| Watermelon fruits contribute to the diets of consumers throughout the world. Sugar content is the key trait for commercial hybrid seeds production. However, watermelon sugar content trait is a complex QTL which can interact with environmental factors. It is hard for traditional biologic and genetic research method to reveal its genetic base and which is the key gene for sugar accumulation and sugar synthesis, which has impeded the watermelon fruit quality improvement. The ellustration for the net work modle for sugar accumulation mechanism in watermelon will make a theory foundation and have commercial breeding values. This research focuses on the QTL fine mapping by SNP high density map and GWAS analysis for sugar content. We try to illustrate the key gene for determination watermelon sugar content and the transcriptional patterns, functional analysis and regulator for its expression.The main research result of this article are listed below.1.The high density integrated genetic map was constructed for sugar content QTL mapping. Four previously described mapping populations derivedfrom independent crosses were used to develop the integrated map. An F8 population consisting of 103 recombinant inbred lines(RILs) derived from a cross betweenthe elite Chinese line 97103 and the U.S. Plant Introduction(PI) 296341-FR, and an F2 populationconsisting of 182 individuals derived from a cross between the elite ZWRM50 and PI 244019(ZWRM× citron) were both elite × citron populations. In addition, an elite × elite [Klondike Black Seeded × New Hampshire Midget(KBS × NHM)] RIL population consisting of 164 lines, and an elite × egusi [Strain II × PI 560023(SII × egusi)] F2 population consisting of 187 individuals were also included. The integrated map contained 1339 markers, spanning 798 cM with an average marker interval of 0.6 cM was constructed from four independent mapping experiments. Fifty-eight previously reported quantitative trait loci(QTL) for 12 traits in these populations were also integrated into the map. In addition, new QTL identified for brix, fructose, glucose and sucrose were added. Three sugar content QTL(QBRIX2-1, QBRIX2-2 and QBRIX2-3)were mapped on Chr2, which overlap with fructose QTL(QFRU2-1, QFRU2-2 and QFRU2-3), and two of them(QBRIX2-1 and QBRIX2-3) overlapped with sucrose QTL(QSUC2-1 and QSUC2-2). These overlapped QTL associated with economically important traits detected in different genetic backgrounds mapped to similar genomic regions of the integrated map, suggesting that such QTL are responsible for the phenotypic variability observed in a broad array of watermelon germplasm.2.Fine mapping of watermelon sugar content by constructing ultra-high density SNP map. A total of 947,400 high quality SNP loci were selected for SNP bin map construction. The skeleton bin map was constructed with a total of 2777 bins, 2539 bins were contained in the final SNP bin map. A total of 386 scaffolds covering approximately 344 Mb(96.9% of the assembled 355 Mb genome) were anchored to the 11 watermelon chromosomes by SNP bin map, only 11 Mb were not anchored and thus were assembled to the Chr0. In total, 314 scaffolds(332.6 Mb) accounting for 93.6% of the assembled genome were oriented, which is big improvement compared to the previous assembly in which only 65% of scaffolds were oriented. This ultrahigh-density SNP bin map was used for fine mapping of watermelon sugar content QBRIX2-1 and QBRIX2-3. To the end, the QBRIX2-1 and QBRIX2-2 was narrowed down to a physical region of 125 Kb(16 genes)and 797 Kb(20 genes) on chromosome 2, respectively. For fine mapping QBRIX2-3, a NIL population was developed and the QBRIX2-3 was narrowed down to two Indel markers WMI251 and WMI233, which contains 87 genes and 6 of them are transcription factors, the different protein sequences between 97103 and PI296341 of one bHLH transcription factor 998 may play a role in sugar accumulation.3.The transcription level, structure variation, tissue specific expression patter of QBRIX2-1 candidate gene 835 and the interaction with QBRIX2-3.The QBRIX2-1, corresponding to the physical position of 125 Kb on chromosome 2, harbours 16 candidate open reading frames(ORFs). Only one candidate of them(835) was highly expressed and annotated as membrane transporters, while the others were unknown or no/low detected expressing proteins by RNAseq transcriptome analysis. To confirm the transcript level of gene 835 is correlated with sugar content, we performed the quantitative PCR(qPCR) in two different populations: the RIL population and 50 germplasms. The low sugar content lines nearly shared the same expression patterns with wild low sugar content line PI296341-FR, while the expression in high sugar content lines were 100-5000 times higher than it in PI296341-FR. Structural viriation shows that a SNP(A/C) in the 5’UTR(up37 bp of ATG) can result 45 ananio acid deletion in 97103, which can be used for molecular marker to test the 130 re-sequencing germplasms. The deleted 45 amino acids in 97103-835 are predicted to be signal peptide(S-score) by SignalP. Different localization of target genes 97103 and PI296341-835 in watermelon fruit cells show that the target genes 97103-835 locate to plasma membrane while the PI296341-835 can mainly located inner membrane, very low signal in plasma membrane even enchanced by 35 S promoter.To study the mechanism why 835 can highly expressed in high sugar content line 97103 while nearly no expression in low sugar content line PI 296341. We find a SNP at up 1187 bp of ATG in MYC(bHLH) E-box(CANNTG)motif. Selection of the yeast one hybrid library generated from 97103 cDNA show that the transcription factor 998 in QBRIX2-3 can bind this motif. Co-expression 998 and the up 1700 bp promoter of 835 in tobacco show that the transcription factor 998 can activate the up 1700 bp promoter of 97103-835 but not the PI296341-835 up 1700 bp promoter. This indicated the SNP in E-box motif can result the different transcription regulating level by the intervaction of with QBRIX2-3. RNA in situ hybridization showed that 97103-835 protein specificly expressed in fruit vascular, indicating the 97103-835 is likely to involve in phloem unloading, rather than loading.4.The transcription level, functional evidence of ClTST2 and the regulator. In the QBRIX2-2 region, one candidate of them(ClTST2) was highly expressed and annotated as vacuole membrane sugar transporters, while the others were unknown or no/low detected expressing proteins by RNAseq transcriptome analysis. To confirm the transcript level of gene ClTST2 is correlated with sugar content, we performed the quantitative PCR(qPCR) in 50 germplasms. The low sugar content lines nearly shared the same expression patterns with wild low sugar content line PI296341-FR, while the expression in high sugar content lines were times higher than that in PI296341-FR. To corroborate the vacuolar localization of the putative watermelon sugar transporter, we fused the ClTST2 gene to YFP(yellow fluorescent protein) gene and expressed the corresponding fusion protein in watermelon fruit protoplasts 10 days after pollination(DAP). The yellow fluorescence was well resolved in vacuoles from fruit cells. The patch clamp technique was applied to prove ClTST2 can uptake sucrose, fructose and glucose in HEK293 T cells. To provide further evidence for sugar transport activity in fruit, we transiently expressed ClTST2 in strawberry fruits at white fruit stage. In sharp contrast, the ClTST2 over expressed(OE) strawberry fruits(sides) can accumulate 1.5 fold sugars than the empty vector control. Yeast one-hybrid and electrophoretic mobility-shift assays(EMSAs) was used to identify the transcription factor which can regulate the expression of ClTST2, the result showing that SUSIBA2 transcription factor of WRKY family can regulate the promoter of ClTST2. To further address if SUSIBA2 is able to activate or inhibite expression of ClTST2 in plants, luciferase(LUC) assay was performed in vivo using tobacco leafs by co-transformation of p35S:: SUSIBA2::tNOS with up 1500 bp p ClTST2::LUC. Significantly reduced LUC activities were detected when 97103-p ClTST2::LUC and PI 296341-FR-p ClTST2::LUC were used.To elucidate how SUSIBA2 regulates ClTST2, we analyzed the RNAseq transcriptome data and found SUSIBA2 was constitutively higher expressed in low sugar fruit(PI 296341-FR) and tissue(97103 rind), in contrast, SUSIBA2 expression was decreased 4 times during 97103 fruit maturating. These results suggested that ClTST2 was derepressed by SUSIBA2 from 10 to 34 DAP in 97103 fruit flesh, while repression existed around the whole stages of 97103 fruit rind and PI 296341-FR fruit.5.The transcription level, structure vaviation, functional study of ClSWT in the Chr1 GWAS loci associated with sugar content. GWAS the sugar content in 130 re-sequencing germplasms by using about one million SNP, we identified a SNP at Chr1 378844. This SNP located in ClSWT, which show 86% agreement with the phenotype. The transcript level of ClSWT is correlated with sugar content trait, we performed the quantitative PCR(qPCR) in four critical stages of fruit development and 50 germplasms. After transporter assay in hexose uptake defection yeast Ysl2-1, the result indicates ClSWT can transport glucose and fructose.6.The modle of sugar accumulation mechanism in watermelon. Stachyose and raffinose are the main transported sugars in phloem, which can be hydrolyzed by galactosidase, then hydrolyzed by invertase into glucose and fructose, this is ths first node for phloem unloading. Glucose and fructose unloading from phloem is the second node which maybe associated with QBRIX2-1 835 and regulated by bHLH transcription factor 998 in QBRIX2-3. The third node is the sugar transporter in plasma membrane, ClSWT may the key transporter. The fouth key node is the antiporter ClTST2 in vacuole, which can be regulated by WRKY transcription factor. This study firstly draws the net work modle for sugar accumulation mechanism in watermelon, which have a theroy foundation and commercial breeding values. |
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