Transcriptome Analysis And Fine Mapping Of Major Genes Controlling Flesh Firmness And Sour Flesh In Watermelon

Watermelon fruit texture is determined by flesh firmness;whereas,fruit flavor is due to the content of organic acids.Both factors determine the sensory quality of watermelon fruit.Like most of the quality traits,flesh firmness and organic acids content are multiplex quantitative traits which controlled by multigenes.Conventional methods for physiology and genetics have limitations to uncover the genetic and molecular mechanisms related to complex traits,thus,identification of key regulatory factors of flesh firmness and accumulation of organic acids can provide a theoretical and technical platform for watermelon quality breeding.In this research,wild watermelon PI271769 was used as donor parent while 203 Z was used as recurrent parent.Molecular markers with combination of phenotypic identification of mature fruit,near-isogenic lines of flesh firmness and sour flesh were obtained by continuous back crossing and selfing.RNA of watermelon fruit samples were used to conduct transcriptome analysis.BSA-Seq analysis with combination of secondary segregation population,fine mapping for major genes controlling flesh firmness and sour flesh were studied.The following preliminary results were obtained:1.Near-isogenic line for watermelon flesh firmness and sour flesh was constructed.Near-isogenic lines of HWF for flesh firmness and SW for sour flesh were obtained through continuous backcrossing for seven times along with selfing for four times.Based on observed botanical characteristics,though there existed significant difference at the target traits,there was no significant phenotypic difference between 203 Z and NILs.2.The content for each cell wall components were measured and comparative transcriptome analysis during fruit development in watermelon was analyzed.Cell wall components,such as protopectin,water soluble pectin(WSP),ion bound pectin(ISP),covalent bound pectin(CSP),cellulose and hemicelluloses were measured during fruit development stages.According to the results,protopectin,ISP,cellulose and hemicelluloses showed similar trend for flesh firmness,while WSP and ISP content continued to rise with decrease in flesh firmness.Comparative transcriptome analysis was conducted on RNA samples of flesh during fruit development and ripening in HWF and 203 Z.The results from the transcriptome analysis revealed that,expression levels of pectinesterase,glycosyl transferase,cellulose synthesis,polygalacturonase and xyloglucan endotransglycosylase were different in HWF and 203 Z.Thus,influencing cell wall components content,that can adjust the changes of flesh firmness in watermelon fruit.3.Fine mapping and identification of major gene controlling flesh firmness in watermelon.Molecular marker tightly linked with flesh firmness was identified on the whole genomic level,which was combined with BSA-Seq analysis for primary mapping of major gene controlling flesh firmness.Ultimately,major gene was delimited in the physical interval between 12780793 and 17658672 bp on chromosome 6.This region includes xyloglucan endotransglycosylase Cla004119 and ethylene response factor Cla004120,the expression levels of both were significantly different among HWF and 203 Z fruits,which indicated that Cla004119 and Cla004220 were key potential candidate genes controlling flesh firmness.4.Soluble sugars and organic acids contents were measured and comparative transcriptome analysis during fruit development in watermelon was analyzed.The soluble sugars and organic acids contents were measured during fruit development and ripening in watermelon.According to this result,the soluble sugars content in 203 Z was higher than that of SW,but it was not significant.However,the organic acids content was significantly lower in 203 Z than in SW.Several differentially expressed genes(DEGs)related to soluble sugar and organic acid accumulation and metabolism were identified on the basis of comparative transcriptome analysis conducted on RNA samples of flesh during fruit development and ripening in SW and 203 Z.These include the DEGs encoding raffinose synthase,sucrose synthase(SuSy),sucrose-phosphate synthase(SPSs),insoluble acid invertases(IAI),NAD-dependent malate dehydrogenase(NAD-cyt MDH),aluminum-activated malate transporter(ALMT),and citrate synthase(CS).5.Fine mapping of major gene controlling sour flesh in watermelon.On the basis of primary mapping of major gene controlling sour flesh by BSA-Seq method,the major gene was delimited in the physical interval between 452916 and 4855093 bp(318 Kb)on chromosome 6,having 28 candidate genes.Combining these findings with comparative transcriptome analysis results,it was concluded that the expression level of Cla009218 and Cla009226 was significant different between SW and 203 Z fruit,which indicated that both genes maybe determine the accumulation of organic acids in watermelon fruit.