Mapping And Functional Analyses Of The Tuberculate Fruit Gene Tu And The Dull Fruit Skin Gene D In Cucumber

Cucumber (Cucumis sativus L.;2n=2x=14), which belongs to thefamily Cucurbitaceae, is an economically important vegetable cropworldwide. Cucumber fruits that have tubercules and spines (trichomes)are known as possessing a warty (Wty) phenotype. Typically, cucumberlines are divided into Wty and non-warty (nWty) fruits, and this externaltrait affects the market value. The Wty fruit trait does not occur in themodel organisms Arabidopsis thaliana，Oryza sativa or in other commonspecies (Cucumis melo, Citrullus lanatus, Benincasa hispida, etc.) of theCucurbitaceae family, and the underlying mechanism is not wellunderstood.The results of the genetic analysis showed that a single dominantgene, Tu (Tuberculate fruit gene), controls the Wty fruit trait. Based onthe preliminary mapping of Tu and the published cucumber genomesequences, we developed332SSR and20SNP markers for map-basedcloning of Tu. Using the large F2population (2808individuals) from thecross of nWty fruit line S06×Wty fruit line S52, Tu was mapped to a41.6kb physical region that included two complete candidate genesCsa016922(encoding phosphoenolpyruvate carboxylase) and Csa016861(encoding C2H2-type zinc finger protein). Csa016922showed100%nucleotide sequence identity between S06and S52. The another geneCsa016861, containing a single exon, existed in Wty fruit line S52,encoded a ZFP with a single zinc finger motif, and featured theplant-specific QALGGH consensus sequence characteristic of C2H2-typeZFP TFs. However, the Csa016861gene in a4888bp region, includingthe promoter and coding sequences (CDS), was completely absent in thenWty line S06. Therefore, we hypothesized that Csa016861corresponded to Tu and controlled the Wty fruit trait. Moreover, based on thedifferences of Csa016861in S52and S06, two dominant markers TY-1and TY-2were designed in the promoter and CDS regions of Csa016861,respectively, and were tested by using94cucumber lines of Wty/nWtyfruits. The results showed that Csa016861existed in all38Wty fruit linesbut was absent in all56nWty fruit lines. Therefore, the results showedthat this transcription factor gene Csa016861, with a single C2H2zincfinger domain, corresponded to Tu and controlled the Wty fruit trait.Southern blot result showed that Tu existed as a single copy within thecucumber genome. Two new developed markers TY-1and TY-2canimprove plant breeding of the Wty/nWty fruit trait in cucumber.We engineered the fusion construct pBI121-CaMV35S-Tu andtransformed it into the nWty line S06using3082cotyledon nodes asexplants with the method of Agrobacterium-mediated transformation.Transgenic cucumber plants strongly confirmed that Tu (Csa016861) wasresponsible for the Wty fruit phenotype. Tobacco leaves transient throughinfilitration showed that the fusion protein GFP-Tu was mainly localizedto the nucleus. Based on analyses of semi-quantitative RT-PCR andquantitative RT-PCR, and mRNA in situ hybridization, we found Tu wasspecifically expressed in fruit spine cells during development of fruittubercules.In a glabrous tubercule-free mutant gl line with Tu, Tu could not beexpressed in the presence of the glabrous fruit gl allele in the mutant glline. Taken together, these results showed that the recessive glabrous fruitgene gl played the role of epistatic effect over Tu. Our study confirmedthat fruit spines are a necessary precondition for the formation of fruittubercules. Taken together, these results can well explain why alltuberculate fruits have spines, fruits with spines do not always havetubercules, and fruits without spines never have tubercules.We measured CTK content in S06fruit epidermis (control), S06T1transgenic fruit epidermis, S06T1transgenic fruit warts, and S52fruitepidermis and warts at two days before flowering (2DBF, the tuberculeinitiation stage) and one day post anthesis. This study found that Wty fruittrait was correlated with high CTK content which could promote cell division leading to an increase in cell numbers. We analyzed thetranscriptomes of S06fruit epidermis and S06T1transgenic fruit warts at2DBF (the tubercule initiation stage), and Tu seemed to regulateCTK-Hydroxylase like genes Csa5M644580and Csa5M224130, bothupstream in the CTK biosynthetic pathway (No. ko00908).We also carried out the study of the melon homolog of Tu (No:MELO3C005347). Our results showed that melon homolog was notexpressed in the melon fruit epidermis without tubercules, furthersuggesting that Tu is responsible for the Wty fruit trait in cucumber. Thisstudy also analyzed the evolutionary characteristic of Tu, and cucumberTu was found to be the orthologue of ZFP6in Arabidopsis lyrata.Fianlly, we proposed the genetic pathway for the Wty fruit trait incucumber.Dull/glossy fruit skin is another highly valuable external quality traitthat affects the market value of cucumbers. Genetic analysis showed thatone dominant gene, D (dull fruit skin), determines the dull fruit skin traitin cucumber. When we examined66published primer pairs, our studyfound that46primer pairs were polymorphic (69.7%) between parentsS94(dull fruit skin line) and S06(glossy fruit skin line). By bulkedsegregant analysis of two DNA bulks, we found that11primer pairscould generate polymorphic products and were linked to the D/d locus.All potential linkage markers were mapped using the216individuals ofthe F2population (S06×S94). A local genetic map of the D/d locus wasconstructed. the D/d gene was preliminarily mapped between markersSCZ69and SSR16203, at genetic distances of0.3and0.6cM,respectively.The genome sequence between SCZ69and SSR16203totaled422.8kb, which was used to develop155SSR markers. Subsequently, the eightnewly developed SSR markers and markers SCZ69and SSR16203werescored on842F2(S06×S94) individuals. Subsequently, a fine geneticmap of the region surrounding the D gene was constructed, and the totalgenetic distance was1.1cM. Finally, the D/d gene was fine-mappedbetween new developed markers SSR37and SSR112, at a physicaldistance of244.9kb (containing31candidate genes). Based on semi-quantitative RT-PCR analysis, the possible candidate gene D wasidentified as Csa016880，Csa016887or Csa016899. Meanwhile, validityanalysis of the markers SSR37and SSR112was performed with72dull/glossy fruit lines, and showed that the two co-dominant SSR markerscould be used for marker-assisted selection of the dull/glossy fruit trait incucumber breeding. Moreover, this study will be helpful for eventualcloning of the D/d gene in cucumber.