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PubMed2007

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (Cobitis: Teleostei), and their ability to establish successful clonal lineages--on the evolution of polyploidy in asexual vertebrates.

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides x C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.

Animals↗

PubMed1985

Comparative plasma levels of androgens and 17 beta-estradiol in the diploid and triploid newt, Pleurodeles waltl.

Seasonal changes in the plasma levels of androgens (testosterone plus dihydrotestosterone) and 17 beta-estradiol in diploid and triploid adult newts, Pleurodeles waltl were studied. In both male and female diploid individuals, large variations were reported with highest levels being found during breeding periods. In triploid newts seasonal variations were also found, similar to the diploid ones, but the plasma concentrations of the 17 beta-estradiol and androgens in triploid females and androgens in triploid males were lower throughout the year than those reported for the diploids. This difference is discussed in relation to the genetic sexual constitution.

Androgens↗

PubMed1979

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.

Animals↗

PubMed1984

Disruptive effects of ethyl alcohol on mitotic chromosome segregation in diploid and haploid strains of Aspergillus nidulans.

To identify, with certainty, the primary genotoxic effects of ethanol, condidia from diploid strains of Aspergillus nidulans were treated during early germination with ethyl alcohol, and all the resulting segregants from large samples were analysed in detail. Results were identical whether technical grade (95%), or highly purified 'absolute', alcohol was diluted to obtain the effective low levels of ethanol (3-6%). This makes it unlikely that trace contaminants, rather than ethanol itself, caused the observed induced segregation. At the most effective concentrations survival was about 50%, but over half of the colonies were abnormal and showed sectoring phenotypes. Higher concentrations were too inhibitory for growth. In practically all cases, when 'abnormals' were replated, aneuploids of various types were recovered. Most aneuploids were hyperdiploid, including a fraction of simple trisomics, and some were even polyploid types. All showed chromosomal-type segregation in diploid sectors, often segregating for genetic markers of many different chromosomes. Mitotic crossing-over was slightly increased, but probably not induced, since an equally high spontaneous frequency was observed among replated aneuploid types. To eliminate conclusively the possibility that chromosome breakage was the primary effect of ethanol, which might indirectly produce aneuploid-like types, haploid conidia were also treated. Up to 8% abnormals, mainly hyperhaploids, were obtained (at about 20-50% survival). When diploid and haploid strains were treated identically with ethanol in liquid media after a few hours of pregermination, frequencies of abnormals were similar for short treatments, but higher in diploid strains for longer ones (10-20% aneuploids). The abnormal colonies from the haploid strain were replated and visually identified: about 2/3 were typical n + 1 hyperhaploids, and most others were n + 2 or 3 or more, including a few 2n + 1 trisomics. It is concluded that as a primary effect, alcohol interferes with, and probably arrests, mitotic segregation, and causes chromosome missegregation and nondisjunction. In most cases, the resulting nuclei contain increased numbers of chromosomes and show high frequencies of chromosome loss.

Aspergillus nidulans↗

PubMed2004

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae.

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.

Amino Acid Transport Systems↗

PubMed2004

Haematological parameters in Umbrina cirrosa (Teleostei, Sciaenidae): a comparison between diploid and triploid specimens.

Haematological features were compared between diploid and triploid specimens of the ray-finned fish Umbrina cirrosa. No significant differences between diploids and triploids were reported in haematocrit and total haemoglobin concentration, but erythrocytes and thrombocytes were significantly greater in size in triploids. Glycaemia was significantly lower in diploids, whereas triploid erythrocytes were more resistant to osmotic stress. In triploids, a greater fraction of leukocytes was positive for alkaline phosphatase activity, when stimulated with Bacillus clausii spores, otherwise no significant increase of oxygen consumption was observed in triploid leukocytes after stimulation, based on assays for superoxide anions. Triploids were characterized by a lower concentration of circulating blood cells with a lower surface/volume ratio when compared with diploids. These features may lead to a general disadvantage of triploids in withstanding stress conditions: a situation that needs to be taken into account in aquaculture practice.

Animals↗

PubMed2006

Flow cytometric S-phase fraction contributes to diagnosis of diploid malignant salivary gland tumours.

DNA ploidy studies on salivary gland tumours have shown that the proportion of aneuploid cases, although confined to the malignant entities, is considerably lower than for other solid malignancies. To analyse whether the S-phase fraction (SPF) may contribute to discrimination of diploid malignant from benign tumours, DNA flow cytometric data from 45 different malignant salivary gland tumours was compared with that of 121 pleomorphic adenomas. All benign tumours were diploid. Twelve malignant tumours contained aneuploid cell populations. The SPF values for diploid malignancies ranged between 0.9% and 11.0% (mean 3.9%), and between 0.5% and 7.9% (mean 2.7%) for pleomorphic adenomas. A 4% cut-off value gained statistical significance for discriminating diploid malignant tumours from pleomorphic adenomas (P<0.01). The sensitivity for SPF>4% was 46% and the positive predictive value was 40%. A sensitivity of 60% and a positive predictive value of 54% was achieved by combining aneuploidy and SPF>4%. These results show that DNA flow cytometry may contribute to diagnostic assessment in salivary gland tumours.

Adenoma, Pleomorphic↗

PubMed2006

Molecular phylogeny of diploid Bulinus sp. (Gastropoda: Planorbidae) populations in Cameroon crater lakes.

Bulinus sp. (2n=36) is a diploid freshwater snail found in Cameroon crater lakes; it belongs to a group of medically important freshwater snails. Some members (Bulinus truncatus, Bulinus tropicus) of this group had been reported to be involved in the transmission of parasites (Schistosoma sp. and Calicophoron microbothrium) to human and livestock in tropical Africa. Yet, understanding of the evolutionary identity of the diploid snail such as its phylogenetic position and the genetic divergence among populations, remains limited. In this study, we constructed the molecular phylogeny of Bulinus sp. using sequences of mitochondrial cytochrome oxydase subunit 1 (CO-1, 365 nucleotides). Partial sequences of CO-1 were obtained and genetic divergences between populations estimated after the alignment of 365 nucleotides from each studied population. The lack of deep molecular divergences between populations of Bulinus sp. from western Cameroon crater lakes may indicate that they belong to the same lineage; therefore, it implies that diploid B. truncatus/tropicus complex snail-like in Cameroon share a common ancestor. The CO-1 of the three studied populations of Bulinus sp., clustered together with other diploid pan-African representatives of the B. truncatus/tropicus complex, showed little evidence of genetic similarities.

Animals↗

PubMed2004

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37.

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.

Animals↗

PubMed1999

Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro.

The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39 degrees C in 5% CO2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37 degrees C or 39 degrees C) in 5% CO2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37 degrees C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes.

Animals↗

PubMed2000

Polysomaty analysis in diploid and tetraploid Portulaca grandiflora.

Polysomaty analysis of the succulent portulaca (Portulaca grandiflora Hook.) plant was carried out using flow cytometry. For both diploid and tetraploid plants, mature leaf tissue was found to have a higher level of polysomaty than young leaf tissue. Mesophyll (MP), bundle sheath (BSP) and water storage protoplasts (WSP) were isolated from leaf tissues of diploid portulaca plants. WSP had a higher degree of endopolyploidization than MP and BSP. The ploidy distribution was also variable in different floral organs. Tetraploid plants artificially induced by colchicine treatment showed a decline in the degree of polysomaty compared to diploid plants. Tetraploid plants had more spherical leaves, a larger number of petals and lower pollen fertility than diploid plants.

Journal Article↗

PubMed1999

Activator protein-1 DNA binding activation by hydrogen peroxide in neuronal and astrocytic primary cultures of trisomy-16 and diploid mice.

The effect of H(2)O(2) on DNA binding activity of activator protein-1 (AP-1) was studied by electrophoretic mobility shift assay (EMSA) in cortical primary cultures of trisomy-16 mice and their diploid littermates. Exposure to 10 microM H(2)O(2) for 15 min elicited a greater and earlier occurring increase of AP-1 DNA binding in neuronal primary cultures of trisomy-16 mice than of diploid mice. When astrocyte-rich primary cultures were exposed to 10 microM H(2)O(2) a two-fold increase of AP-1 DNA binding activity was found in trisomy-16 and diploid mice. Supershift EMSA analysis revealed that c-jun was a component of AP-1 in neuronal and glial cultures of diploid and trisomic mice. A 15-min exposure to 10 microM H(2)O(2) increased c-jun mRNA in cortical neuronal cultures by six-fold, compared with a two-fold increase in cultured astrocytes. The results documented that H(2)O(2)-elicited activation of AP-1 DNA binding in trisomy-16 primary cultures is transcriptionally regulated. Since oxidative stress also activates various stress-inducible protein kinases that may phosphorylate AP-1 dimers, the increase of AP-1 DNA binding may, in part, be triggered by phosphorylation.

Animals↗

PubMed2001

Influence of stage of maturation of bovine oocytes at time of vitrification on the incidence of diploid metaphase II at completion of maturation.

The present study was to verify the incidence of bovine diploid oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughterhouse and then divided into five groups: control group (unvitrified oocytes), 0 h group (composed of oocytes vitrified before the onset of maturation) and 8, 12, and 22 h groups (vitrified respectively at 8, 12 and 22 h after the onset of maturation). The oocytes remained vitrified for 24 h, and then were thawed. In all groups, the oocytes completed 24 h of maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences (P>0.05) in the incidence of diploid metaphase II oocytes were observed between the control, non-vitrified group (2.4%, 1/41) and oocytes vitrified at 12 h (6.9%, 3/43) or 22 h (2%, 1/50). However, significantly (P<0.05) more diploid oocytes were detected after vitrification at 0 h (28.5%, 10/35) or 8 h (35.4%, 11/31) of maturation. These results suggest that the nuclear stage at which bovine oocytes are vitrified may affect the incidence of diploid oocytes, especially in oocytes vitrified before maturation or 8 h after the onset of maturation.

Animals↗

PubMed2006

Evidence of different ploidy eggs produced by diploid F2 hybrids of Carassius auratus (female) x Cyprinus carpio (male).

Based on the presence of three types of eggs with different diameters 0.13, 0.17 and 0.2 cm, we made two crosses: F2 (female) x diploid red crucian carp (male), and F2 (female) x F10 tetraploid (male). The ploidy levels of the progeny of the two crosses were examined by chromosome counting and DNA content measurement by flow cytometer. In the offspring of the former cross, tetraploids, triploids, and diploid were obtained. In the progeny of the latter cross, tetraploids and triploids were observed. The production of the different ploidy level fish in the progeny of the two crosses provided a further evidence that F2 might generate triploid, diploid and haploid eggs. The presence of the male tetraploid found in F2 (female) x diploid red crucian carp (male) suggested that the genotype of XXXY probably existed in the tetraploid progeny. The gonadal structures of the tetraploids and triploids indicated that both female and male tetraploids were fertile and the triploids were sterile. We concluded that the formations of different ploidy level eggs from F2 were contributed by endoreduplication and fusion of germ cells.

Animals↗

PubMed2000

Emergence and maintenance of sex among diploid organisms aided by assortative mating.

Using computer simulations I studied the simultaneous effect of variable environments, mutation rates, ploidy, number of loci subject to evolution and random and assortative mating on various reproductive systems. The simulations showed that mutants for sex and recombination are evolutionarily stable, displacing alleles for monosexuality in diploid populations mating assortatively under variable selection pressure. Assortative mating reduced excessive allelic variance induced by recombination and sex, especially among diploids. Results suggest a novel adaptive value for sex and recombination. They show that the adaptive value of diploidy and that of the segregation of sexes is different to that of sex and recombination. The results suggest that the emergence of sex had to be preceded by the emergence of diploid monosexual organisms and provide an explanation for the emergence and maintenance of sex among diploids and for the scarcity of sex among haploid organisms.

Animals↗

PubMed1998

Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck.

In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN.

Adult↗

PubMed1999

Mating systems of diploid and allotetraploid populations of tragopogon (Asteraceae). I. Natural populations

Although polyploidy is a significant force in the diversification of plants, the evolutionary consequences of polyploidization are not thoroughly understood. One possible consequence of polyploidy predicted by most population genetic theories is that the newly synthesized polyploid will self-fertilize at a greater rate than its diploid progenitors. To test for increased selfing rates in a polyploid, the mating systems of the allotetraploid Tragopogon mirus and one of its diploid progenitors, T. dubius, were compared. Tragopogon mirus is a recently derived species that arose sometime in the last 80 years and thus provides an opportunity to probe how quickly a shift in outcrossing rates might occur. Based on analyses of variation in maternal plants and their progeny arrays, the two tetraploid populations surveyed have higher outcrossing rates than the two diploid populations. This result is the opposite of that predicted by population genetic theory. This discrepancy between theoretical and empirical results may result from bias in the genetic sample, traits in the natural histories of the taxa involved or a lack of sufficient time since the formation of the polyploid (80 years or 40-80 generations) for a shift towards increased selfing to have occurred. Alternatively, the partial dominance model of inbreeding depression typically applied to polyploids may not be appropriate; the overdominance model predicts outcrossing rates in diploids and their tetraploid derivatives that are consistent with those observed in T. dubius and T. mirus.

Journal Article↗

PubMed2001

High levels of diploid male production in a primitively eusocial bee (Hymenoptera: Halictidae).

Under single locus complementary sex determination (sl-CSD), diploid males are produced from fertilized eggs that are homozygous at the sex-determining locus. Diploid males are effectively sterile, and thus their production generates a costly genetic load. Using allozyme electrophoresis, a large number of diploid males were detected in natural populations of the primitively eusocial bee, Halictus poeyi Lepeletier collected in southern and central Florida during May 2000. Estimates for the proportion of diploids that are male ranged from 9.1% to 50%, while the frequency of matched matings ranged from 18.2% to 100%. The effective number of alleles at the sex-determining locus ranged from two to 11, with an average of five alleles. The effective population size of Halictus poeyi was estimated to be 19.6 +/- 2.5 SE. These data are interpreted in the light of the biogeographic history of Florida and the social biology/population dynamics of H. poeyi.

Animals↗