Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Production and cytogenetics of Triticum aestivum L. hybrids with some rhizomatous Agropyron species

Summary Intergeneric hybrids between Triticum aestivum L. and conventional rhizomatous Agropyron species were produced in variable frequencies. They were recovered in high percentage frequencies for T. aestivum cultivars with A. acutum (14.6%), A. intermedium (48.0%), A. pulcherrimum (53.3%), and A. trichophorum (46.6%). The crossability percentages with the highly crossable cultivar Chinese Spring for these Agropyron species accessions were 33.12%, 65.0%, 53.3%, and 65.4%, respectively. Autosyndetic associations of two of their three genomes gave mean meiotic chromosome association data of 17.0 I (univalents) +1.53 II (ring bivalents) + 7.04 II (rod bivalents) +1.43 III (trivalents) +0.05 IV (quadrivalents) +0.01 IV (pentavalents) for A. acutum and of 21.8 I + 1.56 II (rings) +7.22 II (rods) +0.84 III + 0.04 IV for A. intermedium. Chromosome pairing at metaphase I was comparatively lower for A. pulcherrimum (34.4 I + 0.2 II (rings) +3.4 II (rods) +0.14 III) and A. trichophorum (36.7 I + 0.35 II (rings) +2.26 II (rods) + 0.04 III) hybrids with T. aestivum. Hybrids of wheat with A. campestre and A. repens were obtained in low frequency. Direct crossing did not permit T. aestivum/ A. desertorum hybridization. However, by utilizing the 2n=10x=70 A. repens/A. desertorum amphiploid as the pollen source, hybridization with T. aestivum did indeed occur. Aneuploidy was prevalent in this hybrid combination while all other hybrid combinations were apparently normal.     

Identification of instars and species in some larval Polypedilum (Polypedilum) (Diptera: Chironomidae)

Instars II and III of Polypedilum aviceps Townes, Polypedilum convictum (Walker), and Polypedilum illinoense (Malloch) can be identified to species by associating them with instar IV because key taxonomic characters remain relatively unchanged from instar to instar. Instars I cannot be identified to species or genus unless they are associated with older, identifiable larvae reared from the same egg masses. No single character evaluated on slide material can be used to clearly separate instars in all three species. Larvae of P. aviceps can be separated into instars based on any four of seven characters; P. convictum by either of two characters; and, P. illinoense by a combination of two characters. Changes in structures of instars II, III, and IV are described for all three species. Growth ratios for some structures are compared and discussed with regard to Dyar's Rule.     

The pea early nodulin gene PsENOD7 maps in the region of linkage group I containing sym2 and leghaemoglobin

The early nodulin gene, PsENOD7, is expressed in pea root nodules induced by Rhizobium leguminosarum bv. viciae, but not in other plant organs. In situ hybridization showed that this gene is transcribed during nodule maturation in the infected cells of the proximal part of the prefixation zone II. At the transition of zone II into interzone II–III, the level of PsENOD7 mRNA drops markedly. PsENOD7 has no significant homology to other genes. RFLP mapping studies have shown that PsENOD7 is located in linkage group I between the leghaemoglobin genes and sym2.     

Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus

Paxillus involutus (basidiomycetes, Boletales) is a common ectomycorrhizal fungus in the Northern Hemisphere. The fungus displays significant variation in phenotypic characters related to morphology, physiology, and ecology. Previous studies have shown that P. involutus contains several intersterility groups and morphological species. In this study, we have used concordance of multiple gene genealogies to identify genetically isolated species of P. involutus. Fragments from five protein coding genes in 50 isolates of P. involutus collected from different hosts and environments in Europe and one location in Canada were analysed using phylogenetic methods. Concordance of the five gene genealogies showed that P. involutus comprises at least four distinct phylogenetic lineages: phylogenetic species I (with nine isolates), II (33 isolates), III (three isolates), and IV (five isolates). The branches separating the four species were long and well supported compared with the species internodes. A low level of shared polymorphisms was observed among the four lineages indicating a long time since the genetic isolation began. Three of the phylospecies corresponded to earlier identified morphological species: I to P. obscurosporus, II to P. involutus s. str., and III to P. validus. The phylogenetic species had an overlapping geographical distribution. Species I and II differed partly in habitat and host preferences.     

Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils

Within rhizobia, two species nodulating chickpea, Mesorhizobium ciceri and Mesorhizobium mediterraneum, are known as good phosphate solubilizers. For this reason, we have analysed the ability to solubilize phosphate of a wide number of strains isolated from Cicer arietinum growing in several soils in Spain. The aim of this work was to analyse microbial populations nodulating chickpea, that are able to solubilize phosphates, using molecular techniques. In the present work we analyzed 19 strains isolated from effective nodules of C. arietinum growing in three soils from the North of Spain. Nineteen strains showed ability to solubilize phosphate in YED-P medium. These strains were separated into 4 groups according to the results obtained by 879F-RAPD fingerprinting. The 16S rDNA sequencing of a representative strain from each group allowed the identification of strains as belonging to the genus Mesorhizobium. Strains from groups I and II showed a 99.4% and 99.2% similarity with M. mediterraneum UPM-CA142^T, respectively. The strains from group III were related to M. tianshanense USDA 3592^T at a 99.4% similarity level. Finally, the strain from group IV was related to M. ciceri USDA 3383^T with a 99.3% similarity. The LMW RNA profiles confirmed these results. Strains from groups I and II showed an identical LMW RNA profile to that of M. mediterraneum UPM-CA142^T; the profile of strains from group III was identical to that of M.␣tianshanense USDA 3592^T and the profile of strains from group IV was identical to that of M. ciceri USDA 3383^T. Different 879F-RAPD patterns were obtained for strains of the group I, group II and the M.␣mediterraneum type strain (UPM-CA142^T). The 879-RAPD patterns obtained for group III also differed from the pattern shown by M. tianshanense USDA 3592^T. Finally, the patterns between group IV and M. ciceri USDA 3383^T were also different. These results suggest that groups I and II may be subspecies of M. mediterraneum, group III a subspecies of M. tianshanense and group IV a subspecies of M. ciceri. Nevertheless, more studies are needed to establish the taxonomic status of strains isolated in this study.     

Diploidization and chromosomal pairing affinities in the tetraploid wheats and their putative amphiploid progenitor

Summary The genomes of the diploid wheats Triticum boeoticum and T. urartu are closely related, giving 7II in the f₁ hybrid (T^bT^u) and 8.4 (0–14) II + 2.5 (0–7) IV in the derived amphiploid (T^bT^bT^uT^u). The genomes of the tetraploid wheats are also closely related, giving up to 7II at the polyhaploid level (AB) in the absence of the gene Ph but 14II at the tetraploid level (AABB) in the normal presence of Ph. If the amphiploid is the progenitor of the tetraploids, one or the other homoeologue (T^b or T^u) in each of the 7 homoeologous groups (the 7 potential IV) must have differentiated with respect to pairing affinity in order to account for 14II in the tetraploid. Consequently, in tetraploid X amphiploid hybrids (T^bT^uAB) carrying the Ph gene from the tetraploid, the seven differentiated chromosomes (B) would be expected to give 7I while, on the basis of their observed chiasma frequency, T^b, T^u and the less differentiated A would be expected to give 4.17I + 3.57II + 3.23III), assuming homoeologous pairing. The expected chromosomal configuration freqencies at MI (11.17I + 3.57II + 3.23III) closely fit the observed values (11.22I + 3.45II + 3.19III + 0.071IV) for such hybrids (X² = 0.0046; P>0.99). Thus diploidization of the boeoticum-urartu amphiploid clearly could account for the origin of the tetraploid wheats. Furthermore, T. aestivum X amphiploid hybrids (T^bT^uABD) with and without Ph indicated that B as well as A chomosomes tended to pair with their presumed T^bT^u homologues in the absence of Ph. Other tests showed that the tetraploid wheats could not plausibly have originated from any postulated Triticum-Sitopsis (TTSS) parental combinations with or without such chromosomal differentiation.     

The larval development of an Australian limnadiid clam shrimp (Crustacea, Branchiopoda, Spinicaudata), and a comparison with other Limnadiidae

The adult morphology of the Australian Limnadopsis shows some remarkable differences to that of other Limnadiidae. These differences are not reflected in its larval development. In Limnadopsis parvispinus, larval development comprises six stages. In stages I and II only the three naupliar appendages are present: the antennule as a small bud, the biramous antenna as the main swimming organ, and the mandible. The antennal protopod bears two endites, the proximal naupliar process and a more distal endite. In stage III a bifid naupliar process (in earlier stages not bifid) and the first signs of the carapace and trunk limb anlagen (undifferentiated rudiments) appear. In stage IV the carapace anlagen become more pronounced. The number of trunk limb anlagens increases to five, and differentiation has commenced. In stage V the first five pairs of trunk limbs are differentiated to varying degrees. The anterior-most four pairs of trunk limbs are subdivided into five endites, a small endopod, an exopod and an epipod. The bivalved carapace covers the anterior-most limbs. In larval stage VI the carapace is larger and the trunk limbs are further differentiated. A general pattern in the sequence of larval stages is the increasing number of sensilla on the antennules. From the last larval to the first postlarval stage, a significant change in morphology takes place. The trunk limbs are now used for swimming. Typical larval organs are much smaller than in the last larval stage. A comparison with other representatives of the Limnadiidae shows a high degree of correspondence, with most differences explained by the heterochronous appearance of characters during development. Five to seven stages are described for all studied Limnadiidae, including one particular stage in which four fully developed setae, a bifid naupliar process and the first signs of carapace anlagen are present. These characters are found in stage III in L. parvispinus, Limnadia stanleyana, Eulimnadia texana, and Imnadia yeyetta but in stage IV in E. braueriana and L. lenticularis. Based on a comparison of the larval stages of six limnadiid and one cyzicid species, we conclude that at least six naupliar stages belong to the limnadiid ground pattern.     

Effect of zinc and melatonin supplementation on cellular immunity in rats with toxoplasmosis

The effects of zinc (Zn) and/or melatonin supplementation on cellular immunity were investigated in rats infested with Toxoplasma gondii. Fifty Sprague-Dawley male rats were used for this study. All animals were fed a normal diet, ad libitum, containing 97 mg Zn/kg. They were divided into five experimental groups, as follows. Group I (n=10) received intraperitoneal injections of zinc sulfate at a dose of 3 mg/kg/d for 3 wk. Group II (n=10) received intraperitoneal injections of melatonin at a dose of 3 mg/kg/d for 3 wk. Group III (n=10) received intraperitoneal injections of zinc sulfate (3 mg/kg/d) and melatonin (3 mg/kg/d) for 3 wk. Group IV (n=10) was infested controls. Group V (n=10) was healthy controls. There were no differences in the percentage of CD3+ lymphocytes among all groups. For groups I–III, the CD4+ and CD8+ ratios were higher than those of the groups IV and V controls (p<0.01). Similarly, the total lymphocyte ratios in groups I–III were higher than those of infested and healthy controls (p<0.01). The total lymphocyte ratios in group III were significantly higher than those of groups I and II (p<0.01). The plasma Zn levels in the supplemented groups were significantly higher than those of control groups IV and V (p<0.01). These results suggest that melatonin and/or Zn supplementation may activate cellular immunity by stimulating CD4+ and CD8+ production in infected rats with T. gondii.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

Linkage group IV of fish of the genus Xiphophorus (Poeciliidae): Assignment of loci coding for pyruvate kinase-1, glucosephosphate isomerase-1, and isocitrate dehydrogenase-1

Electrophoretic variation ascribable to three enzyme loci, coding for a pyruvate kinase (PK1), a glucose phosphate isomerase (GPI1), and an isocitrate dehydrogenase (IDH1), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in F₁ hybrid heterozygotes confirmed the dimeric structures of GPI and IDH, and indicated a multimeric structure for pyruvate kinase. Variant alleles at the three loci exhibited normal Mendelian segregation in backcross hybrids. Linkage analyses indicate a gene order and estimated recombination of PK1—10%—GPI1—41%—IDH1. No significant interference or sex- or population-specific recombination difference was detected. This group (designated linkage group IV) was shown to assort independently from the nine loci comprising linkage groups I, II, and III and from 23 other informative markers, within the limits of the data. No conclusions with respect to homology of linkage relationships could be reached, due to the presence of presumably duplicated loci in these fish coding for isozymes whose homology with enzymes in other vertebrate species is as yet unestablished.This work was supported in part by Public Health Service Research Grant CA-28909.     

Phylogeny of the genera Trichophyton using mitochondrial DNA analysis

Diversity of mitochondrial DNA (mtDNA) was investigated in 92 Trichophyton rubrum strains, 2 T. mentagrophytes var. mentagrophytes, 2 T. m. vor. interdigitale, 2 T. m. var. goetzii, 1 T. m. var. erinacei, 2 T. quinckeanum, 2 T. schoenleinii, 1 T. tonsurans, 2 T. verrucosum var. album, 2 T. v. var. discoides, 1 T. violaceum var. violaceum, 1 Arthroderma benhamiae, and 1 A. vanbreuseghemii using endonucleases, Hae III, Msp I, Hind III, Xba I, and Bgl II. Trichophyton species were divided into 7 groups, and a phylogenetic tree was produced based on sequence divergence within mtDNA. The following results were obtained: (1) T. rubrum was divided into 2 groups Type I and Type II, and was suggested to be a complex. (2) A. benhamiae was closely related to T. m. var. erinacei. (3) T. rubrum Type II, T. tonsurans, and A. vanbreuseghemii showed identical restriction profiles, and were suggested to be closely related to each other or identical. (4) T. quinckeanum and T. schoenlenii showed identical restriction profiles, which differed slightly from those of A. vanbreuseghemii. (5) mtDNA analysis was useful in identifying pleomorphic strains.     

The phenoloxidases of the ascomycete Podospora anserina: The three forms of the major laccase activity

In Podospora anserina three laccase activities (I, II and III) were identified. Present results show the existence of an additional lacaase (an anodic protein; MW 80,000; R_f 0.07). Laccase IV derived from the dissociation at acid pH (4.5) of a protein which showed identical molecular weight (390,000) and R_f (0.1) to the oligomeric laccase I. The recovery of laccase I activity when starting from laccase IV (purified by means of isoelectric focusing) suggests that laccase I itself was the source of laccase IV. In turn, laccase IV gave rise to the laccase III after electrophoresis or dialysis at basic pH (8.5).     

Interpretation of triose phosphate isomerase isozymes in the cherimoya (Annona cherimola Mill.)

Detailed interpretation of triose phosphate isomerase (TPI) isozymes in seed plants has been restricted to only a few species. Three sets of TPI bands are regularly observed in the cherimoya(Annona cherimola), a primitive angiosperm. The slowest, set I, is expressed as one or three bands; the second-slowest set II, as one or two bands; and the fastest, set IV, as one or three bands. A faint set III, just cathodal to set IV, is detected rarely with overstaining. Set IV bands are expressed in macerated extracted pollen but not in pollen leachate. Dissociation-reassociation experiments reveal that the set II bands are heterodimers involving, in part, the enzymes involved in the set I bands. These data combined with those from full-sib progeny analysis lead us to propose a three-locus model to explain the TPI isozyme banding patterns in cherimoya. Sets I and IV consist of the allelic products of individual, single loci. Sets I and II occur in the cytoplasm. Set IV occurs in organelles. Set II isozymes are the intergenic heterodimers of the locus coding for set I and the locus coding for set III. Our results reported here are contrasted with the TPI isozyme patterns known for other vascular plants and suggest that the locus coding for set III may be a duplication of very ancient origin.This work was supported, in part, by funding from the Elvenia J. Slosson Endowment Fund.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Comparative ultrastructure of the zoospores of nine species ofNeochloris (Chlorophyta)

Nine species ofNeochloris can be divided into three groups on the basis of comparative ultrastructure of the flagellar apparatus, the cell wall and the pyrenoid of zoospores. In Group I,N. wimmeri andN. minuta, zoospores are thin-walled, pyrenoids are penetrated by stromal channels, and the basal bodies are in the clockwise absolute orientation and connected by the distal and two proximal fibers. In Group II,N. aquatica, N. vigenis, N. terrestris, N. pyenoidosa, andN. pseudostigmatica, zoospores are naked or covered by fuzzy material, pyrenoids are covered by a continuous starch sheath or invaginated by cytoplasmic channels, basal bodies are directly opposed, the distal fiber is differentiated into a ribbed structure at the central region, a striated microtubule-associated component (SMAC) is continuous between opposite two-membered rootlets and connected to the ribbed structure, proximal ends of basal bodies are covered by partial caps, each two-membered rootlet and a basal body are connected by a striated fiber to the X-membered rootlet associated with the opposite basal body, and the basal bodies, when oriented at wide angles, are joined at their proximal ends by core extensions. In Group III,N. pseudoalveolaris andN. cohaerens, zoospores are naked, pyrenoids are traversed by parallel thylakoids, basal bodies are in the counterclockwise absolute orientation and overlapped, and each X-membered rootlet is connected to the end of the opposite basal body by a terminal cap. It is suggested that the genusChlorococcopsis gen. nov. be erected for the Group I species. Group II, which includes the type species,N. aquatica, should be preserved asNeochloris. The group appears to be closely related to the coenobial generaPediastrum, Hydrodictyon, andSorastrum, and to have affinities with the coenocytic generaSphaeroplea andAtractomorpha as well. It is also suggested that the genusParietochloris gen. nov. be erected in thePleurastrophyceae for the species of Group III.     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Production,morphology, and cytogenetics of Triticum aestivum (L.) Thell × Elymus scabrus (R. Br.) Love intergeneric hybrids obtained by in ovulo embryo culture

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD), and an apomictic Triticeae species, Elymus scabrus (syn. Agropyron scabrum) (2n=6x=42, HHSSSS), the first successful report of this cross. Nine tiny, underdeveloped, and structureless embryos were obtained in vitro only by in ovulo embryo culture at 4 days after pollination, which gave rise to five mature hybrid plants. All the hybrid plants were vigorous and possessed a phenotype intermediate to the two parents. There were 2n=6x=42 (ABDHSS) somatic chromosomes in the hybrids. There was little or no homology between the parental genomes, as shown by an overall meiotic chromosome association of 32.83 I + 4.08 rod II + 0.21 ring II + 0.18 III + 0.02 IV. The hybrids were completely sterile and so far backcrosses to wheat parent have not been successful. Alternate approaches to induce gene transfer(s) from E. scabrus to wheat are being attempted.Agriculture Canada Contribution No. 398.     

Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species

Karyotypes of 93 individuals belonging to 18 accessions of mandarins, mandarin hybrids and two related species were analysed with the fluorochromes CMA and DAPI, to identify marker chromosomes. The karyotypes revealed highly differentiated banding patterns and could be classified in four groups (I–IV) according to the presence/absence of chromosomes with three bands (type A) or with two bands (one proximal and one terminal, type B, or both terminal, type C). The accessions of group I exhibited the simplest and homozygous karyotypes (lacking chromosome types A, B and C), represented by `Sunki' and `Cleopatra'. Group II (lacking chromosome types A and B) included three accessions of Mediterranean mandarins and `Cravo' mandarin, all of them with very similar and almost completely homozygous karyotypes. All other karyotypes of groups II and III (lacking chromosome type A) were heterozygous for one or more chromosome pairs and most of them seemed to be hybrid derivatives from non-mandarin accessions. Group IV (with chromosome types A and B) was represented only by two heterozygous hybrids (`Murcott' and `King'). The karyotype of most hybrids agrees with one of the possible combinations resulting from chromosome types segregation from their putative ancestor karyotypes, but at least `Orlando' seemed to be a more complex hybrid. Comparing with banding patterns of other Citrus species, those of group I and the Mediterranean mandarins (group II) are the best candidates to represent C. reticulata (sensu Swingle) as a true species.