Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

Differential survival among genotypes of <Emphasis Type="Italic">Daphnia pulex</Emphasis> differing in reproductive mode,ploidy level,and geographic origin

The distributional pattern of geographical parthenogenesis has not yet been clearly explained. In Daphnia pulex, asexuals are found at higher latitude and in more marginal habitats than their sexual relatives. In addition, some asexual lineages, especially northern ones, are polyploid. This study aimed to test if polyploid clones are more resistant than sympatric diploid clones to a wide range of environmental factors and if asexual Daphnia (diploid clones) are more tolerant of extreme environmental conditions than sexual ones. We report significant differences in survivorship after short-term exposure to acute pH, conductivities, and temperature in 12 lineages of the Daphnia pulex complex. Ploidy level, reproductive mode, geographic origin, and heterozygosity level had a significant effect on survival but their effect varied depending on environmental factors.     

Hybrid Strains of brewer’s yeast obtained by protoplast fusion

Hybrid clones obtained by induced fusion of protoplasts of the polyploid strainSaccharomyces uvarum and the haploid strainS. distaticus have properties of both parents that are determined both nuclearly and cytoplasmatically. The strains acquired the ability to produce the killer protein and degrade dextrins in brewer’s wort. Individual clones differ in the size of the cell and some growth characteristics. The differences may be caused by different proportions of the genome of the parent strains in individual hybrids.     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.     

Apogamic induction of haploid plasmodia in a myxomycete,Didymium iridis

Interisolate crosses between haploid (mean DNA = 0.32) CR 5-5 (A²) myxamoebae and polyploid (mean DNA = 1.80) CR 2–25 (A⁵) myxamoebae of the myxomycete Didymium iridis result in plasmodia that have the haploid (mean DNA = 0.32) DNA content rather than the predicted polyploid value. F₁ clones possess the mating type allele of the CR 5-5 clone only, and they also have the same mean DNA content as CR 5-5 myxamoebae. Crosses between these F₁ clones and CR 2–25 myxamoebae again resulted in the production of haploid plasmodia. Hence, the polyploid CR 2–25 clone appears to induce the CR 5-5 clone to produce plasmodia without involving itself in nuclear fusion.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Genetic characterization of an arctic zooplankter: insights into geographic polyploidy

Species of Bosmina from the temperate regions of North America and Europe are diploid and reproduce by cyclical parthenogenesis. By contrast, this study provides evidence that the dominant bosminid taxon in High Arctic lakes reproduces by obligate parthenogenesis and is a polyploid derived from interspecific hybridization. Sinobosmina liederi, a species common in temperate North America, is likely to have been one parent of these hybrids, but the other parent is unknown. As neither parent was detected in the Arctic, it seems unlikely that the hybrid clones that now occupy arctic lakes were synthesized locally. Most habitats contained only one or two clones, despite a total of 38 clones in the region, suggesting that priority effects have been important in restricting diversity within single lakes. The high regional diversity of arctic bosminids could reflect either repeated hybridization between the parent taxa or the genetic instability of newly formed polyploid lineages. These processes would produce hybrid polyploids that are considerably more diverse than their sexual parent taxa, and this difference in genetic diversity may confer an advantage to the polyploid biotype. As many zooplankton taxa from the arctic possess genetic characteristics similar to those of bosminids, these processes may provide a general explanation for the widespread occurrence of polyploids in the Arctic.     

Extreme Environments Facilitate Hybrid Superiority – The Story of a Successful Daphnia galeata × longispina Hybrid Clone

Hybridization within the animal kingdom has long been underestimated. Hybrids have often been considered less fit than their parental species. In the present study, we observed that the Daphnia community of a small lake was dominated by a single D. galeata × D. longispina hybrid clone, during two consecutive years. Notably, in artificial community set-ups consisting of several clones representing parental species and other hybrids, this hybrid clone took over within about ten generations. Neither the fitness assay conducted under different temperatures, or under crowded and non-crowded environments, nor the carrying capacity test revealed any outstanding life history parameters of this hybrid clone. However, under simulated winter conditions (i.e. low temperature, food and light), the hybrid clone eventually showed a higher survival probability and higher fecundity compared to parental species. Hybrid superiority in cold-adapted traits leading to an advantage of overwintering as parthenogenetic lineages might consequently explain the establishment of successful hybrids in natural communities of the D. longispina complex. In extreme cases, like the one reported here, a superior hybrid genotype might be the only clone alive after cold winters. Overall, superiority traits, such as enhanced overwintering here, might explain hybrid dominance in nature, especially in extreme and rapidly changing environments. Although any favoured gene complex in cyclic parthenogens could be frozen in successful clones independent of hybridization, we did not find similarly successful clones among parental species. We conclude that the emergence of the observed trait is linked to the production of novel recombined hybrid genotypes.     

Manifestation of malignancy in somatic hybrids obtained by the cell fusion of 2 tumor lines

Malignancy of 6 independent hybrid clones derived from fusion of two tumor cell lines of Djungarian hamster, which had been transformed with SV40 virus, was studied. In most of the hybrid clones, suppression of the ability to grow progressively in vivo and the increase in the latent period of tumor occurrence were observed. These data bear witness to suggestion about the existence of different genetic alterations in these tumor cells. Suppression of malignancy does not depend on the genome mutations leading to cell polyploidization, since no decrease in tumorigenicity was found in polyploid cell clones of the high tumor cell line. These polyploid cells can actively form colonies in the soft agar medium.     

Puromycin resistance in Chinese hamster cells: Genetic and biochemical studies of partially resistant,unstable clones

Resistance to 10 μg/ml of puromycin has been analyzed in V79 Chinese hamster cells. Clones that were isolated in 10 μg/ml of puromycin and subsequently cultivated in its absence consistently lost their resistance. One clone was analyzed in detail by recloning in the presence and absence of puromycin, and it was found that non-puromycin cultivated sublones also lost their resistance and regained inhibition profiles similar to the V79 parent. Reconstruction experiments between sensitive and resistant cells demonstrated that the yield of mutants was not affected by metabolic cooperation. The mutation rate was calculated to be 1 × 10^?7 per cell per generation, and was the same within the limits of statistical error in a colchicine-produced polyploid derivative of the V79 line. Although a number of resistant clones were found to have polyploid karyotypes, the polyploid V79 line was not more resistant to puromycin, nor did it possess a higher frequency of puromycin resistant cells. Studies employing radiolabeled puromycin established that resistance was due to a lowered uptake of puromycin and that an inverse relationship existed between resistance level and uptake rate.     

Inheritance pattern of tetraploid Dioscorea alata and evidence of double reduction using microsatellite marker segregation analysis

Recent studies have shown that the basic chromosome number of the three major edible yams, Dioscorea alata, Dioscorea rotundata and Dioscorea trifida, is x?=?20, and that the clones with 2n?=?40 chromosomes are diploids. D. alata breeding programmes were limited to the production of diploid hybrids until 2006, when the tetraploids (2n?=?80) were found to be fertile and polyploid hybrids were produced by conventional hybridisation. However, the nature of the polyploidy (autotetraploidy or allotetraploidy) was not known in D. alata tetraploid clones. In the present study, the inheritance pattern of simple sequence repeat markers was determined in a tetraploid progeny using a Bayesian approach and by examining double reduction events. Results obtained confirm the autotetraploid nature of the 2n?=?80 clones of D. alata.     

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased.     

Fuel treatment effects on soil chemistry and foliar physiology of three coniferous species at the Teakettle Experimental Forest, California, USA

A full factorial design crossing overstory (O) and understory (U) thinning and prescribed burning (B) was started at Teakettle Experimental Forest, California, in 2001 with the aim of achieving shifts in species composition to favor fire-resistant pines over fir. The goal of the present study was to evaluate the use of metabolic changes as early indicators for predicting the effects of these treatments on species-specific growth and long-term productivity of Pinus jeffreyi, Pinus lambertiana, and Abies concolor. Foliar polyamines, amino acids, soluble proteins and chlorophyll were quantified and related with foliar and soil chemistry. Most changes in soil chemistry occurred only with combined treatments (BU or BO). Changes in foliar metabolism were not driven by soil chemistry. In general, the pines were more sensitive to water stress as compared to fir as indicated by proline content. Understory thinning had no effect on any species. Both O and BO had species-specific effects on foliar amino acids, chlorophyll, soluble proteins, and inorganic ions. In addition, both O and BO were the most extreme thinning treatments for pines as they greatly increase the time needed to replenish carbon stocks. With the exception of some amino acids, B by itself had no major effects on foliar inorganic ions, chlorophyll content, and polyamines for any of the three species while BU caused a decrease in chlorophyll in white fir, suggesting a decline in photosynthetic capacity. These data point to BU as the best management option to specifically decrease the growth of fire-sensitive white fir without a major impact on pines.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.     

Dormancy, Turion Formation, and Germination by Different Clones of Spirodela polyrrhiza

Some clones of Spirodela polyrrhiza form dormant bodies called turions which require several weeks of chilling treatment before they proceed to renew growth and develop into vegetative fronds. The individual fronds of Spirodela are less than 5 mm long and can be grown aseptically in liquid culture. Turion formation and germination can serve as a bioassay for the various compounds involved in dormancy development.

Turion formation can be induced by manipulation of light intensity during the day, photoperiod, night temperature, day temperature, and concentration of nitrate in the culture medium. Different clones of Spirodela from northeastern United States, Puerto Rico, and Argentina had different requirements for turion formation. The clones from Argentina and Puerto Rico did not form turions under any of the experimental conditions imposed. Turions of some clones required chilling treatments for renewed vegetative growth while others did not. Both gibberellic acid and long photoperiods were required to bypass the chilling requirements of some clones, but not others.

     

Pleistocene glaciations and polyphyletic origins of polyploidy in an arctic cladoceran

RFLP analysis of the ND4-ND5 genes of the mtDNA genome in Daphnia middendorffiana and three closely allied species was used to investigate its origin and age. Populations of D. middendorffiana from arctic Canada were found to possess three distinct mtDNA lineages, only one of which appears unique to this species. The other two mtDNA lineages are either closely allied or identical to haplotypes in D. pulicaria, suggesting that it is the maternal parent of many clones of D. middendorffiana. Within D. pulicaria, mtDNA lineages have largely disjunct distributions, suggesting that populations of this species persisted in three glacial refugia (arctic, western, eastern) during the Pleistocene. Hybridizations between these refugial stocks and other species such as D. melanica and D. pulex likely generated many of the polyploid lineages of D. middendorffiana following the Wisconsinan glaciation. The presence of one unique mtDNA lineage in D. middendorffiana suggests that at least some of its clones are more ancient, but further studies are needed to rule out the possibility of their recent derivation from an as yet undetected sexual species. As a general result, this study suggests that polyploid cladocerans are unlikely to predate the Pleistocene.     

Analysis of Metabolic Capabilities Using Singular Value Decomposition of Extreme Pathway Matrices

It is now possible to construct genome-scale metabolic networks for particular microorganisms. Extreme pathway analysis is a useful method for analyzing the phenotypic capabilities of these networks. Many extreme pathways are needed to fully describe the functional capabilities of genome-scale metabolic networks, and therefore, a need exists to develop methods to study these large sets of extreme pathways. Singular value decomposition (SVD) of matrices of extreme pathways was used to develop a conceptual framework for the interpretation of large sets of extreme pathways and the steady-state flux solution space they define. The key results of this study were: 1), convex steady-state solution cones describing the potential functions of biochemical networks can be studied using the modes generated by SVD; 2), Helicobacter pylori has a more rigid metabolic network (i.e., a lower dimensional solution space and a more dominant first singular value) than Haemophilus influenzae for the production of amino acids; and 3), SVD allows for direct comparison of different solution cones resulting from the production of different amino acids. SVD was used to identify key network branch points that may identify key control points for regulation. Therefore, SVD of matrices of extreme pathways has proved to be a useful method for analyzing the steady-state solution space of genome-scale metabolic networks.     

A Conspectus of Myriophyllum (Haloragaceae) in North America

Thirteen species of the genus Myriophyllum are found in North America; ten are native, three have been introduced. A key to the species is provided and previous conflicting taxonomic treatments are assessed. The possibility of a polyploid series involving four species is considered and dismissed. Eight of the common species are found to exhibit sufficient phenotypic plasticity to account for most if not all the taxonomic problems encountered.     

Dispersal and clonal diversity of North-European parthenogenetic earthworms

At least 15 earthworm species are known to reproduce parthenogenetically. Most of these retain meiosis but the chromosome set is duplicated before meiosis; alternatively there is mitosis instead of meiosis. In both cases the offspring are genetic copies of the parent worm. Parthenogens are always polyploid. Parthenogenesis is associated with a dispersal advantage: a single propagule suffices to establish a new population. We have studied the clone pool structure and dispersal of ecologically dissimilar polyploid parthenogenetic lumbricids in northern Europe using enzyme electrophoresis. The anthropochorous Octolasion cyaneum has a very low number of clones in populations that are located far away from each other. The opposite is the eurytopic Dendrobaena octaedra that has a wide array of clones in each population. The ripicolous Eiseniella tetraedra disperses with flowing water and possibly also through zoochory. On subarctic North-European mountains its clone pool decreases with increasing elevation. At the top there are a few but persistent clones. Small brooks carry propagules downstream, so that at the mouths of brooks clone pools are more diverse than higher up; again larger rivers carry clones downstream. Clone dispersal is relatively free in a freely flowing river, while dams stop propagules in harnessed rivers. The mouths of rivers have high E. tetraedra clone diversity. Clones disperse from these clone centers to islands formed through land uplift along the northern Baltic Sea. The annual turnover of clones is high on these islands. A survey of epigeic and endogeic parthenogens on the Åland islands which serve as stepping stones between Estonia, Finland and Sweden shows an invasion route of clones across the Baltic Sea. Anthropochory (Aporrectodea rosea and Octolasion cyaneum) and hydrochory (E.tetraedra and Dendrobaena octaedra) seem to play important roles in the clone pool formation on the Åland islands. Quite recently an exotic parthenogen Dichogaster bolaui has found a curious habitat in human settlements viz., the sewer pipe system. Many clonal earthworms show significant morphological and morphometric diversity in and between sample localities but we have failed to associate this variation with the clonal variability. It seems that local factors modify the morphometrics and morphology ultimately determined by the genotype of parthenogenetic earthworms.     

Variation in heteroploid reproduction and gene flow across a polyploid complex: One size does not fit all

Whole‐genome duplication is considered an important speciation mechanism in plants. However, its effect on reproductive isolation between higher cytotypes is not well understood. We used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. We backcrossed F1 hybrids derived from 2X‐4X and 4X‐6X crosses in the Campanula rotundifolia autopolyploid complex, measured backcross fitness, and estimated backcross DNA cytotype. We then sampled four natural mixed‐ploidy contact zones (two 2X‐4X and two 4X‐6X), estimated ploidy, and genotyped individuals across each contact zone. Reproductive success and capacity for gene flow was markedly lower for 2X‐4X than 4X‐6X hybrids. In fact, 3X hybrids could not backcross; all 2X‐4X backcross progeny resulted from neotetraploid F1 hybrids. Further, no 3X individuals were found in 2X‐4X contact zones, and 2X and 4X individuals were genetically distinct. By contrast, backcrosses of 5X hybrids were relatively successful, particularly when crossed to 6X individuals. In 4X‐6X contact zones, 5X individuals and aneuploids were common and all cytotypes were largely genetically similar and spatially intermixed. Taken together, these results provide strong evidence that reproduction is low between 2X and 4X cytotypes, primarily occurring via unreduced gamete production, but that reproduction and gene flow are ongoing between 4X and 6X cytotypes. Further, it suggests whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.

To assess the role of ploidy in determining reproductive isolation and speciation in polyploid contact zones, we used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. Reproductive success and capacity for gene flow was markedly higher for 4X‐6X hybrids than 2X‐4X hybrids, which was also seen in natural mixed‐ploidy contact zones. Our results suggest whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.