Transcriptomic analysis reveals the roles of microtubule-related genes and transcription factors in fruit length regulation in cucumber (Cucumis sativus L.)

Abstract

Cucumber (Cucumis sativus L.) fruit is a type of fleshy fruit that is harvested immaturely. Early fruit development directly determines the final fruit length and diameter, and consequently the fruit yield and quality. Different cucumber varieties display huge variations of fruit length, but how fruit length is determined at the molecular level remains poorly understood. To understand the genes and gene networks that regulate fruit length in cucumber, high throughout RNA-Seq data were used to compare the transcriptomes of early fruit from two near isogenic lines with different fruit lengths. 3955 genes were found to be differentially expressed, among which 2368 genes were significantly up-regulated and 1587 down-regulated in the line with long fruit. Microtubule and cell cycle related genes were dramatically activated in the long fruit, and transcription factors were implicated in the fruit length regulation in cucumber. Thus, our results built a foundation for dissecting the molecular mechanism of fruit length control in cucumber, a key agricultural trait of significant economic importance.

Figure 1. Morphological characterization of two near-isogenic cucumber lines with different fruit lengths.

(A)–(D) Cucumber line 408 with long fruit (left) and its near-isogenic line 409 with short fruit (right) at commercially mature fruit stage (A)–(B), anthesis stage (C), and 4 days before anthesis (D). The bars in B represent the standard deviation (n = 10). Asterisk indicates that fruit length is significantly different between 408 and 409 (unpaired t test, P < 0.05). Square brackets in C and D show the lengths of early fruits. Samples at the same developmental stage as in D were used for RNA-Seq analyses. Scale bars represent 1 cm.

Figure 2. Verification of differentially expressed genes by qRT-PCR.

Fourteen DEGs with higher expression and six DEGs with lower expression in line 408 were chosen for qRT-PCR validation. The relative expression level of each gene was expressed as the fold change between two lines in the RNA-Seq data (white bar) and qRT-PCR data (gray bar). The cucumber UBIQUITIN gene was used as an internal control to normalize the expression data. The bars represent the standard deviation (n = 3). Asterisks indicate that the gene transcriptions are significantly different between lines 408 and 409 (unpaired t test, P < 0.05).

Figure 3. Genome-wide distributions of SNPs (A) and DEGs (B) in the cucumber genome.

Number of SNPs and DEGs were calculated over non-overlapping 100 kb and 10 MB bins, respectively.

Figure 4. Significantly enriched Gene Ontology (GO) terms (P < 0.05) in the up-regulated genes in the fruit of line 408 vs 409.

GO terms belong to biological processes (GOBP), molecular functions (GOMF), and cellular components (GOCC) were shown in green, blue, and red, respectively. GO terms were sorted based on p-values.

Figure 5. Significantly enriched Gene ontology (GO) terms (P < 0.05) in the down-regulated genes in the fruit of line 408 vs 409.

GO terms belong to biological processes (GOBP), molecular functions (GOMF), and cellular components (GOCC) were shown in green, blue, and red, respectively. GO terms were sorted based on p-values.

Figure 6. Family assignment of the 130 transcription factors that showed lower expression in the fruit of line 408.

Number of genes assigned to each family is shown behind a comma.

Figure 7. Comparison of cell morphology in lines 408 and 409.

(A)–(C) Microscopic longitudinal sections of the young fruits at 4 days before anthesis (an-4) in line 408 (A) and line 409 (B), and the corresponding quantifications of cell size (C). (D)–(F) Microscopic longitudinal sections of the young fruits at anthesis (an) in line 408 (D) and line 409 (F), and the respective quantifications of cell size (F). (D′)–(E′) are the enlarged view of the boxes in D and E, respectively. The bars in C and F represent the standard deviation (n = 3). Asterisk in F indicates that the cell size in line 408 is significantly larger than that in line 409 at anthesis (unpaired t test, P < 0.05). Bar = 50 um.