| Watermelon(Citrullus lanatus)is among the most vital food crops in the Cucurbitaceae family.Taste has been the subject of human selection in the evolution of agricultural crops.Acidity is among the three key components of developing taste in fleshy fruits,together with sugars and volatile compounds important for flavor.pH,and organic acid contents are multiplex quantitative traits which are controlled by multigenes.Conventional methods for physiology and genetics have limitations to uncover the genetic and molecular mechanisms related to complex traits.Recent advances have led scientists to discover and mine genes that are linked to fruit quality traits by efficient molecular and biochemical analysis coupled with bioinforatics analysis.Gene identification and molecular cloning help researchers and scientists to uncover the hidden genetic and molecular mechanisms of candidate genes regulating important trait of fruit quality.Thus,identification of key regulatory factors of pH,accumulation of sugars,and organic acids can provide a theoretical and technical platform for watermelon quality breeding.Precisely,to investigate the genetic characteristics and hereditary effects of pH and to improve the watermelon fruit quality and breeding efficiency,we used the mixed major gene plus polygene inheritance model with a joint analysis method of multiple generations.Six generations of F1,BC1P1,BC1P2,and F2were derived from a cross between parent P1(SrW‘low pH')and P2(Yul‘high pH')were used in the current study.The results showed that the genetic model of pH was‘one major gene of additive-dominance effects plus additive-dominance-epistasis polygene.The additive effect and dominant effect of the major gene were-0.4775 and-0.4766,respectively.Heritability of the major gene in BC1P1,BC1P2,and F2 populations were 21.76%,3.99%,27.10%and heritability of the polygene were 77.16%,94.64%and 72.09%respectively.These results laid an important theoretical basis for excavation excellent genes and varieties development.A set of 28 F1 hybrids and 28 female parents were used to investigate the accumulation patterns of sugars(Fructose,Sucrose,and Glucose),TSS,pH,and organic acids(Malic acid and Citric acid).Watermelon fruit was divided into nine sub-parts.Higher TSS and sugar contents were observed in non-sour watermelon fruits as compared to sour watermelon fruits.F1 hybrids who were sour have lower pH in the central sub-parts as compared to the edges.Malic acid and citric are the major organic acids in watermelon.The ratios of sugars and organic acids were highest in the heart sections and the peripheral sections of watermelon fruit this ratio goes on decreasing suggesting that the taste of watermelon is the combination of sugars and organic acids ratios.Moreover,watermelon parts having high TSS have high sugars,low acidity,and a higher pH whereas,watermelon parts having low TSS have a lower pH and higher organic acid contents and higher acidity.TSS has no effect on pH,rather pH was affected by the increase or decrease of organic acids contents.Interestingly,we found out that watermelon individuals with a low pH and sour taste have higher contents of organic acids.Two kinds of watermelon materials,namely,sweet watermelon(203Z)with a higher pH of 5.65 and TSS of 12 Brix%and a sour watermelon(SrW)with pH as low as 4.58 and an average TSS of 11.5 Brix%were used to evaluate the variations in sugar and organic acid metabolic contents.Briefly,the pure inbred line watermelon cultivar '203Z‘accumulates high total soluble sugar contents and low total organic acid contents at maturity.'SrW',developed by crossing the inbred cultivar '203Z' and the wild subspecies 'PI271769',accumulates low total soluble sugar contents and high total organic acid contents.Transcriptome profiles were used to investigate the coexpression patterns of gene networks associated with sugars and organic acids metabolism.We identified 3 gene networks/modules containing 2,443 genes highly correlated with sugars and organic acids.Within these modules,based on intramodular significance and Reverse Transcription Quantitative polymerase chain reaction(RT-qPCR),we identified 7 genes involved in the metabolism of sugars and organic acids.Among these genes,'Cla97C01G000640 ','Cla97C05G087120 and 'Cla97C01G018840'(r2=0.83 with glucose content)were identified as sugar transporters(SWEET,EDR6 and STP)and 'Cla97C03G064990'(r2=0.92 with sucrose content)was identified as a sucrose synthase from information available for other crops.Similarly,'Cla97C07G128420','Cla97C03G068240'and 'Cla97C01G008870',having strong correlations with malic(r2=0.75)and citric acid(r2=0.85),were annotated as malate and citrate transporters(ALMT7,CS and ICDH).The expression profiles of these 7 genes in diverse watermelon genotypes revealed consistent patterns of expression variation in various types of watermelon.'Yul'(sweet watermelon with a high pH of 5.66)and 'SrW'(a watermelon with sour taste and low pH of as low as 4.58)were crossed to produce F2 population.Besides,F1 plants were backcrossed with both parental lines to generate BC1P1 and BC1P2.All F1 plants showed low pH,suggesting that the low pH was dominant over the high pH.In spring 2019,it was observed that the ratio of low pH to high pH was 378:127 from 505 F2 population fitting the 3:1 Mendelian ratio(?2=0.10,p=0.76).Moreover,of the 80 plants derived from backcross with low pH parent,37 have a high pH and 43 had a low pH conforming the 1:1 Mendel's segregation ratio(?2=0.51,p=0.47).Both parents and 30 F2 individuals with a high pH and 30 individuals with a low pH were used to construct BSR segregation groups.pH locus was localized within 1,370,000-7,260,000bp(5.89 Mb)mapping region on chromosome 6 having 325 genes,with 96 of them up-regulated.Further,on the basis of functional annotation with other crops three putative genes,auxinresponsive factor 'Cla97C06G112650',transmembrane protein‘Cla97C06G112980',and vATPase‘Cla97C06G113170' were selected.RT-qPCR analysis was performed in both parents at different developmental stages(10,18,26,and 34 DAP).The expression of‘Cla97C06G113170' was higher as compared to the other two genes.A single nucleotide mutation in the low pH parent(C/T)was detected in the vATPase gene‘CIa97C06G113170' via IGV software.These results were also confirmed by sequencing the gene and then alignment via DNAMAN software.Further,we designed a KASP marker on the basis of detected mutation.Fifty-seven watermelon accessions including wild,landraces,and improved,were used to validate the designed KASP marker.KASP results showed two distinct clusters.Wild accessions have TT mutation in vATPase‘Cla97C06G113170' and their pH were low as compared to the landraces and improved accessions.Transient over-expression confirms that vATPase 'Cla97C06G113170' is the candidate gene for low pH.The watermelon fruits inoculated with vATPase gene have a lower pH and higher gene expression as compared to control.Promoter analysis for vATPase‘Cla97C06G113170' revealed that promoter sequence contained various putative cis elements involved in response to auxin transport and regulation,fruit ripening,and vacuolar acidification.In addition,MYB-related DNA binding motifs,the highly conserved binding site for WRKY TFs,were also identified.These results indicated that the expression of vATPase might be regulated by TFs,such as WRKYs and MYBs,and in response to various environmental stimuli,which is consistent with the finding that pH regulation is controlled by environmental and genetic factors.GUS analysis demonstrates that vATPase expression levels play role in pH regulation.GUS driven by the vATPase promoter was highly expressed in fruits injected with vATPase:GUS and not strongly expressed in control fruits.By contrast,CaMV35Spro::GUS was ubiquitously expressed in fruits.Overall,these findings add significantly to our existing knowledge about the genetic and molecular mechanisms regulating pH in watermelon.Further in-depth studies are required for how the transcription factors are involved in the activation of vATPase in watermelon to make pH low. |
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