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BACKGROUND |: VCFs are the most widely used data format for encoding genetic variation. By design, standard VCFs do not include data from sites where all individuals are homozygous for the reference allele ("invariant sites") and thus do not differentiate these from sites where data are completely missing. However, missing data are a key feature of biological datasets across all domains of genomics, and many recent studies have shown that missing data can introduce a variety of statistical biases in the estimation of key population genetic parameters. A solution to this limitation is to include invariant sites in a standard VCF, creating an "all-sites VCF", exposing missing and invariant sites explicitly. One hurdle to the wider adoption of all-sites VCFs is a reliable parameterized simulation framework for generating biologically realistic all-sites VCFs. RESULTS |: Here, we introduce an open-source command line tool, vcfsim, that interfaces with the popular coalescent simulation platform msprime and provides convenience functions for simulating all-sites VCFs with variable levels of ploidy and missing data. We show that the post-processed VCFs generated using vcfsim align precisely with population genetic expectations (i.e. are statistically identical to raw msprime output), accurately introduce missing data, and permit the simulation of data with varying ploidy levels, including the simulation of intraindividual ploidy variation (e.g. heterogametic sex chromosomes) and population structures. CONCLUSIONS |: Our results vcfsim is a useful and easy-to-use tool for the benchmarking of new software tools, performing population genetic inference, training of machine learning models, and the exploration of the effects of missing data in genomics data sets.
Initiation and progression of meiosis was followed in dispersed rat testicular cells by flow cytofluorometry and cytology. The DNA content of dissociated testicular cells of rats 6--30 days old, killed at daily intervals, was analysed by flow cytofluorometry using propidium iodide as a DNA-specific and quantitative fluorochrome. Testicular cells of a 6-day-old rat showed one peak of fluorescence. A second peak, at twice the modal channel number, appeared in testicular cells of 9-day-old animals. The number of cells under this peak increased progressively with age. A third peak, at half the channel number of the original one, appeared at 20 days and accounted for an increasing proportion of cells in testes taken from older rats. Cytological examination of the testicular tissue used for flow cytofluorometric analysis showed that preleptotene spermatocytes first appeared at 8 days after birth. Spermatids were first observed cytologically at 20 days after birth. The close temporal appearance of the fluorescence peaks with that of spermatocytes and spermatids, and the close association of the frequency of diploid and tetraploid cells as derived by flow cytofluorometry and cytology, indicated that the fluorescence peaks correspond--in order of increasing fluorescence--to spermatids, spermatogonia and somatic cells, and to spermatocytes. This conclusion was re-examined by analysing the ploidy levels of testicular cells of hypophysectomized or estradiol-treated by flow cytofluorodmetry. There was a loss of the haploid and tetraploid peaks subsequent to hypophysectomy. Estradiol dipropionate-treated rats, given weekly injections starting at 7 days of age, showed no appearance of the haploid peak and the regression of the tetraploid peak after an initial and transitory appearance. These results indicate that changes in ploidy levels that accompany the progression of meiosis in the testis were reflected in the sequential appearance of three fluorescence peaks as detected by flow cytofluorometry. The close correlation between the frequency of cell types as obtained by cytology and flow cytofluorometry indicates that the latter is a sensitive method for studying selected aspects of spermatogenesis in dissociated testicular cells.
Ploidy level of mesometral cells in experimentaly induced deciduomata is determinated by DNA-Feulgen cytophotometric measures. This tissue shown the first evolutive stages leading to polyploïdy (mono-binucleated cells) it was unable to reach a ploïdy level higher than 4 n.
The dimorphic yeast Candida albicans, as a member of the fungi imperfecti, has been assumed to be in the haploid, or imperfect, state. The deoxyribonucleic acid content of this species has been measured by flow microfluorometry, a technique capable of analyzing single cells. These results were compared with flow microfluorometric deoxyribonucleic acid determinations on a series of strains of Saccharomyces cerevisiae of known ploidy (haploid, diploid, triploid, and tetraploid). These ploidy levels were readily distinguished by the flow microfluorometry procedure. By this criterion, C. albicans was found to contain a diploid amount of deoxyribonucleic acid. Ultraviolet radiation survival and chemical mutagenesis experiments support the conclusion that both clinically isolated and laboratory strains of C. albicans are diploid.
A rapid method for the laser-flow microfluorometry determination of nucleic-acid content per cell is presented. A frequency distribution of fluorescence is obtained from suspensions of living cells treated with ethidium bromide directly in their own medium (or calcium-magnesium-free Hanks' balanced solution). For a fixed number of cells, a frequency distribution of fluorescence is obtained as a function of the amount of ethidium bromide progressively added to the suspension until staturation. At any ratio of added dye per unit of DNA, histograms generated from cells stained with this method give results similar to those generated after fixation and staining by the Feulgen technique, both in terms of cell-cycle phases and ploidy-level determination. The present technique requires a minimal amount of material, is instantaneous, and is conducted directly on living cells. Furthermore, dye concentration-dependence studies of mean fluorescence per cell allow determination of association constant and binding process (primary and secondary) between the intact cell and ethidium bromide. Cells which have the same amount of DNA but vary in the amount of RNA and/or chromatin conformation (like G0 and G1) can then be distinguished.
Ever since meiosis was recognized as a process there has been a continuing interest in its temporal aspects. Two main types of meiotic timing experiments have been conducted: first, experiments to estimate the duration of meiosis (and sometimes its stages); second, experiments to locate the sensitive stage(s) when exposure of meiocytes to various treatments can affect meiotic chromosome behaviour (e.g. pairing or recombination). Such experiments have played an important role in increasing our understanding of the meiotic process. The duration of meiosis has been estimated in about 70 organisms, including two prokaryotes (yeast and Chlamydomonas) and the following eukaryotes: 1 Basidiomycete (Coprinus lagopus), 2 Gymnosperms (Larix decidua and Thuja plicata gracilis). at least 39 angiosperms, and at least 26 animal species. The duration of female meiosis has been estimated in far fewer species than male meiosis. However, estimates of the duration of female meiosis are available for 6 angiosperms. Drosophila melanogaster, Xenopus laevis, and several mammals. Comparison of these data shows that the duration of meiosis is one of the most variable aspects of the meiotic process, ranging from less than 6 h in yeast to more than 40 years in the human female. Developmental holds at different stages of meiosis are common in plants and animals, and inevitably prolong the meiotic division. However, even among species without developmental holds, the duration of meiosis is very variable. For instance, in animals it ranges from about 1-2 days in male Drosophila melanogaster to more than 24 days in male Homo sapiens and several Orthopterans. Despite the large variation in the duration of meiosis three generalizations can be made: (i) first prophase is always very long compared with the remaining meiotic stages, (ii) the rate of meiotic development is very slow compared with the rate of development in dividing somatic meristem cells of the same organisms under the same conditions, (iii) the duration of meiosis is characteristic of the genotype and species. Four main factors have been recognized which effect or determine the duration of meiosis, namely (1) environmental factors (e.g. temperature); (2) nuclear DNA content; (3) ploidy level of the organism; and, (4) the genotype. Because nuclear DNA content plays a major role in determining the duration of meiosis, it has been suggested that DNA influences the rate of meiotic development in two ways: first through its informational content (the genotype), and second indirectly by the physical and mechanical effects of its mass independently of its informational content (i.e. the nucleotype). Thus, the observed duration of meiosis is the result of a complex genotype-nucleotype-environment interaction. With the obvious exception of variation caused by developmental holds, changes in the duration of meiosis usually involve proportional changes in the durations of all its stages...
A quantitative microfluorometric instrument is described that employs a helium cadmium laser (442 nm) as the illumination source. The instrument consists of a double grating monochromator in front of a gallium arsenide photomultiplier that is interfaced with a desktop computer. The versatility of the instrument in making quantitative nucleic acid measurements on acridine orange and Feulgen-Schiff stained cells is demonstrated. The ploidy levels of several populations are easily determined, and the Feulgen fluorescent emissions are considerably greater than those obtained with a standard mercury lamp.
Amid escalating water scarcity and groundwater depletion, halophytes such as Salicornia (Amaranthaceae) represent valuable models for extreme salt tolerance and hold promise for saltwater-based agriculture. Here, we show chromosome-scale genome assemblies for six Salicornia species, revealing four distinct subgenomes, reconciling our assemblies with two existing reference genomes (S. ramosissima UK and S. europaea China), correcting chromosome numbering and orientation. Comparative analyses across ploidy levels demonstrate genome expansion in North American lineages driven by Gypsy retrotransposons, and lineage-specific expansions of two gene families implicated in stress metabolism. Phylogenetic and population-structure analyses of a global resequencing panel of 318 accessions resolve interspecific relationships and establish curated germplasm collections for future crop breeding. Genetic analyses uncover a contrasting population-genetic signal on chromosome 6A between two species, highlighting an OSCA calcium-permeable channel gene as a candidate locus for osmotic adaptation. Together, these resources establish a genomic framework for Salicornia that supports evolutionary studies of halophyte adaptation and crop development.
Polyploidy has repeatedly shaped grass evolution, yet direct observations of how polyploid-derived chromosomes behave when returned to diploidy remain rare. Interspecific crosses between diploid Sorghum bicolor and tetraploid hybrids derived from Sorghum halepense generate mixed-ploidy progeny, providing an opportunity to examine chromosome transmission during the early stages of diploidization. Using genome-wide SNP markers, we characterized chromosomal inheritance patterns in 2 diploid and 2 tetraploid families derived from these crosses. Genotype-dosage profiles alone distinguished diploids from tetraploids with complete accuracy, reflecting strong ploidy-dependent differences in dosage-class distributions. Although diploid progeny retained much of the halepense-derived genomic background, several genomic intervals exhibited extended, nonrandom runs of S. bicolor homozygosity that remained polymorphic in corresponding tetraploid populations. These patterns, together with recurrent segregation distortion across independent families, suggest that the transition from tetraploidy to diploidy can expose allelic combinations that differ in transmission or viability. Analyses of flowering time further indicated that diploid and tetraploid derivatives possess distinct genomic architectures, with major association peaks occurring in different chromosomal regions across ploidy levels. Collectively, these results indicate that early diploidization involves nonrandom retention and loss of parental haplotypes shaped by both selective and structural constraints. The diploid extractions characterized here provide a rare empirical system for investigating the early stages of diploidization and a practical framework for studying and eventually mobilizing polyploid-derived variation for sorghum germplasm development. However, broader integration into elite breeding programs will require additional evaluation of cross-fertility, meiotic behavior, and chromosomal stability across diverse breeding backgrounds.
Simultaneous staining of deoxyribonucleic (DNA) and ribonucleic acid (RNA) in nonfixed, but permeable, cells is described. Cells are made permeable by treatment with non-ionic detergent at low pH. RNA is denatured prior to, or during staining, by exposure of cells to chelating agents to ensure that DNA (native) and RNA (dentured) may be stained differentially with the metachromatic dye, acridine orange. The fluorescence of individual cells is measured in a flow cytofluorometer. A comparison between various staining procedures employing acridine orange or other intercalating dyes in unfixed cells is discussed in terms of staining specificity, cell permeability and preservation. Evidence is provided that acridine orange staining of unfixed cells may be used as a simple, fast means of obtaining information on cell ploidy levels and cell cycle status from DNA measurements (green fluorescence), and cell transcriptional activity from RNA staining (red fluorescence), in human and murine cells lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phytohemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures. Exposure of cells to detergent at low pH as an alternative to cell fixation or hypotonic treatment is proposed as a fast, convenient method of making cells permeable to dyes.
Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.
BACKGROUND: Centromeres are crucial for precise chromosome segregation and maintaining genome stability during cell division. However, their evolutionary dynamics, particularly in polyploid organisms with complex genomic architectures, remain largely enigmatic. Allopolyploid wheat, with its well-defined hierarchical ploidy series and recent polyploidization history, serves as an excellent model to explore centromere evolution. RESULTS: In this study, we perform a systematic comparative analysis of centromeres in common wheat and its corresponding ancestral species, utilizing the latest comprehensive reference genome assembly available. Our findings reveal that wheat centromeres predominantly consist of five types of centromeric-specific retrotransposon elements (CRWs), with CRW1 and CRW2 being the most prevalent. We identify distinct evolutionary trajectories in the functional centromeres of each subgenome, characterized by variations in copy number, insertion age, and CRW composition. By utilizing CENH3-ChIP data across various ploidy levels, we uncover a series of CRW invasion events that have shaped the evolution of AA subgenome centromeres. Conversely, the evolutionary process of the DD subgenome centromeres involves their expansion from diploid to hexaploid wheat, facilitating adaptation to a larger genomic context. Integration of complete einkorn centromere assemblies and Aegilops tauschii pan-genomes further revealed subgenome-specific centromere evolutionary trajectories. By inclusion of synthetic hexaploid from S2-S3 generations, alongside 2x/6 × natural accessions, we demonstrate that DD subgenome centromere expansion represents a gradual evolutionary process rather than an immediate response to polyploidization. CONCLUSIONS: Our study provides a comprehensive landscape of centromere adaptation, evolution, and maturation, along with insights into how retrotransposon invasions drive centromere evolution in polyploid wheat.
BACKGROUND: Characterizing population structure and admixture events between ancestral groups plays a key role in understanding the evolutionary history of species and crops. Most tools for inferring admixture have been developed for diploids and are not suitable for polyploids, in particular those with high and mixed ploidy such as Saccharum. RESULTS: Here we present AdmixPoly, an R-package designed to infer admixture in polyploid species both at the genome-wide scale and locally along chromosomes. We compare AdmixPoly with state-of-the-art methods using simulations, demonstrating its precision and computational efficiency. Notably, local admixture inference in complex scenarios, such as high ploidy levels, large numbers of ancestral groups and alleles per marker is enabled through efficient approximations of emission and transition probabilities within a hidden Markov model framework. We apply this approach to characterize the contributions of wild Saccharum species to the complex polyploid genome of modern sugarcane cultivars. A panel of wild and cultivated Saccharum accessions is genotyped for 80K genomic regions, each revealing approximately 50 read-scale haplotypes. CONCLUSIONS: The results reveal that most of the approximately 12 copies of each basic chromosome in modern cultivars are derived from the domesticated species Saccharum officinarum, with one to four copies typically contributed by distinct subgroups of the wild species Saccharum spontaneum. In addition, contributions from an unknown wild Saccharum group originating from the Pacific were identified in most cultivars. The conserved pattern of these introgressions suggests that they can be traced back to the early stages of sugarcane breeding approximately a century ago.
The section Alatae of genus Lemna of the monocotyledonous aquatic duckweed family (Lemnaceae) consists of rather diverse accessions with unknown phylogeny and unclear taxonomic assignment. In contrast to other duckweeds, some Alatae accessions, in addition to mainly vegetative propagation, produce readily flowers and viable seeds. We analyzed the genomic diversity and phylogenetic relationship of 52 Alatae accessions. For this purpose, we applied multiple molecular and cytogenetic approaches, including plastid and nuclear sequence polymorphisms, chromosome counting, genome size determination, and genomic in situ hybridization in combination with geographic distribution. We uncovered ploidy variation, recurrent hybridization, and backcrosses between species and their hybrids. The latter successfully spread over three continents. The results elucidate the evolution of Alatae accessions and explain the difficult taxonomic assignment of distinct accessions. Our study might be an example for analogous studies to resolve the hitherto unclear relationships among accessions of the duckweed genera Wolffiella and Wolffia.
The polyploid strains of Pullularia pullulans differ from each other and from the haploid strain in the amount of the polysaccharide pullulan liberated into the cultural broth. The highest pullulan synthesizing activity (per unit of the assimilated carbon source and the synthesized biomass) was manifested by the diploid strain of P. pullulans 1125(13) whose cells produced more pullulan (by 75%) within three days of growth than the cells of the haploid culture. The content of pullulan calculated per unit area of the cell surface increased with the level of ploidy: 1.5-1.8 times in the diploid cultures and 3.4 times in the tetraploid culture cf. the parent haploid culture. Apparently, the polyploidy of the P. pullulans culture was accompanied with mutations involved in the synthesis of the extracellular polysaccharide.
Hybridization blurs species boundaries and leads to intertwined lineages resulting in reticulate evolution. Polyploidy, the outcome of whole genome duplication (WGD), has more recently been implicated in promoting and facilitating hybridization between polyploid species, potentially leading to adaptive introgression. However, because polyploid lineages are usually ephemeral states in the evolutionary history of life it is unclear whether WGD-potentiated hybridization has any appreciable effect on their diploid counterparts. Here, we develop a model of cytotype dynamics within mixed-ploidy populations to demonstrate that polyploidy can in fact serve as a bridge for gene flow between diploid lineages, where introgression is fully or partially hampered by the species barrier. Polyploid bridges emerge in the presence of triploid organisms, which despite critically low levels of fitness, can still allow the transfer of alleles between diploid states of independently evolving mixed-ploidy species. Notably, while marked genetic divergence prevents polyploid-mediated interspecific gene flow, we show that increased recombination rates can offset these evolutionary constraints, allowing a more efficient sorting of alleles at higher-ploidy levels before introgression into diploid gene pools. Additionally, we derive an analytical approximation for the rate of gene flow at the tetraploid level necessary to supersede introgression between diploids with nonzero introgression rates, which is especially relevant for plant species complexes, where interspecific gene flow is ubiquitous. Altogether, our results illustrate the potential impact of polyploid bridges on the (re)distribution of genetic material across ecological communities during evolution, representing a potential force behind reticulation.
The olfactory receptor (OR) genes constitute the molecular basis of fish olfaction, mediating survival behaviors and environmental adaptation while coevolving with habitat-driven evolution. Schizothorax, a cyprinid genus endemic to the Qinghai-Tibetan Plateau, exhibits remarkable dietary divergence and ploidy variation in response to plateau environmental changes, which presumably facilitates the adaptive evolution of OR genes. However, the evolutionary patterns of OR genes associated with trophic divergence and ploidy variation in this genus remain unclear. In this study, three species were selected: the herbivorous diploid S. macropogon, the carnivorous diploid S. lantsangensis, and the herbivorous tetraploid S. curvilabiatus. S. macropogon possessed 142 OR genes (92.25% functional), primarily located on chromosomes 14 and 24, with the fewest sequence clusters. Such compact gene repertoire and highly overlapping chromosomal clusters indicated specialization for a herbivorous olfactory niche. S. lantsangensis contained 127 OR genes (93.70% functional), concentrated on chromosomes 4 and 5, with fewer sequence clusters and a scattered distribution, reflecting evolution of OR genes under carnivorous feeding habits. The herbivorous tetraploid S. curvilabiatus exhibited striking features: 316 OR genes (94.30% functional), the most subfamilies, unique ε and κ OR subfamilies, and species-specific motifs. These characteristics revealed that ploidy, rather than herbivory, dominated OR gene evolution. In conclusion, dietary differentiation and ploidy variation together drove olfactory adaptive evolution in Schizothorax, providing new insights into vertebrate OR gene ecological adaptation.