Transcriptomic analysis of short-fruit 1 (sf1) reveals new insights into the variation of fruit-related traits in Cucumis sativus

Abstract

Fruit size is an important quality trait in different market classes of Cucumis sativus L., an economically important vegetable cultivated worldwide, but the genetic and molecular mechanisms that control fruit size are largely unknown. In this study, we isolated a natural cucumber mutant, short fruit 1 (sf1), caused by a single recessive Mendelian factor, from the North China-type inbred line CNS2. In addition to significantly decreased fruit length, other fruit-related phenotypic variations were also observed in sf1 compared to the wild-type (WT) phenotype, indicating that sf1 might have pleiotropic effects. Microscopic imaging showed that fruit cell size in sf1 was much larger than that in WT, suggesting that the short fruit phenotype in sf1 is caused by decreased cell number. Fine mapping revealed that sf1 was localized to a 174.3 kb region on chromosome 6. Similarly, SNP association analysis of bulked segregant RNA-Seq data showed increased SNP frequency in the same region of chromosome 6. In addition, transcriptomic analysis revealed that sf1 might control fruit length through the fine-tuning of cytokinin and auxin signalling, gibberellin biosynthesis and signal transduction in cucumber fruits. Overall, our results provide important information for further study of fruit length and other fruit-related features in cucumber.

Figure 1

Phenotypes of WT and sf1. (A) Fruit of WT, F1 and sf1 at 11 days after anthesis (DAA). (B) Fruit cracking in sf1 at 20 DAA. (C,D) Distribution of bloom trichomes on the pericarp of WT and sf1 at 9 DAA. (E,F) Fruit spines and tubercules of WT and sf1 at 11 DAA. (G,H) Seed cavities of WT and sf1 fruits at 9 DAA. (I,J) Stigmas from WT and sf1 at 0 DAA. Fruit hardness (K) and crispness (L) in WT and sf1 at 0, 9 and 23 DAA. Acid-soluble lignin content (M) in WT and sf1 cucumbers at 9 DAA. Scale bars represent 2 cm in (A),(B),(G) and (H), 2 mm in (E),(F),(I) and (J), and 500 μm in (C) and (D). ‘**’and ‘*’ indicate significant differences from WT at the 0.01 and 0.05 probability levels, respectively. Vertical bars represent standard deviation (n = 3).

Figure 2

Kinematic analysis of fruit growth and pericarp cell size and number in WT and sf1. Fruit length (A) and daily increase in fruit length (B) were determined in WT and sf1 from 0 to 22 DAA. Pericarp cell size of WT (C–E) and sf1 (F–H) at 0 (C and F), 9 (D and G) and 23 DAA (E and H). Cell number per square millimetre (I) of WT and sf1 pericarp was measured at three time points. ‘**’ indicates significant difference from WT at the 0.01 probability level. Scale bars represent 50 μm in (C) and 10 μm in (D–H). Vertical bars represent standard deviation (n = 18 for A and B, and n = 6–70 for I).

Figure 3

Mapping of the sf1 gene. The sf1 gene was fine-mapped to a 174.3 kb region between markers SNP1 and SNP2 on chromosome 6 in an F2 population containing 6,720 individuals from a cross between sf1 and ‘Chinese long’ 9930.

Figure 4

Chromosomal distributions of SNPs and DEGs in the cucumber genome. The SNPs and DEGs of all genes expressed in the fruit samples of the mutant vs. WT groups at 2 DBA (A) and 9 DAA (B), which came from the F2 population from a cross between sf1 and ‘Chinese long’ 9930, are shown as the up direction (○) and the down direction (○) from the zero point on the same y-axis, respectively. The purple rectangles indicate the overlapping region (11,603,968 to 11,631,493) between the fine-mapped 174.3 kb region and the SNP-enriched region on chromosome 6. The number of SNPs and DEGs was calculated based on non-overlapping 50-kb bins.

Figure 5

Significantly enriched GO terms (p-value < 0.01) in the cucumber fruits of the mutant vs. WT groups at 6 DBA (A), 2 DBA (B) and 9 DAA (C). GO terms were categorized into biological processes, cellular components and molecular functions based on their p-values.

Figure 6

DEG-enriched pathways from the KEGG analysis. (A) DEG-enriched auxin signalling in the cucumber fruits of the mutant vs. WT groups at 6 DBA (A1), 2 DBA (A2) and 9 DAA (A3). (B) DEG-enriched cytokinin signalling in the cucumber fruits of the mutant vs. WT groups at 6 DBA (B1), 2 DBA (B2) and 9 DAA (B3). (C) DEG-enriched gibberellin biosynthesis and signalling in the cucumber fruits of the mutant vs. WT group at 9 DAA. The red closed rectangles represent the genes that were up-regulated, and the green closed rectangles represent the down-regulated genes. The number of rectangles indicates the fold gene expression difference between the mutant and WT fruits. Aux/IAA, Auxin/indole-3-acetic acid protein; ARF, auxin-response factor; SAUR, small auxin up RNA; CRE1, cytokinin response 1; HP, histidine phosphotransfer protein; B-RR, type-B response regulator; A-RR, type-A response regulator; KAO, ent-kaurenoic acid oxidase; GA, gibberellin; GA20ox, GA 20-oxidase; GA2ox, GA 2-oxidase; GID1, gibberellin insensitive dwarf 1.

Figure 7

Heatmap showing the differential expression of selected transcriptional factors in the mutant vs. WT group fruits at 6 DBA, 2 DBA and 9 DAA. Genes highly or weakly expressed in the mutant vs. WT group fruits are shown in red and green, respectively. Genes without expression in the mutant vs. WT group fruits are shown in black. The heatmap was generated using cluster 3.0 software.