Initial Ploidy Research Articles

Phylogenetic placement of whole genome duplications in yeasts through quantitative analysis of hierarchical orthologous groups

Background: Whole genome duplications (WGD) are genomic events leading to formation of polyploid organisms. Resulting duplicated genes play important roles in driving species evolution and diversification. After such events, the initial ploidy is usually restored, complicating their detection across evolution. With the advance of bioinformatics and the rising number of new well-assembled genomes, new detection methods are ongoingly being developed to overcome the weaknesses of different approaches. Results: Here we propose a novel method for detecting WGD in yeast lineages based on the quantitative and comparative analysis of hierarchical orthologous groups (HOGs) of duplicated genes for a given set of organisms. We reconstruct ancestral genomes to obtain evolutionary information for each phylogenetic branch. This reconstruction relies on the inference of HOGs from the selected species’ proteomes. To estimate WGD events, the number of HOGs of duplicated genes across all taxonomic ranges are adjusted according to the molecular clock hypothesis and by the average genome size. Branches with a significant increase in the adjusted number of duplicated gene families are kept as candidates for WGD placement. The developed method was tested on two real datasets and showed promising results in phylogenetic WGD placements on the yeast lineage.

Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

P29 Developmental potential of day 3 embryos diagnosed by array CGH analysis on the blastomere

Introduction: To investigate if chromosome complement as determined by analysis of a single blastomere from a day 3 embryo by array CGH, is indicative of blastocyst developmental potential. Method: 283 day 3 embryos from 45 PGS patients were included in the study. Patients were undergoing PGS testing for a number of recognised indications such as advanced maternal age, recurrent implantation failure and recurrent miscarriage. Embryos were cultured until day 3 and a single blastomere was biopsied as per protocol using calcium/ magnesium free medium (Cook) and a laser to make a small breach in the zona. Embryos with a minimum of 5 cells were included. All cells were tubed individually and analysed using aCGH (Genesis24) for their ploidy status. Results were analysed based on initial ploidy and the rate of blastulation, and secondly to the extent of aneuploidy and the rate of blastulation. Cells with more than 4 aneuosomies were deemed to be complex aneuploid. Results were analysed using Chi squared two tailed test. Results: 58 embryos were inferred to be euploid following analysis of the blastomere. Of these 39 (67.2%) developed to blastocysts on day 5 and a further 1 by day 6 totalling a 69.0% blastocyst rate. 225 embryos were inferred to be aneuploid following aCGH testing on the blastomere; 100 (44.4%) reached a blastocyst stage by day 5 followed by a further 33 by day 6. This gave a total blastocyst rate of 59.1%. No statistical difference was seen with reference to the developmental stage of blastocysts. When analysing the extent of aneuploidy in terms of the number of chromosomes affected, a trend towards a higher blastocyst rate when fewer aneusomies were inferred from the blastomere was observed. When only one aneusomy was present, 46 of the 60 embryos developed to a blastocyst stage whereas when more than 4 aneusomies were inferred only 31 of the 92 embryos reached a blastocyst stage (p 4 aneusomies the development to blastulation is significantly decreased.

P30 Atypical cases of pre-case haplotyping analysis in GENNET

Introduction: To investigate if chromosome complement as determined by analysis of a single blastomere from a day 3 embryo by array CGH, is indicative of blastocyst developmental potential. Method: 283 day 3 embryos from 45 PGS patients were included in the study. Patients were undergoing PGS testing for a number of recognised indications such as advanced maternal age, recurrent implantation failure and recurrent miscarriage. Embryos were cultured until day 3 and a single blastomere was biopsied as per protocol using calcium/ magnesium free medium (Cook) and a laser to make a small breach in the zona. Embryos with a minimum of 5 cells were included. All cells were tubed individually and analysed using aCGH (Genesis24) for their ploidy status. Results were analysed based on initial ploidy and the rate of blastulation, and secondly to the extent of aneuploidy and the rate of blastulation. Cells with more than 4 aneuosomies were deemed to be complex aneuploid. Results were analysed using Chi squared two tailed test. Results: 58 embryos were inferred to be euploid following analysis of the blastomere. Of these 39 (67.2%) developed to blastocysts on day 5 and a further 1 by day 6 totalling a 69.0% blastocyst rate. 225 embryos were inferred to be aneuploid following aCGH testing on the blastomere; 100 (44.4%) reached a blastocyst stage by day 5 followed by a further 33 by day 6. This gave a total blastocyst rate of 59.1%. No statistical difference was seen with reference to the developmental stage of blastocysts. When analysing the extent of aneuploidy in terms of the number of chromosomes affected, a trend towards a higher blastocyst rate when fewer aneusomies were inferred from the blastomere was observed. When only one aneusomy was present, 46 of the 60 embryos developed to a blastocyst stage whereas when more than 4 aneusomies were inferred only 31 of the 92 embryos reached a blastocyst stage (p 4 aneusomies the development to blastulation is significantly decreased.

Genomic Convergence toward Diploidy in Saccharomyces cerevisiae

Genome size, a fundamental aspect of any organism, is subject to a variety of mutational and selection pressures. We investigated genome size evolution in haploid, diploid, and tetraploid initially isogenic lines of the yeast Saccharomyces cerevisiae. Over the course of ~1,800 generations of mitotic division, we observed convergence toward diploid DNA content in all replicate lines. This convergence was observed in both unstressful and stressful environments, although the rate of convergence was dependent on initial ploidy and evolutionary environment. Comparative genomic hybridization with microarrays revealed nearly euploid DNA content by the end of the experiment. As the vegetative life cycle of S. cerevisiae is predominantly diploid, this experiment provides evidence that genome size evolution is constrained, with selection favouring the genomic content typical of the yeast's evolutionary past.

An hypothesis about genome structures in mammalian polyploid cells based on a new concept that genome is fractal of six hierarchies

A new model for the spatial configurations of DNA is proposed to solve the problem of DNA loss in mammalian polyploid cells. The ordinary concept that chromosomes are situated independently in nuclei cannot account well for the DNA loss in polyploid cells. A new concept about the DNA configurations in diploid cells is constructed based on observations that have been reported. Briefly, the DNA structure is self-similar fractal with a unit of opposite-handed twin-circles. In human diploid cells, DNA is constructed with six hierarchies whose sizes are 32(5), 32(4), 32(3), 32(2), 32(1) and 32(0) with a unit of 200 DNA base pairs, corresponding to a genome, a chromosome, a chromosome band, a replicon, a rosette loop (a gene) and a nucleosome, respectively. A model assuming particular spatial configurations of chromosomes in polyploid cells is deduced from this new concept about chromosome configurations in diploid cells. It can account satisfactorily for the problem of DNA loss in polyploid cells. When cell division is inhibited and DNA synthesis progresses, replicated DNA will be stacked. When inhibitors are removed, the polyploidized cells may return to the initial ploidy, because the stacked DNA loops have not been linked. When the stacked DNA twin-loops are linked with a proper configuration, the cells may become polyploid cells. There is a distinct difference in genome structure between polyploidized and polyploid cells. The homologous chromosomes of polyploid cells are arrayed mirror-symmetrically and they can come close to each other in the folded structure. If DNA synthesis is bypassed at the paired homologous chromosomes, DNA content is lost at every cell division. As the DNA loss progresses, the chromosome configuration of polyploid cells deviates gradually from mirror-symmetry and the DNA loss ceases, resulting in the establishment of semi-stable hypoploid.

DNA Loss and Related Alterations in Long-term Cultures of Diploid, Tetraploid and Octaploid Meth-A Cells

The DNA content of polyploid cells sometimes decreases during subculturing. The mechanism of DNA reduction is not yet known. Precise measurements of DNA decay and related alterations in polyploid cells will be required to understand the mechanism. Diploid, tetraploid and octaploid Meth-A cells were continuously cultured for 244 d and the cellular DNA content was measured from the DNA histograms. The DNA content decays gradually with day t as expressed by f(t)=Ip exp{−G(t)}, where Ip is the initial ploidy, G(t)=at/{exp(−bt)+ct}, and a, b and c are the following parameters: a=0.026, b=0.01 and c=0.0175 (for tetraploid cells) or c=0.01 (for octaploid cells). The DNA content of diploid Meth-A cells was constant within the experimental errors. The DNA loss of polyploid cells was confirmed by a decrease in chromosome number. The cellular morphology changed in diploid and octaploid Meth-A cells, but not in tetraploid cells, suggesting that DNA loss and morphology alteration are independent. The doubling time shortened with culture time in the tetraploid and octaploid cells. The findings suggested that chromosomes are not independent in polyploid cells.

Human hepatocyte polyploidization kinetics in the course of life cycle.

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86-92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C x 2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.

Enzyme gene markers as indicators of the initial ploidy in anther cultures of trees

Enzyme gene markers proved effective as indicators of the initial ploidy of invitro material from anther cultures. The suggested methods were tested by using embryoids or calli of the tree species Aesculushippocastanum L., Fagussylvatica L., and Quercuspetraea (Mattuschka) Liebl. Three categories of problems were considered: one identified donor tree, a no longer existing donor tree, and donor trees of uncertain identity. Only haploids (dihaploids) were identified in F. sylvatica, haploids and a few diploids were identified in Q. petraea, and frequently occurring diploids were identified in A. hippocastanum. The results demonstrate the need to determine the initial ploidy of plantlets derived from anther cultures. Limitations in the case of chimeric tissues are outlined briefly.

The Prognostic Value of Modal Deoxyribonucleic Acid in Low Grade, Low Stage Untreated Prostate Cancer

We selected for a prospective surveillance study 167 patients with untreated grades 1 and 2 prostate cancer. The tumors were classified regarding modal deoxyribonucleic acid value (ploidy) by flow cytometry, cytological grade by transrectal fine needle aspiration biopsy and local tumor stage. Of the patients 146 could be evaluated. Mean followup was 50 months.The initial ploidy was statistically correlated to cytological grade but not to initial local tumor stage, prostatic acid phosphatase activity or patient age. Initial ploidy and cytological grade had a prognostic value regarding local tumor progression when considered as single predictors and in combination. Two patients with diploid and 8 with nondiploid tumors initially had metastases during the surveillance. Five patients (1 with diploid and 4 with nondiploid disease) died of prostatic cancer. Modal deoxyribonucleic acid value and cytological grade were of prognostic value in untreated prostate cancer.

Flow cytometric and karyological analysis of polysomaty and polyploidization during callus formation from leaf segments of various potato genotypes

Flow cytometry and karyological analysis were used to study polysomaty and polyploidization during the first 15 days of callus formation in leaf segments from shoot cultures and greenhouse-grown plants of various lines and genotypes of Solanum tuberosum and S. phureja. The greenhouse-grown plants showed a higher degree of polysomaty (77% and 49% of polyploidized nuclei) than the shoot cultures (< 3%). During the in vitro culture period, polyploidization occurred through endoreduplication. Segments of the five shoot cultures showed up to 87%, 53%, 59%, 45% and 56% polyploidization, respectively; the DNA content of corresponding interphase nuclei amounted to 8C, 16C, 16C, 16C and 8C, and the chromosome numbers to 96. Segments from the two greenhouse-grown genotypes showed up to 87% and 84% polyploidization; the DNA content amounted to 32C and 16C, and the chromosome numbers to 192 and 96. The number of reduplication cycles was species-dependent; the degree of polyploidization was dependent on the initial ploidy level of the genotypes. Cell proliferation did not take place at a constant rate. The maximum frequencies of metaphases (52-171 per segment) occurred after 1 week of culture and were correlated with the ploidy level of the genotypes. Cells were triggered to mitosis rather than to endoreduplication. Cell cycles with normal monochromosomes could be shorter than 1 day, and those with diplochromosomes lasted at least 1 day. Polysomaty, degree of polyploidization and abnormal nuclear processes are discussed in relation to the origin of genetic instability early in culture.

Variation in Phenotype and Chromosome Number of Plants Regenerated from Protoplasts of Dihaploid and Tetraploid Potato

AbstractAn investigation was made on the origin and basis of somaclonal variation in potato. Plants were regenerated from protoplasts of dihaploid and tetraploid potato, and the degree of variation in phenotype and chromosome number was analysed. All the regenerants from dihaploid potato showed variation in chromosome number, while only part of these varied in plant morphology. In tetraploid potato, 60 % of the regenerants showed variation in chromosome number, all of which exhibited phenotypic changes. The data suggest that the explant source and the initial ploidy level of the genotype play an important role in influencing the degree of somaclonal variation.

Cytological Studies in the Endosperm and Embryo ofCoffeaL.

SUMMARYThe variation in chromosome number found within one and the same endosperm in species of Coffea can probably be ascribed to: 1) endoreduplication producing cells with euploid chromosome numbers, multiple of the initial ploidy level; 2) endoreduplication of only some of the chromosomes; 3) unequal anaphases. The mechanisms 2 and 3 would produce cells with aneuploid chromosomes numbers, at any ploidy level. Such phenomena occur either independently or concomitantly.The numerical variation was detected in a much higher degree in endosperms of the artificial tetraploid of Coffea canephora (2 n = 44) and C. eugenioides (2 n = 44), the supposed interspecific hybrid C. arabica × C. dewevrei (PL « 387 »—2 n = 44) and polyploid forms of C. arabica (bullata—2 n = 50 and bullata −2 n = 66) than in the diploid species C. canephora (2 n = 22) and C. dewevrei (2 n = 22) and in the natural tetraploid C. arabica.As pointed out by various authors for other plants, aneuploidy results probably from a genetic unbalance, which can cause disorders in the physiological process controlling mitosis.Chromosome number variations were also detected in embryos, although of smaller amplitude than in the endosperms.Some discrepancies found between the chromosome number of some endosperms and their respective embryos are discussed.