High-resolution mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.)

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.     

Identification and mapping of molecular markers linked to the tuberculate fruit gene in the cucumber (Cucumis sativus L.)

Warty fruit is one of the highly valuable external quality traits related to the market values of cucumber. Genetic analysis has shown that a single dominant gene, Tu (Tuberculate fruit), determines the warty fruit trait in the cucumber plant. An F₂ population (247 individuals) from the cross of S06 × S52 was used for the mapping of the Tu/tu locus. By combining bulked segregant analysis with the sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers, 15 markers (9 SRAPs and 6 SSRs) linked to the Tu/tu locus were identified. Of nine SRAP markers, three closely linked to the Tu/tu locus were successfully converted into sequence characterized amplified region (SCAR) markers. The Tu/tu locus was mapped between the co-dominant SSR marker SSR16203 and the SCAR marker C_SC933, at a genetic distance of 1.4 and 5.9 cM, respectively. Then the linked SSR markers in the study were used as anchor loci to locate the Tu/tu locus on cucumber chromosome 5. Moreover, the validity analysis of the C_SC69 and C_SC24 markers was performed with 62 cucumber lines of diverse origins, showing that the two SCAR markers can be used for marker-assisted selection (MAS) of the warty fruit trait in cucumber breeding. The information provided in this study will facilitate the map-based cloning of the Tu/tu gene.     

Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.)

Zucchini yellow mosaic virus (ZYMV) routinely causes significant losses in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). ZYMV resistances from the cucumber population TMG1 and the melon plant introduction (PI) 414723 show different modes of inheritance and their genetic relationships are unknown. We used molecular markers tightly linked to ZYMV resistances from cucumber and melon for comparative mapping. A 5-kb genomic region (YCZ-5) cosegregating with the zym locus of cucumber was cloned and sequenced to reveal single nucleotide polymorphisms and indels distinguishing alleles from ZYMV-resistant (TMG1) and susceptible (Straight 8) cucumbers. A low-copy region of the YCZ-5 clone was hybridized to bacterial artificial chromosome (BAC) clones of melon and a 180-kb contig assembled. One end of this melon contig was mapped in cucumber and cosegregated with ZYMV resistance, demonstrating that physically linked regions in melon show genetic linkage in cucumber. However the YCZ-5 region segregated independently of ZYMV resistance loci in two melon families. These results establish that these sources of ZYMV resistances from cucumber TMG1 and melon PI414723 are likely non-syntenic.     

Effect of ovular receptivity on seed set and fruit development in cucumber (Cucumis sativus L.)

Summary Seed set and fruit development in cucumber (Cucumis sativus L.) were studied in relation to female flower receptivity from day — 2 before anthesis to day + 2 after anthesis. The female cucumber flower is protogynous. The pistil was receptive 2 days before anthesis. The iso-electric focusing (IEF) patterns of the stigma/style proteins, were identical from day -5 to day +2. In pollinated flowers in vivo germination and pollen-tube growth in the ovary were affected by pistil age from day -2 to day +2. In addition, differences in sectorial filling in full seeds were observed within the fruits. A negative correlation was observed between the frequency of fertilized ovules in the pedoncular part of the fruit and ovary length at the time of pollination. In the whole fruit, significant differences in the number of full seeds and fruit size at maturity were found, and these were observed to be correlated with the various stages of female flower maturation at pollination. The day -2 and day +2 stages yielded the smallest fruits with few full seeds compared to the day -1, day 0 and day +1 stages, which had the biggest fruits and a large number of full seeds. A strong positive correlation was found between total seed number (including full and empty seeds), fruit length and weight at maturity. All these results suggest that both seed set in the different parts of the fruit and fruit development are controlled by ovular receptivity rather than by stigma/style receptivity.     

Molecular cloning and expression analysis of spermidine synthase gene during sex reversal induced by Ethrel in cucumber (Cucumis sativus L.)

A full-length cDNA encoding spermidine synthase CsSPDS of 317 amino acids was isolated from cucumber. CsSPDS showed high similarity to SPDSs of other species, with the greatest similarity to SPDS of Coffea arabica (identity, 87%). Northern blot analysis revealed that CsSPDS was expressed predominantly in rapid growing tissues, such as roots, young leaves and flower buds. The response of CsSPDS to the sex reversal of three-leaf stage androecious cucumber seedlings by Ethrel treatment was also examined. Our results showed that the CsSPDS was up-regulated in shoot apex during sex reversal process, which correlated with increases of spermidine synthase activity and free spermidine (Spd) content, and a decrease of putrescine (Put) content in shoot apices.     

Transformation of cucumber (Cucumis sativus L.) plants with Agrobacterium rhizogenes

Summary Transgenic cucumber plants (Cucumis sativus L., cv. Straight Eight) were regenerated from roots induced by inoculation of inverted hypocotyl sections with Agrobacterium rhizogenes containing the vector pARC8 in addition to the resident Ri-plasmid. The DNA transferred to the plant from the vector (T-DNA) included a gene which encoded the enzyme neomycin phosphotransferase II, and thus conferred on the plant cells resistance to kanamycin. The transgenic plants looked normal and were positive for the neomycin phosphotransferase II. Southern blot analysis of the transgenic plants revealed that all plants contained vector DNA, but only some of them contained DNA from the Ri plasmid.     

Ploidy instability of embryogenic cucumber (Cucumis sativus L.) callus culture

Embryogenic callus cultures were established from immature cucumber(Cucumis sativus L.) embryos on E20A (Dumas de Vaulxet al. 1981) or MS (Murashige and Skoog 1962) media supplemented with 6-benzylaminopurine (BAP), α-naphthylacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration of plants was observed after a transfer to culture media either without growth regulators or supplemented with kinetin and NAA. Flow cytometry was employed to estimate DNA ploidy levels. Most of cell nuclei in young leaf tissues were found in G₁ phase with 2C DNA content. Callus cultures were mixoploid with DNA content ranging from 2C to 32C. The frequency of polyploid cells was increasing with the age of culture and the polyploidization was accompanied by a gradual loss of regeneration ability. Plants regenerated from callus cultures were classified as diploid (57 %), tetraploid (18 %), octoploid (4 %) and mixoploid (2n/4n, 4 %) and (4n/8n, 17 %). The results of this study confirmed a close link between the polyploidization and the loss of totipotencyin vitro. Tetraploid plants obtained in this study have a potential to be used in interspecific crosses where their tetraploid status could help in overcoming existing breeding barriers due to differences in chromosome number.     

QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.)

A population of F₇ recombinant inbred lines (RILs) was made from a cross between susceptible (‘Santou’) and resistant (PI197088-1) lines of cucumber in order to study powdery mildew resistance loci. Susceptibility to powdery mildew in the F₇ RIL individuals showed a continuous distribution from susceptible to resistant, suggesting that powdery mildew resistance is controlled by quantitative trait loci (QTLs). A QTL analysis identified two and three loci for powdery mildew resistance under 26 and 20°C conditions, respectively. One QTL was found in the same position under both temperature conditions. Therefore, it is more likely that one major QTL acts under both temperature conditions and that other QTLs are specific to the two temperature conditions. The above results suggest that the four QTLs are controlled in a different temperature manner, and that their combination played an important role in expressing a high level of resistance to powdery mildew in this cucumber population. Sequence-tagged site (STS) markers associated with each QTL were developed and would be useful for breeding a cucumber line with a high level of powdery mildew resistance. Y. Sakata and N. Kubo contributed equally to this work and are considered as first authors.     

黄瓜性型分化的分子机制

黄瓜(Cucumis sativus)是雌雄异花植物性型分化研究的重要模式植物,近年来虽然其性型分化的分子机制研究取得了一定的成果,但其性型分化的调控机制尚未完全阐明。该文综合花器官发育基因、性别决定基因、内源激素、环境因子、性型分化假说,在分子水平构建了黄瓜性型分化的表达调控网络。同时对激素和性别决定基因协控的黄瓜单性花器官凋亡机制进行了阐述,并就miRNA在黄瓜性型分化调控中的作用进行了探讨。     

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.)

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.     

Virus particles in guttate and xylem of infected cucumber (Cucumis sativus L.)

Particles of plant viruses representing ten genera were visualised by electron microscopy in samples of guttate from systemically infected cucumber (Cucumis sativus L.) plants. For the majority of viruses tested, guttation samples tested positively by ELISA and infectivity was also demonstrated. Virus particles of zucchini yellow mosaic potyvirus were also observed in xylem in sections of infected root tissue. Since guttate originates from xylem exudate, this study demonstrates the potential for xylem tissue to act as a conduit for a diverse group of plant viruses in planta     

Inheritance and linkage relationships of isozyme loci in cucumber (Cucumis sativus L.)

Summary Genetic analyses were conducted among 18 provisionary isozyme loci in Cucumis sativus L. Fourteen loci demonstrated simple Mendelian inheritance while observed variation at four loci (Gpi2, Gr2, Pgm3, Skdh2) was determined not to have a predictable genetic basis. Joint segregation analyses among the 14 genetically predictable polymorphic loci resulted in the assignment of 12 loci to four linkage groups. Linkage groups contain the following loci: (1) Gr1, Pgm1, Idh, Pgd1; (2) Pep-pap, Mdh2, Mdh3, Gpi1; (3) Pep-la, Per4; (4) Pgd2, G2dh. Mpi2 and Mdh1 segregated independently. Recombination fractions for linked loci ranged between 0.051 (Pgm1-Idh) to 0.385 (Pep-la-Per4). Some practical applications of isozyme marker loci for cucumer improvement are discussed.     

Aluminium effect on nitrate assimilation in cucumber (Cucumis sativus L.) roots

In vivo effect of aluminium on nitrate uptake and reduction by cucumber seedlings was investigated. The high-performance liquid chromatography was used to analyse the rate of nitrate uptake. Low (0.5 mM) concentration of AlCl₃ in the nutrient solution stimulated nitrate uptake during the first 3 h. On the other hand, 6 h exposure of the cucumber seedlings to 1 or 5 mM of AlCl₃ resulted in inhibition of nitrate uptake and at 5 mM concentration of AlCl₃ the efflux of nitrate was observed. Furthermore, the amount of nitrate accumulated in cucumber roots after aluminium treatment was decreased. The noteworthy fact was observed, that at all concentrations of aluminium tested on increase of the nitrate reductase activity. This stimulation was concentration depended, but independent of the source of the enzyme. The activity of both the cytosolic and the plasma membrane bound nitrate reductase activity was enhanced in vivo. On the other hand, AlCl₃ applied in vitro only slighty decreased nitrate reductase activity.