Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.     

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome).     

A method for increased plant regeneration from immature F1 and BC1 embryos of Cucurbita maxima Duch. x C. pepo L. hybrids

Plants have been regenerated from abnormal embryos with spongy cotyledons and albino sectors, derived from Cucurbita maxima and C. pepo F₁ and BC₁ hybrids. Shoot regeneration was induced directly from the cotyledons without an intervening callus phase on the medium without hormones. On the rooting medium, shoots continued to proliferate, which allowed for further multiplication in vitro. The number of plants obtained varied with genotype and ranged up to 65 plants per embryo.     

Heterogeneity and organization of the ribosomal RNA genes ofCucurbita maxima

Thirty-six clones were recovered fromCucurbita maxima genomic DNA which had been enriched for rDNA and cleaved at the unique repeat unitHind III site. Twenty-nine of these, which contain complete rDNA units, were compared to a standard whose intergenic spacer (IGS) nucleotide sequence has been determined. Twenty-one are identical in length and restriction site pattern. Eight which differ from the standard in length do so because of addition or deletion of varying numbers of IGS subrepetitive units of two different classes, with four of the length variants being different in both of these classes. Seven clones were isolated which contain incomplete repeat units, six of which are composites of rDNA and non-rDNA material. They have been cleaved at the unique rDNAHind III site at one end and at a non-rDNAHind III site at the other. We consider it most likely that these are derived from the termini of repeat unit tandem arrays, although other explanations are possible. Twelve individual plants of two different cultivars were examined for heterogeneity of IGS length distribution. They all appear to be identical in this regard.     

Differential homogenization and amplification of two satellite DNAs in the genus Cucurbita (Cucurbitaceae)

Two different satellite DNAs exist in the genus Cucurbita which are different with respect to repeat length (350 by and 170 bp), array size, and sequence homogenization. Whereas the 350-bp satellite DNA is prominent and very homogeneous in all species investigated except for C. maxima and C. lundelliana, the 170-bp satellite is rather evenly distributed in all species. In C. maxima and C. lundelliana the 350-bp satellite is present only in small amounts, but detectable by the sensitive PCR method. These repeats are also very homogeneous, reflecting a silent stage of satellite DNA. In contrast, the 170-bp satellite DNA is intra- and interspecifically heterogeneous. It is striking that the species with no detectable amount of 350-bp satellite contain 170-bp satellite DNA clusters with the highest degree of homogeneity. The evolution of satellite DNA repeats within cultivated and wild species in the genus Cucurbita is elucidated using the sequence data of both satellite DNAs from all species investigated. The value of satellite DNA for phylogenetic analysis between closely related species is discussed. Correspondence to: V. Hemleben     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in <Emphasis Type="Italic">Urvillea chacoensis</Emphasis> Hunz. (Paullinieae,Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72–73% and the studied clones showed an identity of 92.5–95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.     

The Cucurbita maxima ribosomal DNA intergenic spacer has a complex structure

The nucleotide (nt) sequence of the 5508-nt intergenic spacer (IGS), between the 25S- and the 18S-coding regions of Cucurbita maxima rDNA, was determined. The fragment sequenced is 6142 nt long and includes 472 nt of 25S- and 162 nt of 18S-coding regions. The IGS has a complex primary structure, composed of five repetitive families (A-E) and three unique domains. It is dominated by the presence of nine, tandemly-repeating units of approximately 250 nt (repeat D), each unit containing four copies of an internal subrepeat (repeat E). The repetitive units show sequence variability consisting of nt changes, insertions and deletions. Upstream of the nine D repeats and between two copies of the B repeat is a 575-nt region, highly G + C rich (83%) and heavily biased toward C (58%) in the sense strand. Within this region are six repetitive units, averaging 42 nt (repeat C) each, containing but a single A nt. Downstream from the terminus of the 25S-coding sequence, are two tandem copies of the 103-nt A repeat. The IGS of C. maxima is longer and more complex than that of other plant IGSs described to date. The 600 nt at the 5' portion of cucurbit IGS is more conserved in evolution than the remainder, as revealed by comparison of C. maxima and C. pepo IGS restriction maps and by nucleotide sequence comparison of C. maxima and Cucumis sativa IGSs.     

Heat shock pre-treatment enhances the response of Arabidopsis thaliana leaves and Cucurbita pepo cotyledons to benzyladenine

The effects of heat shock (HS) pre-treatment on the response tobenzyladenine were studied in two plant model systems (1) retardation ofsenescence of Arabidopsis thaliana L. Heyhn rosette leavesand (2) induction of greening of detached Cucurbita pepoL.cotyledons. N⁶-benzyladenine (BA) retarded senescence of rosetteleaves of Arabidopsis thaliana (L) Heyhn and briefpre-treatment with HS (3 at 37)essentially enhanced this cytokinin effect. BA stimulated cotyledon greening inCucurbita pepo L due to the activation of chlorophyllsynthesis. Brief cotyledon pre-heating at moderate temperatures (3 at 33–35) also enhanced thiscytokinin effect.     

Pollen gene expression analysed by micro-isoelectric focusing of proteins from isolated pollen grains in Cucurbita pepo L.

Summary Isoelectric focusing of proteins from single pollen grains of Cucurbita pepo L. has been developed for large scale study of pollen grain populations' heterogeneity. Forty to forty-five protein bands from one pollen grain are revealed after silver staining. Applications of this technique to pollen grain populations from different genotypes are described in this paper. Possible applications and limits of this technique are discussed with respect to plant breeding especially for the measure of gene frequencies in pollen grain populations.The work was partly supported by a grant from LIMAGRAIN and AGPM     

Ultrastructural localization of glutathione in Cucurbita pepo plants

Summary. Electronmicroscopic immunogold cytochemistry was used to investigate the cellular and subcellular distribution of glutathione in root and leaf cells of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) plants. Gold particles bound to glutathione were found in various cell structures. Statistical evaluation of the gold particle density was made for different cell compartments including nuclei, mitochondria, plastids, peroxisomes, and the cytosol. In each cell type the highest level of glutathione immunoreactivity occurred in mitochondria, for which the labeling density was found to be higher in mesophyll cells of the youngest fully developed leaves (younger leaves) than in the 5th leaves (older leaves) or in root tip cells. Additionally, a statistically significant increase of gold particles bound to glutathione was observed in nuclei (22%) and the cytosol (14%) of the root cells in comparison with mesophyll cells of older (17% and 9%, respectively) and younger leaves (11% and 6%, respectively). The relevance and specificity of glutathione labeling is discussed with respect to difficulties of immunolocalization of low-molecular-weight compounds.     

Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

Phylogenetic relationship and germplasm evaluation of different taxa of the genus Cucurbita using seed storage protein profiling

Seed protein profiling of 34 lines of Cucurbita pepo L. from different geographic regions of the world were evaluated for their polypeptide patterns and their phylogenetic relationship with other taxa of the genus Cucurbita. Considerable variations were observed in the polypeptide patterns of various lines on the SDS-polyacrylamide gels under reduced conditions, i.e. seed protein extract with 2-mercaptoethanol. The variations were observed in five different molecular weight regions, i.e. in the range 64–70, 53–60, 30–41, 20–26 and 18–20 kDa. Cluster analysis showed 100% genetic similarity between C. pepo and C. pepo var. pepo whereas it is quite distinct from C. pepo var. texana and C. pepo var. fraterna. On the basis of electrophoretic profiling C. pepo var. texana and C. pepo var. fraterna should be considered as different species and also supports the origin of C. pepo from C. texana.     

Plastid differentiation during Cucurbita pepo (Cucurbitaceae) pollen grain development

The development of plastids in the pollen of Cucurbita pepo was followed from meiosis to pollen maturation by quantitative light and electron microscopy. Plastids are initially undifferentiated, then divide, and at late microspore stage differentiate into amyloplasts containing starch. Later the amyloplasts form lobes and divide. Amyloplasts containing a single starch grain are present from the early bicellular stage. Plastid development is considered in relation to such cytoembryological features as the pollen does not dehydrate at anthesis and germination begins 3 min after pollination.     

Chloroplast DNA diversity among wild and cultivated members of Cucurbita (Cucurbitaceae)

Summary Cladistic analysis of 86 chloroplast DNA restriction-site mutations among 30 samples representing 15 species of Cucurbita indicates that annual species of the genus are derived from perennials. The Malabar Gourd, C. ficifolia, is placed as a basal, sister taxon relative to other domesticated species and allied wild-types. The pattern of variation supports three species groups as monophyletic: (1) C. fraterna, C. pepo, and C. texana, (2) C. lundelliana, C. martinezii, C. mixta, C. moschata and C. sororia, and (3) C. foetidissima and C. pedatifolia. Domesticated samples representing subspecies of C. pepo are divided into two concordant groups, one of which is allied to wild-types referable to C. texana and C. fraterna. The data failed to resolve relationships among cultivars of C. moschata and C. mixta and their association to the wild C. sororia. The South American domesticate, C. maxima, and its companion weed, C. andreana, show close affinity and alliance to C. equadorensis.     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

An endogenous RNA-synthesis-promoting oligopeptide from Cucurbita pepto var. patissonina

The effect of CPPTI (Cucurbita pepo patissonina trypsin inhibitor) on the growth of Cucurbita pepo var patissonina (White bush) was examined. Plants treated with CPPTI grew faster than untreated plants and similarly to those treated with gibberellic acid. Isolated cell nuclei from plants treated with CPPTI showed that of the three DNA-dependent RNA polymerases assayed, RNA polymerase I (EC2.7.7.6) activity was preferentially elevated.Abbreviations CPPTI Cucurbita pepo patissonina trypsin inhibitor - GA₃ gibberellic acid - IAA indole-3-acetic acid - kDa kilodalton - RP I DNA-dependent RNA polymerase I     

Identification and characterization of Cucurbita gummy stem blight fungi in Northeast China

Cucurbita maxima (pumpkin) is a typical cucurbit that is susceptible to gummy stem blight. In recent years, this major fungal disease has decimated pumpkin yields in Northeast China (Heilongjiang Province) in increasingly numerous outbreaks with more rapid spread in recent years. After culturing fungi on potato dextrose agar medium, we conducted a systematic study of the growth and morphological characteristics of various purified strains of Cucurbita gummy stem blight (GSB) fungus from across Northeast China. Subsequently, DNA samples of 30 isolates with distinct hyphal variations were subjected to internal transcribed spacer (ITS) sequencing. Sequence analysis resulted in identification of the isolates as Stagonosporopsis cucurbitacearum and demonstrated that this is a dominant and widely distributed fungal species in this region. Subsequently, multi‐site phylogenetic analysis assigned the 30 aforementioned strains to two genotypes that aligned to seven phenotypic types of Cucurbita GSB fungi. By analysing ITS conserved sequences of these phenotypically diverse groups, we found that Cucurbita GSB pathogens broadly shared two motifs that contained sequence variations unique to two groups in addition to common identical motifs. Ultimately, this study provided useful data for rapid and accurate identification of S. cucurbitacearum and diagnosis in early symptoms of Cucurbita GSB. This work also provides tools to explore the distribution and regularity of GSB outbreaks spatially and temporally across Northeast China.