Levels of seed proteins in Citrullus and Praecitrullus accessions

Variation among 17 accessions of Citrullus lanatus from different geographic regions and interspecific relationships of six taxa of Citrullus and Praecitrullus were studied using electrophoretic patterns of their seed storage proteins. Globulins, the salt soluble proteins, represented the major fraction with their proportion varying between 56.6% and 67.0%. These were followed by albumins (16.6–20.8%) and glutelins (13.5–18.5%) with prolamins as the lowest (2.2–4.1%) of the four fractions. Two-dimensional gel electrophoresis under nonreducing conditions in the first dimension and reducing conditions in the second revealed disulphide-bonded subunit pairs of molecular weight 53, 52, 50 and 41 kDa, unlike the single subunit pair generally reported in different cucurbits, each consisting of a large and a small subunit. In the UPGMA dendrogram based on polypeptide patterns, the occurrence of C. lanatus var. lanatus, C. lanatus var. citroides and C. lanatus accession PI 482318 in one subcluster suggested that phylogenetically C. lanatus var. citroides and C. lanatus var. lanatus are closely related. The recently described annual wild species, Citrullus rehmii, occurred independently nearest to the subcluster of these cultivated and wild taxa. Citrullus colocynthis, the perennial wild species occurred farther from this cluster showing relatively more genetic distance from the watermelons. Praecitrullus fistulosus was outclustered and appeared genetically distant from all the Citrullus taxa; this supported its placement in a separate genus unlike its nomenclature as a botanical variety of watermelon or as a separate species of Citrullus proposed in certain earlier studies.     

An analysis of interspecific hybrids and phylogenetic implications inCucumis (Cucurbitaceae)

Investigations on interspecific crossability in 8Cucumis species (2n = 24) and chromosome pairing and pollen fertility of their hybrids from 15 combinations have been utilized for tracing the phylogenetic relationships among these taxa and factors responsible for their differentiation. A collective evaluation of data suggests that there are three broad groups of species, one of the spiny fruited interfertile species, whose hybrids show varying degree of chromosome associations and low to high pollen fertility; the second of species with non-spiny fruits, which are completely incompatible with the former but weakly compatible with the cultivated species,C. melo L. to produce partly developed seeds, and the third group ofC. metuliferus E. Mey. exSchrad. andC. melo and its different botanical varieties. The species with spiny fruits can be further divided based on karyomorphological similarities and/or on relative genomic affinity, indicated by chromosome pairing and hybrid pollen fertility.Cytogenetics inCucumis III.     

Meiotic analyses ofCucumis hybrids and an evolutionary evaluation of the genusCucumis (Cucurbitaceae)

Meiosis in seven interspecificCucumis hybrids has been analysed i.a. inC. metuliferus ×C. zeyheri, where the parents belong to different sections. In the triploid hybrids a remarkably high number of trivalents has been found. Additional data from literature on geographical distribution, cucurbitacins, flavonoid patterns, isozymes, C-banding, genome size, DNA amount and chloroplast DNA are used to discuss species relationships and evolution. The African cross-compatible group is divided into theMyriocarpus subgroup with the diploid speciesC. africanus, C. myriocarpus subsp.leptodermis and subsp.myriocarpus, and theAnguria subgroup withC. anguria, C. dipsaceus, C. ficifolius, C. prophetarum, C. zeyheri and all polyploids (exceptC. heptadactylus). It is argued that the Asian subg.Melo with x = 7 is derived from the African subg.Cucumis with x = 12; the latter contains all the polyploid species and has the most common basic chromosome number of theCucurbitaceae. This phylogenetic advance is interpreted with concepts of the quantum model of evolution.     

Phylogenetics of <Emphasis Type="Italic">Cucumis</Emphasis> (Cucurbitaceae): Cucumber (<Emphasis Type="Italic">C. sativus</Emphasis>) belongs in an Asian/Australian clade far from melon (<Emphasis Type="Italic">C. melo</Emphasis>)

Background  

Melon, Cucumis melo, and cucumber, C. sativus, are among the most widely cultivated crops worldwide. Cucumis, as traditionally conceived, is geographically centered in Africa, with C. sativus and C. hystrix thought to be the only Cucumis species in Asia. This taxonomy forms the basis for all ongoing Cucumis breeding and genomics efforts. We tested relationships among Cucumis and related genera based on DNA sequences from chloroplast gene, intron, and spacer regions (rbcL, matK, rpl20-rps12, trnL, and trnL-F), adding nuclear internal transcribed spacer sequences to resolve relationships within Cucumis.     

Collecting cultivated and wild Cucurbits in Mexico

The authors spent approximately 5 weeks in Mexico (October 13–November 15, 1979) collecting Cucurbitaceae in 5 areas: 1) Tampico- Valles- Ocampo; 2) San Luis Potosi; 3) Celaya- Queretaro; 4) Veracruz; 5) Zacatecas- Durango- Mazatlan. A few supplemental collections were made in the vicinity of Tapachula, Chiapas. A grand total of 183Cucurbita collections was made, distributed as follows:C. pepo-49;C. moschata-47;C. mixta- 41;C. ficifolia-11;C. foetidissima-13;Cucurbita sp. wild, unidentified- 22. A few collections were made in the following genera:Sechium-4;Lagenaria-2;Sicana-1;Momordica-4;Sicyos-2;Cucumis melo-2;C. anguria-1. The ecological distribution of the 4 cultivated species is reported and discussed. Seed of these materials was divided equally between the participating countries, and will be made available to interested plant breeders.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Comparison of the Efficacy of Ground Wild Cucumber Fruits, Aldicarb and Fenamiphos on Suppression of Meloidogyne incognita in Tomato

During the present study the efficacy of ground Cucumis myriocarpus fruits and synthetic non‐fumigant nematicides on suppression of Meloidogyne incognita numbers and effect on growth of tomato was evaluated and compared in a microplot experiment. All microplots were artificially inoculated with second‐stage juveniles of M. incognita. Treatments included an (i) untreated control, application of (ii) Cucumis, (iii) aldicarb (a. i. = 150 g/kg), (iv) fenamiphos (a. i. = 100 g/kg) each at the product quantity of 42 kg/ha. The study was repeated in the second year using randomized complete block design with 10 replications. The application of products reduced nematode numbers in tomato roots (83–99%) and increased tomato fresh fruit yield (72–94%). Generally, the effect of the three applications on the parameters measured were not significantly (P = 0.05) different from one another, confirming the potent nematicidal properties of ground C. myriocarpus fruits.     

Two nuclear-coded subunits of mitochondrial complex I are similar to different domains of a bacterial formate hydrogenlyase subunit

A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.     

A comparative analysis of phloem exudate proteins from Cucumis melo,Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing

Summary Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.Abbreviations DTT dithiothreitol - Bis N,N-methylenebisacrylamide - SDS Sodium dodecyl sulphate - TCA trichloroacetic acid     

Chemical and immunological similarities between the phloem proteins of three genera of the Cucurbitaceae

Phloem exudates from Cucurbita, Cucumis, and Citrullus were gelled by oxidative formation of disulphide bridges between the phloem filaments. Gellation could be inhibited by dithiothreitol or iodoacetamide and did not require the presence of the phloem lectin. Each exudate contained a dimeric lectin of similar relative molecular mass and purified specific activity; these were all specific for oligomers of N-acetyl-glucosamine, and shared antigenic determinants. The similarity of the phloem proteins between Cucurbita, Cucumis, and Citrullus implied that they served the same function in each genus. This is postulated to be the sealing of wounded sieve-tubes, with the lectin on the filaments binding and preventing the entry of micro-organisms. The phloem lectin and the filament-forming protein from Cucurbita shared sequence homologies as judged by amino-acid-composition comparisons, but antibodies raised against each showed no cross-reactivity with the other protein. The exudates from Cucurbita and Cucumis may contain a high concentration of phloem proteins because the large diameter of their sieve-pores does not allow rapid blocking by callose synthesis on wounding, and a chemical mechanism of gellation is required.     

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50–200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ?OmpA and ?OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.     

Purification and functional characterization of bovine RP-A in an<Emphasis Type="Italic">in vitro</Emphasis> SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.     

Development of novel chloroplast microsatellite markers for Cucumis from sequence database

The development of chloroplast microsatellite (cpSSR) markers in Cucumis species and analysis of their polymorphism and transferability were reported. Fifteen microsatellite markers, represented by mononucleotide repeats, were developed from the complete sequence of Cucumis sativus chloroplast genome. Intraspecific variation was successfully detected in C. sativus and C. melo and revealed mean 1.6 and 1.9 alleles per cpSSR locus, respectively. With the exception of two exon region-located cpSSR markers being monomorphic, each of the others amplified polymorphic fragments in C. sativus or C. melo. A total of 34 polymorphic loci were detected with these cpSSR markers in the two species. Transferability of the newly developed cpSSR markers was checked on an additional set of 41 Cucurbitaceae accessions (belonging to 12 different species), and except for two markers with no amplification in Cucurbita maxima, the others could be transferable to all the accessions tested. Of the 15 cpSSR markers, 14 markers generated fragments with expected band sizes and 13 markers detected interspecific polymorphism among the accessions. Intraspecific polymorphism was also observed within four Cucurbitaceae species excluding C. sativus and C. melo.     

Sucrose metabolism and accumulation in developing fruit of Cucumis

Sucrose, reducing sugars and starch content were measured in developing cucumber (C. sativus cv Delilah), sweet melon (C. melon cv Galia and cv Noy Yizre'el) and non-sweet melon (C. melo cv Bird's Nest) fruit. Sweet melon were characterized by accumulation of sucrose in the maturing fruit, while cucumber and non-sweet melon had a low sucrose content at all stages studied. Soluble acid invertase activity (EC 3.2.1.26) dramatically decreased in sweet melon, concomitant with the onset of sucrose accumulation. Significant activity of soluble acid invertase was retained in mature cucumber and non-sweet melon. Insoluble acid invertase, determined not to be an artifact of extraction, constituted a significant portion of total invertase activity (ca 25% in young sweet melon and ca 50% in young cucumber). In sweet melon sucrose synthase activity (EC 2.4.1.13), measured in both the cleavage and synthesis direction, increased during the sucrose accumulation period. The results are discussed in terms of the roles of invertase and sucrose synthase in sucrose accumulation in Cucumis.     

An assessment of some molecular parameters of mevalonate kinase from plant and animal sources

The molecular weights, diffusion coefficients, and sedimentation coefficients of mevalonate kinase in partially purified preparations from Hevea brasiliensis latex, Cucumis melo cotyledons, Phaseolus vulgaris cotyledons, bakers yeast, chicken liver, and rabbit liver have been determined by gel filtration in Sephadex G-100 and G-200 and by sucrose density gradient centrifugation. The enzyme had similar molecular weights (94800–103500), diffusion coefficients (5.39–5.62 × 10^?7 cm²/sec), and sedimentation coefficients (5.85–6.00 S) in the six preparations.     

Characterization of complement 1q binding protein of tiger shrimp,Penaeus monodon,and its C1q binding activity

The receptor for the globular heads of C1q, C1qBP/gC1qR/p33, is a multicompartmental, multifunctional cellular protein with an important role in infection and in inflammation. In the present study, we identified and characterized the complement component 1q subcomponent binding protein (C1qBP) from the tiger shrimp Penaeus monodon (designated as PmC1qBP). The open reading frame of PmC1qBP encodes 262 amino acid residues with a conserved MAM33 domain, an arginine-glycine-aspartate cell adhesion motif, and a mitochondrial targeting sequence in the first 53 amino acids. PmC1qBP shares 32%–81% similarity with known C1qBPs and clusters with lobster gC1qR under phylogenetic analysis. The temporal PmC1qBP mRNA expression in the hepatopancreas was significantly enhanced at 9 h after Vibrio vulnificus challenge. The native PmC1qBP was expressed in the gills, hepatopancreas, ovaries, and intestines as a precursor (38 kDa) and the active peptide (35 kDa). The recombinant PmC1qBP protein was expressed in Escherichia coli BL21, and was purified using nickel–nitrilotriacetic acid agarose. A complement 1q binding assay indicated that the rC1qBP protein competitively binds to C1q in mouse serum. The data reveal that PmC1qBP is not only involved in shrimp immune responses to pathogenic infections, but also cross-binding to the mouse C1q.     

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S–5.8S–26S and 5S ribosomal RNA gene families,and the two tandemly repeated DNA sequences

In the genus Carthamus (2n = 20, 22, 24, 44, 64; x = 10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S–26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S–26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n = 20, and the two botanical varieties of B genome C. tinctorius (2n = 24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S–26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n = 20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n = 44), and C. lanatus is one of the progenitors for the hexaploid (2n = 64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2–10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.     

Fine genetic mapping of a locus controlling short internode length in melon (Cucumis melo L.)

Compact and dwarfing vining habits in melon (Cucumis melo L.; 2n = 2x = 24) may have commercial importance since they can contribute to the promotion of concentrated fruit set and can be planted in higher plant densities than standard vining types. A study was designed to determine the genetics of dwarfism associated with a diminutive (short internodes) melon mutant line PNU-D1 (C. melo ssp. cantalupensis). PNU-D1 was crossed with inbred wild-type melon line PNU-WT1 (C. melo ssp. agrestis), and resultant F₁ progeny were then self-pollinated to produce an F₂ population that segregated as dwarf and vining plant types. Primary stem length of F₂ progeny assessed under greenhouse conditions indicated that a single recessive gene, designated mdw1, controlled dwarfism in this population. To identify the chromosomal location associated with mdw1, an simple sequence repeat (SSR)-based genetic linkage map was constructed using 94 F₂ progeny. Using 76 SSR markers positioned on 15 linkage groups spanning 462.84 cM, the location of mdw1 was localized to Chromosome 7. Using the putative dwarfing-associated genes, fine genetic mapping of the mdw1 genomic region was facilitated with 1,194 F₂ progeny that defined the genetic distance between mdw1 and cytokinin oxidase gene, a candidate gene for compact growth habit (cp) in cucumber, to be 1.7 cM. The candidate gene ERECTA (serin/threonine kinase) and UBI (ubiquitin) were also mapped to genomic regions flanking mdw1 at distances of 0.6 and 1.2 cM, respectively.     

Genetic differentiation and postglacial migration of the Dactylorhiza majalis ssp. traunsteineri/lapponica complex into Fennoscandia

Eight variable regions (microsatellites, insertion/deletion and duplication regions) from the plastid DNA genome were analyzed for 91 populations belonging to Dactylorhiza majalis ssp. traunsteineri and closely related taxa. A total of 36 composite plastid haplotypes were found. The two dominating haplotypes had a clear geographic distribution suggesting at least two separate immigration routes into Scandinavia after the last glaciation: one southwestern route and one or two southeastern routes. D. majalis ssp. traunsteineri could not be clearly separated from any of the other taxa included in the study except for D. majalis ssp. sphagnicola. The morphologically similar taxa D. majalis ssp. traunsteineri, D. majalis ssp. lapponica and D. majalis ssp. russowii showed no genetic differentiation, and therefore we suggest an amalgamation of the three taxa into one broadly circumscribed subspecies; D. majalis ssp. lapponica. The plastid data also revealed incidents of hybridization and possible introgression between D. majalis ssp. lapponica and other members of the genus, e.g., D. incarnata.     

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.