Figure 7

The clustering pattern of DEGs generated with the MapMan tool. (A) Overview of DEG regulation in MM1. vs-MM_2. (B) Overview of DEG regulation in 20965_1-vs-20965_2. (C) Cellular response of DEGs in MM_1-vs-MM_2. (D) Cellular response of DEGs in 20965_1-vs-20965_2. (E) Transcription of DEGs in MM_1-vs-MM_2. (F) Transcription of DEGs in 20965_1-vs-20965_2. Each square represents a separate gene. Red represents upregulation and blue represents downregulation. The color brightness represents the degree of difference (see scale).

Figure 7

The clustering pattern of DEGs generated with the MapMan tool. (A) Overview of DEG regulation in MM1. vs-MM_2. (B) Overview of DEG regulation in 20965_1-vs-20965_2. (C) Cellular response of DEGs in MM_1-vs-MM_2. (D) Cellular response of DEGs in 20965_1-vs-20965_2. (E) Transcription of DEGs in MM_1-vs-MM_2. (F) Transcription of DEGs in 20965_1-vs-20965_2. Each square represents a separate gene. Red represents upregulation and blue represents downregulation. The color brightness represents the degree of difference (see scale).

Figure 8

FPKM values obtained via RNA-seq and relative mRNA levels obtained via qRT-PCR for 8 DEGs. Three technical replicates were performed for each biological replicate of each sample. Error bars represent standard deviation. p < 0.05 means significant difference: a was significantly higher than b and c; b was significantly higher than c. There was no significant difference between the same letters.

Figure 9

Network analysis of DEGs in the “circadian rhythm-plant” category. The small rectangular squares represent genes or proteins.The small dots represent chemical molecules. Straight lines and arrows represent activation. Dotted lines and arrows represent indirect effects. T-bars stand for inhibition. +u represents ubiquitination. +p represents phosphorylation. The purple squares represent DEGs enriched in this pathway in the comparison groups MM_1-vs-MM_2 and 20965_1-vs-20965_2.