Fig 1.
Overview of mGWAS results in this study.
(A) The strength of the association of metabolites is indicated as the negative logarithm of the P value for the CMLM model. All metabolite–SNP associations with P values < 2.4 × 10−7 are plotted against genome location at 1-Mb intervals. Red and blue horizontal dashed lines in the manhattan plots indicate the genome-wide suggestive (P values < 2.4 × 10−7) and significant (P values < 1.2 × 10−8) threshold, respectively. Amino acids are indicated by olive green circles. Organic acids are indicated by light green circles. Sugars are indicated by light blue circles. Other compounds are indicated by pink circles. (B) Distribution density of mGWAS signals across the tomato genome (SL2.50). mGWAS, metabolomic genome wide association analyses.
Table 1.
Summary of significant loci–trait associations identified in GWAS population.
Fig 2.
Genome-wide association results for ascorbate content in tomato fruits.
(A) Manhattan plot displaying the GWAS results for ascorbate content in fruits (CMLM, N = 302). Negative log10-transformed P values from the compressed mixed linear model are plotted on the y-axis. Horizontal dashed line indicates a genome-wide suggestive threshold of 2.4×10−7. (B) Quantile-quantile plot for ascorbate content in the GWAS population. (C) Box plot for ascorbate content, plotted as a function of genotypes at SNP ch09_64101874. The metabolic data for ascorbate were log2 transformed. (D) Detailed plot from 63.1–65.1 Mb on chromosome 9 (x-axis). Lead SNP is indicated in purple. A representation of pairwise r2 values (a measure of LD) among all SNPs in region 63.1–65.1 Mb, where the colour of each box corresponds to the r2 value according to the legend. (E) A representation of the pairwise r2 values (a measure of LD) among all polymorphic sites in the 830 kb genomic region corresponding to (D), where the darkness of the color of each box corresponds to the r2 value according to the legend. The 145 haploblocks are represented by inverted triangle. Haploblock 20 (marked by red star) contains Lead SNP associated with fruit AsA content. (F) Gene structure of two genes in the haploblock 20. Filled grey, blue and blacklines represent coding sequence, promoter & 3’UTR and introns respectively. (G, H) The ascorbate content (G) and relative expression of candidate gene (SlbHLH59) (H) in fruits from different selected accessions. Data represent means ±s.d. (n = 3). The dashed horizontal lines represent the average ascorbate content and expression levels of SlbHLH59 in fifteen low-AsA accessions and fifteen high-AsA accessions.
Fig 3.
Haplotype and functional analysis of SlbHLH59 in tomato.
(A) Structural variations of the four SlbHLH59 haplotypes. Blue represent the promoter and 3’ UTRs, grey boxes represent coding sequences and the line between the grey boxes represents the intron. The 11 nucleotide polymorphisms are indicated at their corresponding positions. Four haplotypes of SlbHLH59 are examined in 367 tomato accessions. (B, C) The relative ascorbate content (B) and expression of SlbHLH59 (C) in fruits of four SlbHLH59 haplotypes. (D) Transient expression assay of promoter activity in Arabidopsis protoplasts. The SlbHLH59 promoter fragments were inserted into the reporter vector (pCAMBIA1304-GUS). Both vectors of reporters and internal control (pGreen II 0800, 35S+LUC) were transformed into Agrobacterium cells and used for Arabidopsis protoplast transformation. Left, constructs with site-directed mutations at the four polymorphisms in the promoter region. Right, relative GUS/LUC values. Values are represented as means ± SD (n = 3). (E, G, I) The relative expression of SlbHLH59 in SlbHLH59SNP9A overexpression (E) SlbHLH59SNP9G overexpression (G) and SlbHLH59 RNAi (I) transgenic tomato lines. (F, H, J) The ascorbate content in SlbHLH59SNP9A (F) SlbHLH59SNP9G (H) and SlbHLH59 RNAi (J) transgenic tomato lines. The background of SlbHLH59 overexpression and RNAi transformation is TS-186 and TS-265 respectively. Asterisks indicate significant differences by t test: **P< 0.01.
Fig 4.
The characterization of SlbHLH59.
(A) The transcript levels of SlbHLH59 indifferent tomato organs: R, root; S, stem; L, leaf; FL, flower; IMG, immature fruit; MG, mature green fruit; BR, breaker stage fruit; YR, yellow stage fruit; RR, red ripe stage fruit. TS-186 is a low-AsA accession; and TS-265 is a high-AsA accession. (B, C) Subcellular co-localization of transiently expressed SlbHLH59SNP9A-YFP (B) and SlbHLH59SNP9G-YFP (C) fusion protein with a nuclear marker (ERF) in N. benthamiana leaves. Bars = 50 μm. (D-F) Phenotype comparisons of SlbHLH59-OE and wild-type plants after methyl viologen (MV) treatment. The SlbHLH59-OE transgenic plants had less yellow lesion (D, E) and H2O2 accumulation (F) than WT after MV treatment. Bar = 10 cm (D), 5cm (E) and 2cm (F).
Fig 5.
Identification of SlPMM, SlGMP2 and SlGMP3 as direct targets of SlbHLH59.
(A, B) Relative expression of ascorbate (AsA) biosynthesis, recycling and oxidation-related genes in fruits of SlbHLH59 overexpression (A) and RNAi (B) lines. (C) The promoter cis-elements of SlPMM, SlGMP2 and SlGMP3. The sequences in black boxes represent E-box cis-elements. (D) Yeast-one hybrid (Y1H) assay of SlbHLH59 binding to promoter fragments of SlPMM, SlGMP2 and SlGMP3. The bait vectors SlPMMpro, SlGMP2pro and SlGMP3pro (the fragments containing the region 3000 bpupstream from initiation codon) and the prey vector containing SlbHLH59 were introduced into yeast strain Y187, and interaction between bait and prey enhanced ABA resistance. Yeast cells spread on SD-Leu media with various concentrations of ABA (0, 10, 15, and 20 mM). The bait vector (SlPMMpro, SlGMP2pro and SlGMP3pro) + pGADT7 were also transformed into Y187 as a negative control. (E) Binding of SlbHLH59 to promoters of SlPMM, SlGMP2 and SlGMP3 assayed by dual luciferase system. The SlbHLH59 ORF was cloned into the effector vector (pGreen II62-SK) and promoter fragments of SlPMM, SlGMP2 and SlGMP3 were inserted into the reporter vector (pGreen II 0800 LUC). Both vectors of effectors and reporters were transformed into Agrobacterium cells and used to infiltrate tobacco leaves. LUC, firefly luciferase activity; RLU, Renilla luciferase activity; PG, the empty vector of pGreenII 62-SK. The promoters of SlPMM, SlGMP2 and SlGMP3 plus PG were used as control. (F-J) Dynamics of ascorbate content and SlbHLH59 expression during tomato fruit development. Ascorbate (AsA) concentration (F) and expression of SlbHLH59 (G), SlPMM (H), SlGMP2 (I), SlGMP3 (J) at different fruit developmental stages of wild-type (TS-186) and SlbHLH59 transgenic lines. Experiments were performed in immature (IMG), mature green (MG), breaker (BR), yellow ripe (YR) and red ripe (RR) fruit, respectively, with three replicates. All data in the graphs are presented as means ± SE. Asterisks indicate significant differences by t test: *P < 0.05; ** P< 0.01.
Fig 6.
Nucleotide diversity analysis of TFA9 loci during tomato domestication and improvement.
(A) Frequency of derived and ancestral InDel_8 allele in tomato subpopulations. n = number of accessions. (B) Comparison of ascorbate content of Hap 1 accession TS-186 and six Hap 4 accessions in BIG subgroup. The data are presented as means ± SE (n = 3). The asterisks represented significant differences from TS-186 (Hap 1), as indicated by the t-test (** P< 0.01). (C) Distribution of nucleotide diversity (π) of the PIM (red line), CER (purple line), and BIG (blue line) within the 10-kb region of TFA9. Grey lines indicate the locations of indel_8 (TCTCTTTC/-). The X-axis denotes the position of SlbHLH59 and the Y-axis indicates average p values. (D) The ratio of nucleotide diversity (π) is calculated from the SlbHLH59 sequence of PIM with CER (red line) and CER with BIG (blue line). In total, 331 tomato accessions were used for analysis, including 53 PIM accessions, 112 CER accessions, and 166 BIG accessions.