Chromosome-specific painting reveals the Y genome origin and chromosome rearrangements of the St genome in Triticeae.

Karyotypes provide key cytogenetic information on phylogenetic relationships and evolutionary origins in related plant species. The St genome of Pseudoroegneria contributes to 8 alloploid genera, representing over half of the species that are highly valuable for wheat (Triticum aestivum) breeding and for understanding Triticeae species evolution. However, St chromosome characterization is challenging due to limited cytogenetic markers and DNA information. We developed a complete set of St genome-specific chromosome painting probes for identification of the individual chromosomes 1St to 7St based on the genome sequences of Pseudoroegneria libanotica and wheat. We revealed the conservation of St chromosomes in St-containing species by chromosome painting, including Pseudoroegneria, Roegneria, Elymus, and Campeiostachys. Notably, the Y genome showed hybridization signals, albeit weaker than those of the St genome. The awnless species harboring the Y genome exhibited more intense hybridization signals compare to the awned species in Roegneria and Campeiostachys, yet weaker than the hybridization signals of the St genome in autotetraploid Pseudoroegneria strigosa. Although awnless species were morphologically more similar to each other, phenotypic divergence progressively increased from awnless to awned species. Our results indicate that the Y genome originated from the St genome and shed light on the possible origin of the Roegneria and Campeiostachys species, enhancing our understanding of St-genome-containing species evolution.

Viable offspring derived from single unfertilized mammalian oocytes.

In mammals, a new life starts with the fusion of an oocyte and asperm cell. Parthenogenesis, a way of generating offspring solelyfrom female gametes, is limited because of problems arising fromgenomic imprinting. Here, we report live mammalian offspringderived from single unfertilized oocytes, which was achieved by tar-geted DNA methylation rewriting of seven imprinting control regions.Oocyte coinjection of catalytically inactive Cas9 (dCas9)-Dnmt3a ordCpf1-Tet1 messenger RNA (mRNA) with single-guide RNAs (sgRNAs)targeting specific regions induced de novo methylation or demethyla-tion, respectively, of the targeted region. Following parthenogeneticactivation, these edited regions showed maintenance of methylationas naturally established regions during early preimplantation develop-ment. The transfer of modified parthenogenetic embryos into fostermothers resulted in significantly extended development andfinally inthe generation of viable full-term offspring. These data demonstratethat parthenogenesis can be achieved by targeted epigenetic rewrit-ing of multiple critical imprinting control regions.

Maternal mRNAs with distinct 3' UTRs define the temporal pattern of Ccnb1 synthesis during mouse oocyte meiotic maturation.

The final stages of female gamete maturation occur in the virtual absence of transcription, with gene expression driven by a program of selective unmasking, translation, and degradation of maternal mRNAs. Here we demonstrate that the timing of Ccnb1 mRNA translation in mouse oocytes is dependent on the presence of transcripts with different 3' untranslated regions (UTRs). This 3' UTR heterogeneity directs distinct temporal patterns of translational activation or repression. Inclusion or exclusion of cis-acting elements is responsible for these divergent regulations. Our findings reveal an additional layer of translation control through alternative polyadenylation usage required to fine-tune the timing of meiosis progression.

Chromosome-specific painting in Thinopyrum species using bulked oligonucleotides.

KEY MESSAGE: Chromosome-specific painting probes were developed to identify the individual chromosomes from 1 to 7E in Thinopyrum species and detect alien genetic material of the E genome in a wheat background. The E genome of Thinopyrum is closely related to the ABD genome of wheat (Triticum aestivum L.) and harbors genes conferring beneficial traits to wheat, including high yield, disease resistance, and unique end-use quality. Species of Thinopyrum vary from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70), and chromosome structural variation and differentiation have arisen during polyploidization. To investigate the variation and evolution of the E genome, we developed a complete set of E genome-specific painting probes for identification of the individual chromosomes 1E to 7E based on the genome sequences of Th. elongatum (Host) D. R. Dewey and wheat. By using these new probes in oligonucleotide-based chromosome painting, we showed that Th. bessarabicum (PI 531711, EbEb) has a close genetic relationship with diploid Th. elongatum (EeEe), with five chromosomes (1E, 2E, 3E, 6E, and 7E) maintaining complete synteny in the two species except for a reciprocal translocation between 4 and 5Eb. All 14 pairs of chromosomes of tetraploid Th. elongatum have maintained complete synteny with those of diploid Th. elongatum (Thy14), but the two sets of E genomes have diverged. This study also demonstrated that the E genome-specific painting probes are useful for rapid and effective detection of the alien genetic material of E genome in wheat-Thinopyrum derived lines.

Precisely mapping a major QTL for grain weight on chromosome 5B of the founder parent Chuanmai42 in the wheat-growing region of southwestern China.

KEY MESSAGE: QTgw.saas-5B was validated as a major thousand-grain weight-related QTL in a founder parent used for wheat breeding and then precisely mapped to a 0.6 cM interval. Increasing the thousand-grain weight (TGW) is considered to be one of the most important ways to improve yield, which is a core objective among wheat breeders. Chuanmai42, which is a wheat cultivar with high TGW and a high and stable yield, is a parent of more than 30 new varieties grown in southwestern China. In this study, a Chuanmai42-derived recombinant inbred line (RIL) population was used to dissect the genetic basis of TGW. A major QTL (QTgw.saas-5B) mapped to the Xgwm213-Xgwm540 interval on chromosome 5B of Chuanmai42 explained up to 20% of the phenotypic variation. Using 71 recombinants with a recombination in the QTgw.saas-5B interval identified from a secondary RIL population comprising 1818 lines constructed by crossing the QTgw.saas-5B near-isogenic line with the recurrent parent Chuannong16, QTgw.saas-5B was delimited to a 0.6 cM interval, corresponding to a 21.83 Mb physical interval in the Chinese Spring genome. These findings provide the foundation for QTgw.saas-5B cloning and its use in molecular marker-assisted breeding.

Cyclin B2 is required for progression through meiosis in mouse oocytes.

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption.

During oocyte maturation, changes in gene expression depend exclusively on translation and degradation of maternal mRNAs rather than transcription. Execution of this translation program is essential for assembling the molecular machinery required for meiotic progression, fertilization, and embryo development. With the present study, we used a RiboTag/RNA-Seq approach to explore the timing of maternal mRNA translation in quiescent oocytes as well as in oocytes progressing through the first meiotic division. This genome-wide analysis reveals a global switch in maternal mRNA translation coinciding with oocyte re-entry into the meiotic cell cycle. Messenger RNAs whose translation is highly active in quiescent oocytes invariably become repressed during meiotic re-entry, whereas transcripts repressed in quiescent oocytes become activated. Experimentally, we have defined the exact timing of the switch and the repressive function of CPE elements, and identified a novel role for CPEB1 in maintaining constitutive translation of a large group of maternal mRNAs during maturation.

DNA methylation analysis and editing in single mammalian oocytes.

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy )-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy -related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.

Embryonic poly(A)-binding protein is required at the preantral stage of mouse folliculogenesis for oocyte-somatic communication.

Embryonic poly(A)-binding protein (EPAB)-deficient mice are infertile due to defects in both the oocyte and the somatic cells of the ovary. Since EPAB is oocyte specific, the abnormalities in the somatic compartment of Epab−/− mice are likely due to factors inherent to the oocyte. Herein, we investigated whether oocyte­somatic communication is disrupted as a result of EPAB deficiency. We found that gap junctions are disrupted at the late preantral stage of folliculogenesis in Epab−/­ mice and remain disrupted in cumulus-enclosed oocytes (COCs) from antral follicles. Consistent with the timing of gap junction dysfunction, F-actin staining of transzonal processes (TZPs) is lower in Epab−/− follicles at the late preantral stage and completely absent in Epab−/− COCs. Epab−/− oocytes express significantly lower levels of the junction protein E-cadherin, which is likely to be a contributing factor leading to premature TZP retraction. Overall, these results demonstrate that EPAB is important for oocyte­somatic communication by maintaining TZPs and gap junctions at the preantral stage of folliculogenesis.

Genomic constitution and intergenomic translocations in the Elymus dahuricus complex revealed by multicolor GISH.

Multicolor genomic in situ hybridization was used to investigate the genomic constitution and intergenomic translocations in the Elymus dahuricus complex. The genomic constitution of species of the E. dahuricus complex was confirmed as StYH. H/Y and H/St intergenomic translocations were identified in the present study, with 7H and 1Y chromosomes involved in reciprocal translocations for all the accessions investigated in the complex. We propose that the translocations in the E. dahuricus complex are species-specific, associated with allopolyploidy, and may serve as important structural alterations for allopolyploid stability. Furthermore, they may help to restore fertility and nucleocytoplasmic compatibility in a newly formed polyploid and facilitate the successful establishment of E. dahuricus as a stable species. It was found that more chromosomes were involved in translocations and more types of intergenomic translocations were observed in the high altitude (4150 m) population Y 2228 than in populations from relatively lower altitudes (2600-3800 m). We speculate that more complicated genomic changes were associated with escalating altitudes in the Tibetan Plateau. These genomic changes contribute to promote the genetic variability and enable the newly formed allopolyploids to adapt to more changeable and harsher environments during the evolution of a polyploid species, thus facilitating their rapid and successful establishment in nature.

Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals.

The global prevalence of prediabetes and type 2 diabetes (T2D) is increasing, and it is contributing to the susceptibility to diabetes and its related epidemic in offspring. Although the impacts of paternal impaired fasting blood glucose and glucose intolerance on the metabolism of offspring have been well established, the exact molecular and mechanistic basis that mediates these impacts remains largely unclear. Here we show that paternal prediabetes increases the susceptibility to diabetes in offspring through gametic epigenetic alterations. In our findings, paternal prediabetes led to glucose intolerance and insulin resistance in offspring. Relative to controls, offspring of prediabetic fathers exhibited altered gene expression patterns in the pancreatic islets, with down-regulation of several genes involved in glucose metabolism and insulin signaling pathways. Epigenomic profiling of offspring pancreatic islets revealed numerous changes in cytosine methylation depending on paternal prediabetes, including reproducible changes in methylation over several insulin signaling genes. Paternal prediabetes altered overall methylome patterns in sperm, with a large portion of differentially methylated genes overlapping with that of pancreatic islets in offspring. Our study uniquely revealed that prediabetes can be inherited transgenerationally through the mammalian germ line by an epigenetic mechanism.

Enriched environment-induced maternal weight loss reprograms metabolic gene expression in mouse offspring.

The global prevalence of weight loss is increasing, especially in young women. However, the extent and mechanisms by which maternal weight loss affects the offspring is still poorly understood. Here, using an enriched environment (EE)-induced weight loss model, we show that maternal weight loss improves general health and reprograms metabolic gene expression in mouse offspring, and the epigenetic alterations can be inherited for at least two generations. EE in mothers induced weight loss and its associated physiological and metabolic changes such as decreased adiposity and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, decreased plasma and hepatic lipid levels, and improved glucose tolerance and insulin sensitivity. Maternal weight loss altered gene expression patterns in the liver of offspring with coherent down-regulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling of offspring livers revealed numerous changes in cytosine methylation depending on maternal weight loss, including reproducible changes in promoter methylation over several key lipid biosynthesis genes, correlated with their expression patterns. Embryo transfer studies indicated that oocyte alteration in response to maternal metabolic conditions is a strong factor in determining metabolic and epigenetic changes in offspring. Several important lipid metabolism-related genes have been identified to partially inherit methylated alleles from oocytes. Our study reveals a molecular and mechanistic basis of how maternal lifestyle modification affects metabolic changes in the offspring.

Morphological, cytological, and molecular evidences for natural hybridization between Roegneria stricta and Roegneria turczaninovii (Triticeae: Poaceae).

Some plants with low fertility are morphologically intermediate between Roegneria stricta and Roegneria turczaninovii, and were suspected to be natural hybrids between these species. In this study, karyotype analysis showed that natural hybrids and their putative parents were tetraploids (2n = 4x = 28). Meiotic pairing in natural hybrids is more irregular than its putative parents. Results of genomic in situ hybridization and fluorescence in situ hybridization indicate that natural hybrids contain the same genome as their putative parents. The nuclear gene DNA meiotic recombinase 1 (DMC1) and the chloroplast gene rps16 of natural hybrids and their putative parents were analyzed for evidence of hybridization. The results from molecular data supported by morphology and cytology demonstrated that the plants represent natural hybrids between R. stricta and R. turczaninovii. The study is important for understanding species evolution in the genus since it demonstrates for the first time the existence of populations of natural homoploid hybrids in Roegneria. The study also reports for the first time that the composition of the genomic formula of R. turczaninovii is StY, confirming that the current taxonomic status is correct.

JNK2 participates in spindle assembly during mouse oocyte meiotic maturation.

It is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.

Preparation and Properties of Fractionated Soybean Protein Isolate Films.

Soybean protein isolate (SPI) and its four fractionated products (7S globulin, 11S globulin, upper soybean residue, and lower soybean residue) were compared by fabricating films and film liquids. The separation and grading effects, rheological properties of the film liquids, and difficulty in uncovering the films, in addition to the mechanical properties, water vapor permeability, oil permeability, and surface morphology of the films, were investigated. Results showed that the centrifugal precipitation method could be used to produce fractionated products. The 7S and 11S globulin films exhibited better hydrogels at lower shear rates than the other SPIs; however, they were more difficult to uncover. The tensile strength of the graded films decreased by varying degrees. However, the elongation at the break of the upper soybean residue film considerably increased, reaching 70.47%. Moreover, the permeability and surface morphology of the film were enhanced or weakened. The fractionated products, 7S and 11S globulin films, exhibited better performance. Overall, this study provides a basis for the improved development and use of fractioned SPI products.

Postharvest biochemical characteristics and ultrastructure of Coprinus comatus.

BACKGROUND: Coprinus comatus is a novel cultivated edible fungus, hailed as a new preeminent breed of mushroom. However, C. comatus is difficult to keep fresh at room temperature after harvest due to high respiration, browning, self-dissolve and lack of physical protection. METHODS: In order to extend the shelf life of C. comatus and reduce its loss in storage, changes in quality, biochemical content, cell wall metabolism and ultrastructure of C. comatus (C.c77) under 4 °C and 90% RH storage regimes were investigated in this study. RESULTS: The results showed that: (1) After 10 days of storage, mushrooms appeared acutely browning, cap opening and flowing black juice, rendering the mushrooms commercially unacceptable. (2) The activity of SOD, CAT, POD gradually increased, peaked at the day 10, up to 31.62 U g-1 FW, 16.51 U g-1 FW, 0.33 U g-1 FW, respectively. High SOD, CAT, POD activity could be beneficial in protecting cells from ROS-induced injuries, alleviating lipid peroxidation and stabilizing membrane integrity. (3) The activities of chitinase, ß-1,3-glucanase were significantly increased. Higher degrees of cell wall degradation observed during storage might be due to those enzymes' high activities. (4) The fresh C. comatus had dense tissue and every single cell had the number of intracellular organelles which structure can be observed clearly. After 10 d storage, the number of intracellular organelles was declined and the structure was fuzzy, the nucleus disappeared. After 20 d storage, C. comatus's organization was completely lost, many cells were stacked together and the cell wall was badly damaged.

The RNA-binding protein DAZL functions as repressor and activator of mRNA translation during oocyte maturation.

Deleted in azoospermia-like (DAZL) is an RNA-binding protein critical for gamete development. In full-grown oocytes, the DAZL protein increases 4-fold during reentry into the meiotic cell cycle. Here, we have investigated the functional significance of this accumulation at a genome-wide level. Depletion of DAZL causes a block in maturation and widespread disruption in the pattern of ribosome loading on maternal transcripts. In addition to decreased translation, DAZL depletion also causes translational activation of a distinct subset of mRNAs both in quiescent and maturing oocytes, a function recapitulated with YFP-3'UTR reporters. DAZL binds to mRNAs whose translation is both repressed and activated during maturation. Injection of recombinant DAZL protein in DAZL-depleted oocytes rescues the translation and maturation to MII. Mutagenesis of putative DAZL-binding sites in these mRNAs mimics the effect of DAZL depletion. These findings demonstrate that DAZL regulates translation of maternal mRNAs, functioning both as the translational repressor and activator during oocyte maturation.

Physiological and Biochemical Mechanisms and Cytology of Cold Tolerance in Brassica napus.

Cold damage has negatively impacted the yield, growth and quality of the edible cooking oil in Northern China and Brassica napus L.(rapeseed) planting areas decreased because of cold damage. In the present study we analyzed two Brassica napus cultivars of 16NTS309 (highly resistant to cold damage) and Tianyou2238 (cold sensitive) from Gansu Province, China using physiological, biochemical and cytological methods to investigate the plant's response to cold stress. The results showed that cold stress caused seedling dehydration, and the contents of malondialdehyde (MDA), relative electrolyte leakage and O2 - and H2O2 were increased in Tianyou2238 than 16NTS309 under cold stress at 4°C for 48 h, as well as the proline, soluble protein and soluble sugars markedly accumulated, and antioxidant enzymes of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were higher in 16NTS309 compared with in Tianyou2238, which play key roles in prevention of cell damage. After exposure to cold stress, the accumulation of the blue formazan precipitate and reddish brown precipitate indicated that O2 - and H2O2, respectively, were produced in the root, stem, and leaf were higher than under non-cold conditions. Contents of O2 - and H2O2 in cultivar Tianyou2238 were higher than 16NTS309, this is consistent with the phenotypic result. To understand the specific distribution of O2 - in the sub-cellular, we found that in both cultivars O2 - signals were distributed mainly in cambium tissue, meristematic cells, mesophyll cytoplasm, and surrounding the cell walls of root, stem, leaves, and leaf vein by morphoanatomical analysis, but the quantities varied. Cold stress also triggered obvious ultrastructural alterations in leaf mesophyll of Tianyou2238 including the damage of membrane system, destruction of chloroplast and swelling of mitochondria. This study are useful to provide new insights about the physiological and biochemical mechanisms and cytology associated with the response of B. napus to cold stress for use in breeding cold-resistant varieties.

Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

[A case of parapharyngeal space infection followed abdominal pain].

Descending necrotizing mediastinitis that has an abdominal pain as a main clinical manifestation is seldom. Here one case is reported. At the beginning, the patient had pharyngalgia and his swallowing was not smooth. After that, abdominal pain became a main symptom. Pharyngalgia relieved . However CT showed mediastinal infection. Surgical drainage,antibiotics treatment and nutritional support were performed. The patient was cured.