The evolution of the land plant alternation of generations has been an open question for the past 150 years. Two hypotheses have dominated the discussion: the antithetic hypothesis, which posits that the diploid sporophyte generation arose de novo and gradually increased in complexity, and the homologous hypothesis, which holds that land plant ancestors had independently living sporophytes and haploid gametophytes of similar complexity. Changes in ploidy levels were unknown to early researchers. The antithetic hypothesis is contradicted by generation cycles in Lower Devonian Rhynie chert plants, whose sporophytes and gametophytes have similar morphologies and by some Silurian sporophytes whose complexity exceeds that of Rhynie chert sporophytes. The oldest unambiguous bryophyte gametophytes (thalli) are from the upper Middle Devonian, with an unconnected sporophyte nearby. Based on the 2024 discovery that conjugate algae are paraphyletic to land plants, we present a new hypothesis for the evolution of the land plant generation cycle, focusing on labile ploidy levels and types of reproduction found in conjugate algae. Our \'sexual lability\' hypothesis assumes a period of unstable generation cycles (as regards ploidy), likely with predominant clonal growth, as is common in conjugate algae, resulting in sporophytes and gametophytes of similar morphology. When sexual reproduction became stabilized, the timing of gamete fusion, meiosis, and resistant wall formation, which are heterochronic in some conjugate algae, became standardized, with wall formation permanently delayed. In our scenario, independently living adult sporophytes are the land plant ancestral condition, and life-long sporophyte retention on the gametophyte is a bryophyte apomorphy.

Ovarian high-grade serous carcinoma (HGSC) originates in the fallopian tube, with secretory cells carrying a TP53 mutation, known as p53 signatures, identified as potential precursors. p53 signatures evolve into serous tubal intraepithelial carcinoma (STIC) lesions, which in turn progress into invasive HGSC, which readily spreads to the ovary and disseminates around the peritoneal cavity. We recently investigated the genomic landscape of early- and late-stage HGSC and found higher ploidy in late-stage (median 3.1) than early-stage (median 2.0) samples. Here, to explore whether the high ploidy and possible whole-genome duplication (WGD) observed in late-stage disease were determined early in the evolution of HGSC, we analysed archival formalin-fixed paraffin-embedded (FFPE) samples from five HGSC patients. p53 signatures and STIC lesions were laser-capture microdissected and sequenced using shallow whole-genome sequencing (sWGS), while invasive ovarian/fallopian tube and metastatic carcinoma samples underwent macrodissection and were profiled using both sWGS and targeted next-generation sequencing. Results showed highly similar patterns of global copy number change between STIC lesions and invasive carcinoma samples within each patient. Ploidy changes were evident in STIC lesions, but not p53 signatures, and there was a strong correlation between ploidy in STIC lesions and invasive ovarian/fallopian tube and metastatic samples in each patient. The reconstruction of sample phylogeny for each patient from relative copy number indicated that high ploidy, when present, occurred early in the evolution of HGSC, which was further validated by copy number signatures in ovarian and metastatic tumours. These findings suggest that aberrant ploidy, suggestive of WGD, arises early in HGSC and is detected in STIC lesions, implying that the trajectory of HGSC may be determined at the earliest stages of tumour development. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BACKGROUND: Despite the oral cavity being readily accessible, oral cancer (OC) remains a significant burden. The objective of this study is to develop a DNA ploidy-based cytology test for early detection of high-risk oral lesions.
METHODS: This retrospective study was conducted using 569 oral brushing samples collected from 95 normal and 474 clinically abnormal mucosa with biopsy diagnosis of reactive, low-grade or high-grade precancer or cancers. Brushing cells were processed to characterize DNA ploidy. A two-step DNA ploidy-based algorithm, the DNA ploidy oral cytology (DOC) test, was developed using a training set, and verified in test and validation sets to differentiate high-grade lesions (HGLs) from normal. The prognostic value of the test was evaluated by an independent outcome cohort, including progressed and non-progressing normal, reactive and low-grade lesions. Classification performance was assessed by accuracy, sensitivity, and specificity, while the prognostic value was evaluated by using the Cox proportional hazards analysis on 3-year progression-free survival (PFS).
RESULTS: The developed DOC test exhibited high accuracy for detecting HGLs in the test and validation sets, with a sensitivity of 0.97 and 0.96, respectively. Its application to the Outcome cohort demonstrated significant prognostic value for 3-year PFS (log rank, p < 0.001). Multivariate analysis showed that high-grade pathology was the only variable explaining positive DOC test, not age, smoking, or lesional site.
CONCLUSIONS: Clinical implementation of the DOC test could provide an effective screening method for detecting HGLs for biopsy and lesions at risk of progression.

Radioresistant cancer cells are risk factors for recurrence and are occasionally detected in recurrent tumors after radiotherapy. Intratumor heterogeneity is believed to be a potential cause of treatment resistance. Heterogeneity in DNA content has also been reported in human colorectal cancer; however, little is known about how such heterogeneity changes with radiotherapy or how it affects cancer radioresistance. In the present study, we established radioresistant clone SW480RR cells after fractionated X-ray irradiation of human colorectal cancer-derived SW480.hu cells, which are composed of two cell populations with different chromosome numbers, and examined how cellular radioresistance changed with fractionated radiotherapy. Compared with the parental cell population, which mostly comprised cells with higher ploidy, the radioresistant clones showed lower ploidy and less initial DNA damage. The lower ploidy cells in the parental cell population were identified as having radioresistance prior to irradiation; thus, SW480RR cells were considered intrinsically radioresistant cells selected from the parental population through fractionated irradiation. This study presents a practical example of the emergence of radioresistant cells from a cell population with ploidy heterogeneity after irradiation. The most likely mechanism is the selection of an intrinsically radioresistant population after fractionated X-ray irradiation, with a background in which lower ploidy cells exhibit lower initial DNA damage.

Flow cytometry has made a significant contribution to the study of several complex fundamental mechanisms in plant cytogenetics, becoming a useful analytical tool to understand several mechanisms and processes underlying plant growth, development, and function. In this study, the genome size, DNA ploidy level, and A-T/G-C ratio were measured for the first time for two genotypes of chia, Salvia hispanica, an herbaceous plant commonly used in phytotherapy and nutrition. This study also evaluated, for the first time by flow cytometry, the capacity to produce organic acids of tissues stained with LysoTracker Deep Red after elicitation with either yeast extract or cadmium chloride. Rosmarinic acid content differed between the two chia varieties treated with different elicitor concentrations, compared with non-elicited plant material. Elicited tissues of both varieties contained a higher content of rosmarinic acid compared with non-elicited cultures, and cadmium chloride at 500 μM was much better than that at 1000 μM, which led to plant death. For both genotypes, a dose-response was observed with yeast extract, as the higher the concentration of elicitor used, the higher rosmarinic acid content, resulting also in better results and a higher content of rosmarinic acid compared with cadmium chloride. This study demonstrates that flow cytometry may be used as a taxonomy tool, to distinguish among very close genotypses of a given species and, for the first time in plants, that this approach can also be put to profit for a characterization of the cytoplasmic acid phase and the concomitant production of secondary metabolites of interest in vitro, with or without elicitation. KEY POINTS: • Genome size, ploidy level, A-T/G-C ratio, and cytoplasm acid phase of S. hispanica • Cytometry study of cytoplasm acid phase of LysoTracker Deep Red-stained plant cells • Yeast extract or cadmium chloride elicited rosmarinic acid production of chia tissues.

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Ploidy is relevant to numerous biological phenomena, including development, metabolism, and tissue regeneration. Single-cell RNA-seq and other omics studies are revolutionizing our understanding of biology, yet they have largely overlooked ploidy. This is likely due to the additional assay step required for ploidy measurement. Here, we developed a statistical method to infer ploidy from single-cell ATAC-seq data, addressing this gap. When applied to data from human and mouse cell atlases, our method enabled systematic detection of polyploidy across diverse cell types. This method allows for the integration of ploidy analysis into single-cell studies. Additionally, this method can be adapted to detect the proliferating stage in the cell cycle and copy number variations in cancer cells. The software is implemented as the scPloidy package of the R software and is freely available from CRAN.

OBJECTIVE: Are human embryos arising from two plus one small pronucleated zygotes, called 2.1 pronuclei (PN), clinically useful?
METHODS: In a retrospective embryo cohort study and prospective experimental study, a total of 287 cycles in which at least one 2.1PN was identified in the fertilization check were included. Embryonic development and clinical outcome were compared for the 1395 2PN zygotes and 304 2.1PN zygotes that were siblings. All embryos were individually cultured in time-lapse systems. Twenty-five 2.1PN-derived blastocysts, donated for research, were used in focused single-nucleotide variant ploidy analysis to identify the distribution pattern of heterozygosity.
RESULTS: The average diameter of PN was 24.9 ± 2.4 µm for large PN and 10.2 ± 2.4 µm for small PN; 79.9% of small PN was derived from female pronuclei. Blastocyst formation rate and good-quality blastocyst rate were significantly lower with 2.1PN embryos than with 2PN embryos (40.0% vs. 57.7%, 21.4% vs. 33.5%, respectively). A total of 13 embryos derived from 2.1PN were transferred, and three healthy babies were born. In ploidy constitutions of trophectoderm (TE), 2.1PN-derived blastocyst TE was shown to be mostly diploid (95.8%, 23/24), and only one blastocyst showed triploid.
CONCLUSIONS: It was suggested that 2.1PN embryos have lower embryonic developmental potential than 2PN embryos, but most of the 2.1PN were diploid, indicating that they are likely to be clinically usable. It is recommended to perform embryo transfer following a combination of PGT-A and ploidy analysis.

Enhancement of shellfish populations has long been discussed as a potential nutrient reduction tool, and eastern oyster aquaculture was recently approved as a nutrient reduction best management practice (BMP) in Chesapeake Bay, USA. This study addressed BMP-identified data gaps involving variation in nutrient concentration related to ploidy, effects of reproductive development, and a paucity of phosphorus concentration data. Diploid and triploid oysters were collected from farms in Maryland and Virginia across the typical local reproductive cycle. The nutrient concentration of tissue and shell was consistent with the currently implemented BMP. Minor variation observed in nitrogen and phosphorus concentration was within the previously reported range, for farm location, ploidy, and reproductive cycle timing. Ploidy-based differences in tissue dry weight were not observed at either farm, which contrasts with current nutrient reduction estimates. These results suggest separate crediting values for diploids and triploids may need further investigation and potential re-evaluation.

Given the universality of autopolyploid species in nature, it is crucial to develop genomic selection methods that consider different allele dosages for autopolyploid breeding. However, no method has been developed to deal with autopolyploid data regardless of the ploidy level. In this study, we developed a modified genomic best linear unbiased prediction (GBLUP) model (polyGBLUP) through constructing additive and dominant genomic relationship matrices based on different allele dosages. polyGBLUP could carry out genomic prediction for autopolyploid species regardless of the ploidy level. Through comprehensive simulations and analysis of real data of autotetraploid blueberry and guinea grass and autohexaploid sweet potato, the results showed that polyGBLUP achieved higher prediction accuracy than GBLUP and its superiority was more obvious when the ploidy level of autopolyploids is high. Furthermore, when the dominant effect was added to polyGBLUP (polyGDBLUP), the greater the dominance degree, the more obvious the advantages of polyGDBLUP over the diploid models in terms of prediction accuracy, bias, mean squared error and mean absolute error. For real data, the superiority of polyGBLUP over GBLUP appeared in blueberry and sweet potato populations and a part of the traits in guinea grass population due to the high correlation coefficients between diploid and polyploidy genomic relationship matrices. In addition, polyGDBLUP did not produce higher prediction accuracy than polyGBLUP for most traits of real data as dominant genetic variance was not captured for these traits. Our study will be a significant promising method for genomic prediction of autopolyploid species.