Vertebrates, including mammals, are considered to have evolved by whole genome duplications. Although some fish have been reported to be polyploids that have undergone additional genome duplication, there have been no reports of polyploid mammals due to abnormal development after implantation. Furthermore, as the number of physiologically existing tetraploid somatic cells is small, details of the functions of these ploidy-altered cells are not fully understood. In this present study, we aimed to clarify the details of the differentiation potency of tetraploids using tetraploid embryonic stem cells. To clarify the differentiation potency, we used mouse tetraploid embryonic stem cells derived from tetraploid embryos. We presented tetraploid embryonic stem cells differentiated into neural and osteocyte lineage in vitro and tetraploid cells that contributed to various tissues of chimeric embryos ubiquitously in vivo. These results revealed that mouse embryonic stem cells maintain differentiation potency after altering the ploidy. Our results provide an important basis for the differentiation dynamics of germ layers in mammalian polyploid embryogenesis.

The distribution of genetic diversity in invasive plant populations can have important management implications. Alligatorweed [Alternanthera philoxeroides (Mart.) Griseb.] was introduced into the United States around 1900 and has since spread throughout much of the southern United States and California. A successful biological control program was initiated in the late 1960s that reduced A. philoxeroides in the southern United States, although control has varied geographically. The degree to which variation among genotypes may be responsible for variation in control efficacy has not been well studied due to a lack of genetic data. We sampled 373 plants from 90 sites across the United States and genotyped all samples at three chloroplast regions to help inform future management efforts. Consistent with clonal spread, there was high differentiation between sites, yet we found six haplotypes and high haplotype diversity (mean h = 0.48) across states, suggesting this plant has been introduced multiple times. Two of the haplotypes correspond to previously described biotypes that differ in their susceptibility to herbicides and herbivory. The geographic distribution of the three common haplotypes varied by latitude and longitude, while the other haplotypes were widespread or localized to one or a few sites. All the haplotypes we screened are hexaploid (6n = 102), which may enhance biological control. Future studies can use these genetic data to determine whether genotypes differ in their invasiveness or respond differently to control measures. Some states, for instance, have mainly a single haplotype that may respond more uniformly to a single control strategy, whereas other states may require a variety of control strategies. These data will also provide the basis for identifying the source regions in South America, which may lead to the discovery of new biological control agents more closely matched to particular genotypes.

Hydrilla is one of the most serious aquatic weed problems in the United States, and fluridone is the only U.S. Environment Protection Agency (USEPA)–approved herbicide that provides relatively long-term systemic control. Recently, hydrilla biotypes with varying levels of fluridone resistance have been documented in Florida. Fluridone-susceptible (S) and several fluridone-resistant (R1 to R5) hydrilla biotypes varying in resistance levels were maintained in 950-L tanks under ambient sunlight and day-length conditions. To correlate varying levels of fluridone resistance to ploidy in hydrilla, flow cytometric analysis was performed. Differential ploidy levels (diploid, 2n = 2x = 16; triploid, 2n = 3x = 24; and tetraploid, 2n = 4x = 32) were reported among different hydrilla biotypes, plants within each biotype, and within shoot tissues of the same plant. Triploid plants were predominant in all biotypes. Diploid plants were observed in all hydrilla biotypes except the susceptible hydrilla (S). Plants with tetraploidy were rare within biotypes. The diploid, triploid, and tetraploid plants had nuclear DNA contents of 2.43 to 2.73 pg, 3.44 to 3.71 pg, and 4.64 to 4.90 pg, respectively, and no differences were observed among plants with same ploidy for nuclear DNA content in different hydrilla biotypes. Endoreduplication patterns were observed in diploid plants of R1 and R3 biotypes. However, no plant with higher ploidy levels (triploid or tetraploid) in any hydrilla biotypes showed endoreduplication.

Hybridization between two introduced plant species can influence the invasion capabilities of the exotic taxa, but the role of hybridization will likely differ in different invasions, even of the same species. Until now, studies concerning the ploidy of Japanese knotweed, giant knotweed, and their hybrids have been conducted in Europe or native ranges in Asia. Here, we assess the role of hybridization and ploidy in a U.S. invasion. We use flow cytometry to characterize DNA content in (1) established families in a common garden, (2) seedlings grown from common garden parents, and (3) wild populations. We also measured fertility in the garden and the field and vegetative growth traits in the garden. Although the majority of our parental and hybrid samples had ploidy levels previously documented in Europe (4X and 8X for parental species; 6X for hybrids), we found a wider range of knotweed cytotypes established in our garden (4X, 6X, 7X, 8X, 9X, and 10X) and additionally detected 5X, 11X, 12X, and possibly 14X ploidy levels in progeny from garden seed parents. The unexpected cytotypes were not confined to the greenhouse or common garden, in that all < 11X ploidy levels were also found in field populations in Massachusetts. In several cases, these data contradicted our expectations on the basis of morphological and molecular analysis, suggesting both significant introgression and the introduction of multiple cytotypes from Asia. With one exception (14X), we found all cytotypes were capable of strong vegetative growth, seed set, and the production of viable pollen. Without barriers to sexual reproduction, introgression is expected to progress, creating a progressively more diverse swarm of invasive genotypes.

Diploid (2n = 2x = 20) and triploid (2n = 3x = 30) Fasciola hepatica have been reported in the UK, and in Asia diploid, triploid and mixoploid (2x/3x) Fasciola spp. exist but there is little information to indicate how common triploidy is, particularly in UK fluke. Here the ploidy of 565 adult F. hepatica from 66 naturally infected British sheep and 150 adult F. hepatica from 35 naturally infected British cattle was determined. All 715 of these parasites were diploid, based on observation of 10 bivalent chromosomes and sperm (n = 335) or, since triploids are aspermic, sperm alone (n = 380). This constitutes the first extensive analysis of the ploidy of F. hepatica field isolates from Great Britain and shows that most F. hepatica isolated from cattle and sheep are diploid and have the capacity to sexually reproduce. These data suggest that triploidy, and by extension parthenogenesis, is rare or non-existent in wild British F. hepatica populations. Given that F. hepatica is the only species of Fasciola present in Britain our results indicate that the parasite is predominantly diploid in areas where F. hepatica exists in isolation and suggests that triploidy may only originate in natural populations where co-infection of F. hepatica and its sister species Fasciola gigantica commonly occurs.

Cultivars of Miscanthus used as bioenergy crops or tested in trials are largely clonally propagated, wild sourced genotypes or clonally propagated F1 hybrids. One of the most productive taxa is the sterile triploid M. × giganteus. Little domestication or breeding has been undertaken and there is huge potential to utilize the extensive genetic resources of the genus for crop improvement. The challenge is to generate new highly adapted genotypes suitable for a range of environments. Production on marginal land, not used for food crops, is particularly desirable, but presents many barriers to crop breeders, as these are largely unproductive and/or stressful environments. This article outlines progress made in characterizing natural genetic variation in Miscanthus including next-generation single-nucleotide polymorphism genotyping, quantitative trait locus analysis and association mapping. It also explains how this knowledge is being used to develop novel genotypes suited for growth in a broad range of agricultural and marginal lands by defining breeding pools, generating novel crosses, manipulating polyploidy and applying genomic selection approaches.

Psyllids harbor the primary symbiont, Carsonella ruddii (γ-Proteobacteria), within the cytoplasm of specialized cells called bacteriocytes. Carsonella has the smallest known cellular genome (160 kb), lacking numerous genes that appear to be essential for bacterial life. This raises the question regarding the genetic mechanisms of the host which supports the survival of Carsonella. Our preceding analyses have indicated that some of the genes that are encoded in the psyllid genome and which are highly expressed in the bacteriocyte are of bacterial origin. This implies that psyllids acquired genes from bacteria by lateral gene transfer (LGT) and are using these genes to maintain the primary symbiont, Carsonella. To reveal the complete picture of LGT from symbiotic bacteria to the genome of psyllids, whole genome analysis of psyllids is essential. In order to assess the feasibility of whole genome analysis of the host psyllid, the genome size of the hackberry petiole gall psyllid, Pachypsylla venusta, was estimated. Feulgen image analysis densitometry and flow cytometry demonstrated that the haploid genome size of P. venusta is 0.74 pg (724 Mb), verifying the feasibility of whole genome analysis. Feulgen image analysis densitometry further revealed that bacteriocytes of P. venusta are invariably 16-ploid. This higher ploidy may be essential to facilitate the symbiotic relationship with bacteria, as it appears to be a feature common to insect bacteriocytes. These results provide a foundation for genomics-based research into host-symbiont interactions.

The objective of this study was to determine developmental pattern and cell allocation to the inner cell mass and trophectoderm in haploid and diploid embryos following parthenogenetic activation. In vitro matured porcine oocytes were activated by ethanol treatment and cultured in the presence or absence of cytochalasin B for 5h. The oocytes were then cultured in the NCSU23 for 9 days. The combined treatment with cytochalasin B following ethanol treatment did not increase (p >0.1) the incidence of activation. The incidence of development to the blastocyst stage was higher (p <0.05) in the combined treatments of ethanol and cytochalasin B as compared with ethanol treatment alone. The percentage of oocytes with two female pronuclei was higher (p < 0.01) in oocytes treated with cytochalasin B than that in ethanol treatment alone. Treatment with both ethanol and cytochalasin B increased (p <0.01) the incidence of diploid chromosome spread over just the ethanol treatment alone. The average numbers of total cells and inner cell mass were significantly reduced (p <0.05) in the ethanol treatment alone as compared with the combined cytochalasin B and ethanol treatment. These results suggested that the ploidy may affect blastocoele formation and cell allocation to inner cell mass and trophectoderm in the pig.

In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

In a previous study of mouse tetraploid[harr ]diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in postimplantation stage tetraploid[harr ]diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid[harr ]diploid chimaeric blastocysts.

Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.