Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species–specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats.Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of  NMU was first identified in the porcine spinal cord by the uterine smooth muscle contractile activity assay in 19853. The mature form of NMU consists of 25 amino acid residues in humans and 23 amino acid residues in mice and rats, each containing the highly conserved core active C-terminal domain of eight amino acid residues6. This domain is highly conserved among various species of mammals, birds, amphibians, and fish, suggesting that NMU plays an important role in endocrine functions8.Excessive energy intake is a cause of obesity. Feeding and energy metabolism are complexly regulated by various factors, including hypothalamic anorexigenic and orexigenic neuropeptides. One of these neuropeptides, Neuromedin U (NMU), is an anorexigenic factor and is expected to be a potential target for anti-obesity agents12, and moderately expressed in the testis, ovary, thyroid gland, spleen, lymphocytes, adipose tissue, endothelial cells, and pancreas13. The wide distribution of expression suggests that NMU has multiple physiological functions in addition to smooth muscle contraction.NMU is highly expressed in the gastrointestinal tract, brain, and pituitary gland2, stress responses14, circadian rhythmicity15, tumorigenesis16, and inflammation18. In particular, many reports have shown that NMU plays an essential role in metabolic homeostasis, such as regulating body weight, feeding behavior, activity, and energy metabolism, which are closely related to the pathophysiology of obesity.A series of subsequent studies has revealed many physiological roles of NMU, including feeding and energy expenditure19. Furthermore, to investigate essential roles of endogenous NMU by genetic analyses, NMU-deficient mice, which developed hyperphagia and obesity, were generated20. Additionally, transgenic mice overexpressing NMU are hypophagic and leaner than wildtype (WT) mice21. The exact physiological functions of NMU are yet to be clearly defined, but the phenotypes of genetically modified mice strongly support a role of NMU in energy balance.The role of NMU in regulation of energy homeostasis in mammals has been well established by both pharmacological and genetic analyses in mice. Intracerebroventricular (ICV) administration of NMU in mice suppresses dark-phase food intake and fasting-induced feeding24. Moreover, acute central administration of NMU reduces food intake in rats25, chickens26, and quails27.Pharmacological studies in other vertebrates have shown that NMU inhibits food intake and increases energy expenditure. ICV administration of NMU in rats reduces food intake and body weight, but increases body temperature, motor activity, and heat production28. One study has also reported that chronic injection of NMU into the paraventricular nucleus (PVN) of free feeding rats does not influence food intake or body weight29. Taken together, the anorexigenic effect of central NMU administration may be a pharmacological effect that is exerted only temporarily under certain conditions.In contrast to these reports, inconsistent results have been obtained from administration experiments, especially in rats. ICV injection of NMU into 24 h-fasted rats decreases subsequent food intake and body weight gain, but not in free-feeding ratsNmu mRNA in the rat pars tuberalis (PT) with higher expression levels during the light phase and lower levels during the dark phase due to melatonin-induced suppression30. Although further investigations are needed to prove our hypothesis, we suggest that NMU in PT is related to the feeding rhythm such as rats eating food mostly during the night period.We previously reported circadian rhythmic expression of Nmu knockout (KO) rats using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and genome editing via the oviductal nucleic acid delivery (GONAD) method. The resulting NMU KO rats unexpectedly showed no hyperphagia, obesity, or impaired circadian feeding patterns compared with WT littermates. Furthermore, expression of Nmu and its receptor, Neuromedin U receptor 2 (Nmur2), was hardly detectable in hypothalamic regions, which are involved in the regulation of feeding and energy metabolism. Our data indicate that NMU is not critically required for appetite or fat deposition and is likely not a central regulator of food intake in rats.In this study, to investigate the role of endogenous NMU in rats, especially its functions related to energy homeostasis and food intake, including the circadian pattern, we generated 32. Sequence analysis of the founder rat genome revealed that tandem stop codons were integrated 27 bases downstream of the first ATG , dorsomedial hypothalamic nucleus (DMH), and arcuate nucleus (ARC)35. Because non-RI detection sensitivity using the DIG-labeled cRNA probe was not very high, we next examined expression by RT-qPCR using LMD samples. In addition to high levels in PT, Nmu expression was detected at low levels in SCN, which were close to the detection limit in DMH, ventromedial hypothalamic nucleus (VMH) and ARC, and was not detected in PVN   Fig. A. Althou V) Fig. B.Nmu and its receptor, Nmur2, was not detected or very low, which was probably close to the detection limit, in rat hypothalamic nuclei involved in regulation of feeding and energy metabolism. Our data indicate that rats NMU is not critically required for regulation of feeding and energy metabolism, and is likely not a central regulator of food intake.By generating NMU KO rats using the CRISPR/Cas9 system and rGONAD method, we demonstrated that NMU deficiency in rats does not cause hyperphagia, hypertrophy, or fat deposition compared with WT littermates. Additionally, the combination of in situ hybridization and RT-qPCR confirmed that expression of 20. Hanada et al. also demonstrated that NMU KO mice have decreased Crh mRNA expression in the PVN, suggesting that CRH neurons are the main target of the NMU signaling pathway. CRH in the PVN is well known to reduce appetite and increase energy expenditure by stimulating sympathetic nerve activity. Therefore, obesity caused by NMU deficiency in mice may be attributed in part to the reduction of this anorectic peptide CRH20. Additionally, Graham et al. reported high expression of Nmu in the DMH of obese ob/ob mice, and the Nmu mRNA level in the DMH of WT mice is elevated by fasting35. These reports support the hypothesis that in mouse NMU is involved in the hypothalamic feeding regulation network.Although NMU is a multifunctional peptide, it is believed that the main function of NMU is to regulate energy balance, which is strongly supported by the phenotypes of genetically modified mice. NMU-deficient mice develop significant obesity caused by hyperphagia and low energy metabolism, including reduced locomotor activity, energy expenditure, and decreased body temperatureNmu and Nmur2. In the mouse hypothalamus, Nmu is expressed in ARC, DMH, and VMH, and Nmur2 is expressed in PVN, DMH, VMH, and ARC35. These distributions of expression suggest that a high probability that NMU regulates feeding behavior through the neuronal circuits constituted by these hypothalamic regions. However, our study indicated that Nmu and Nmur2 mRNAs in these hypothalamic nuclei, which are related to regulation of food intake and energy balance, were hardly detectable. Our data agree with those of Graham et al. (2003) who reported important differences in Nmu and Nmur2 expression sites between the mouse and rat hypothalamus35. They demonstrated that Nmu mRNA is abundantly expressed in the mouse DMH and VMH, but only weakly in the rat DMH. Nmu is expressed in the ARC and SCN in both mice and rats, although its expression is much lower in rats. Thus, it appears reasonable that NMU deficiency in rats impairs neither body weight nor food intake. Although the peptide sequence of NMU is highly conserved among species, the species-specific expression pattern may allow NMU to have distinct functions across species. Nevertheless, we still need to take several points into consideration, functional protein levels could be different from the mRNA expression levels, especially for receptors. In this study, we detected Nmur2 mRNA expression at very weak level in the PVN and ARC. Further understanding of NMUR2 in the PVN and ARC requires detailed analysis of NMUR2 protein expression, such as immunohistochemistry. Furthermore, feeding regulatory neuronal network is constructed not only by the hypothalamus but also by the cerebral cortex, midbrain, pons and medulla oblongata. Distribution studies in the rat brain revealed a wide distribution of NMU and NMU receptor other than in hypothalamus. Detailed ISH analysis showed Nmu expression in the nucleus of the solitary tract (NTS), which is in the control of feeding via the gastric and intestinal signals, and NMU expression in the NTS decreased in the obese Zucker fatty rats28. The NMUR2 expression in the dorsal raphe nucleus (DRN) was also demonstrated by immunostaining, suggesting that DRN is the site of NMU action36. The DRN is involved in the regulation of energy expenditure by brainstem GABA neurons37. The possibility of NMU in these nuclei for the regulation of energy intake/expenditure needs to be addressed in future studies.Our results in rats were, however, inconsistent with those in mice. The cause of this discrepancy may be due to species-specific differences in the expression regions of Nmu expression was restricted in the rat brain and abundant in the PT of the pituitary39. Interestingly, in mice, Nmu expression in the PT has not been reported. Although NMU in the rat PT appears abundant, its physiological function remains to be clarified. We previously revealed the circadian rhythm of Nmu expression in the rat PT and proposed that NMU may be involved in the feeding rhythm such as rats eating food mostly at night30. In contrast to our previous expectation, this study showed that NMU deficiency did not impair the circadian pattern of feeding. Thus, in rats, our data suggest that endogenous NMU has no physiological function in the regulation of food consumption or the circadian feeding pattern.Consistent with other studies in rats, we confirmed that 40, Nmur2 expression in the ependymal cell layer of 3 V was abundant in rats, which has also been reported in mice. The ependymal cell layer of the 3 V is composed with tanycytes that project onto several hypothalamic nuclei42. Although it must be elucidated in future studies, this indicates that endogenous NMU may have a common function in rats and mice. This study highlighted not only the similarities but also the considerable differences in the expression patterns for NMU and NMU2R in the hypothalamus of rats and mice.Regarding the central NMU receptorNmu and Nmur2 expression in the brain. The RT-qPCR analysis for Nmu expression in human CNS showed that in addition to the pituitary, several cortical areas of the brain such as the cingulate gyrus and medial frontal gyrus also express moderate levels of Nmu. Low to moderate levels on Nmu expression were also observed in many other brain regions, including the hypothalamus43. Nmur2 expression in the substantia nigra and thalamus were observed by northern blotting25 and that in the hippocampus, hypothalamus, and cerebral cortexthat were detected by RT-qPCR40. At present, the lack of detail information , such as an absolutely clear picture image, on human sites of expression makes it impossible to identify which animal model may be more appropriate to infer physiological and behavioral responses to NMU in humans8. Together with our observations that NMU KO rats lacked significant differences in phenotypes regarding growth and appetite, we propose that it is necessary to carefully examine whether NMU is a critical factor involved in hypothalamic regulation of energy homeostasis and whether an NMU agonist could be an efficacious anti-obesity agent.In humans, there are limited data obtained on Several limitations of our study need to be considered, and we cannot rule out the possibility of compensatory responses for NMU deficiency in NMU KO rats. Further investigations of other rat models, such as conditional NMU knockout rats and injection of neutralizing antibody against NMU into wild type rats, may be necessary to fully support our conclusions.Nmu and Nmur2 in the rat hypothalamus, we propose that NMU in rats is not essential for regulation of food intake or energy balance.In conclusion, we have demonstrated that NMU deficiency in rats does not impair their body weight, amount of food intake, or energy balance. Taken together with the expression pattern of F344 and WKY/NCrl rats were obtained from Charles River Laboratories Japan, Inc. . The rats were maintained in a 12-h light/dark cycle (light switched on at 08:00) at room temperature (23 ± 2 °C) with food and water provided ad libitum. Animal experiments were approved by the Animal Care and Use Committee at Okayama University  and Shigei Medical Research Institute , and were performed in accordance with the Guidelines for Animal Experimentation at Okayama University and Shigei Medical Research Institute. During the study and the care, housing and use of animals were strictly performed in accordance with the appropriate guidelines and regulations. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. Reporting of animal data in this study followed the recommendations set out in the ARRIVE guidelines.32. Pre-estrous F344 female rats were mated with male rats overnight and then copulation plugs were visually inspected the following morning and used for electroporation experiments. Electroporation experiments were operated on anesthetized female rats . Single-stranded DNA containing three stop codons (5ʹ-TAGCTAGCTAGAATTCCCGG-3ʹ), Cas9 guide RNA against the first exon of Nmu (5ʹ-CGAGCAGCTAATCGCCGCCCAGG-3ʹ) . Gene alterations were verified by PCR and then DNA sequencing. Genomic DNA was isolated from an ear piece. Sequencing was performed using the PCR products. The PCR products were purified and directly sequenced and analyzed on an Applied Biosystems 3500 DNA sequencer (Thermo Fisher Scientific) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). The selected founder generation rat was crossed with a wildtype rat to generate F1 generation Nmu + /- rats. WT and NMU KO rats were generated by crossing Nmu + /- rats. Genotyping was performed by PCR with genomic DNA isolated from an ear piece. PCR amplification was performed using EmeraldAmp MAX PCR Master Mix  in accordance with the manufacturer’s instructions. Oligonucleotide-specific primers for WT and KO alleles were Nmu-Fw (5′-GATTTAAAAGTTGGTGGCGCG-3′) and Nmu-Rv (5′-GACAGGAGAGGAGATGCAGTT-3′). Amplicon sizes were confirmed by 2% agarose gel electrophoresis   Fig. A, and CaRats were maintained as two animals per cage after weaning. Body weight was measured weekly from 4 to 24 weeks of age during normal chow feeding  and from 4 to 15 weeks of age during high fat chow feeding .Rats were maintained in an individual cage from 8 weeks of age and with food and water provided ad libitum. The amounts of food and water intake were measured weekly from 9 to 24 weeks of age to calculate average daily amounts. To analyze circadian patterns of food and water intake, we assessed light and dark phases of food intake for 4 days continuous and calculated the average amounts.Locomotor activity and body temperature were measured using the E-mitter telemetry system . Rats were subcutaneously implanted with G2 E-transmitters under isoflurane anesthesia. Rats were individually maintained in plastic home cages  with food and water provided ad libitum. Data were collected for 4 continuous days from 5 days after the operation to calculate average activity volumes and mean body temperature.44. Briefly, a synthetic peptide of 19 amino acid residues with the sequence YKVNEYQGPVAPSGGFFLF-Cys, which corresponds to a part of the C-terminal side of rat mature NMU, was conjugated to keyhole limpet hemocyanin and an antigen emulsion was injected into WKY/NCrl rats anesthetized with sevoflurane. The treated rats were euthanized 17 days after the injection, and lymphocytes were fused with SP2/0-Ag14 myeloma cells. After the cell fusion, culture supernatants were screened to confirm positive clones by a solid-phase enzyme-linked immunosorbent assay (ELISA).We produced a rat monoclonal antibody against rNMU as described previouslyThe specificity for the newly raised antibody was confirmed by western blot analysis to recognize synthetic rat NMU peptide . Synthetic rat NMU peptide (3 ng) was subjected to 16% SDS-PAGE and transferred onto PVDF blotting membranes. The membranes were blocked with 5% dry skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. Immunoblotting was performed with the anti-NMU antibody, followed by a horseradish peroxidase-conjugated secondary antibody (1:5000 dilution). Immune complexes were visualized by enhanced chemiluminescence (Thermo Fisher scientific) using a ImageQuant 800 (Cytiva). Single bands corresponded to the predicted sizes  and photographed with a digital camera .Immunohistochemical detection of NMU was carried out by the avidin–biotin complex (ABC) method. Male WT and NMU KO rats were sacrificed by decapitation under isoflurane anesthesia and brains were quickly removed. Brain tissues fixed with 4% paraformaldehyde were dehydrated in an ascending ethanol series, immersed in xylene, embedded in paraffin , and sectioned at a thickness of 10 µm. The sections were dewaxed, incubated for 2 h with 10 mM sodium citrate (pH 6.0) for antigen retrieval, treated with 0.5% sodium metaperiodate to block endogenous peroxidases for 15 min, and then incubated with TNBS  for 1 h. After washing with PBS, the sections were incubated overnight in a humid chamber with the anti-NMU antibody diluted at 1:10 in TNBS. The ABC method was carried out with a staining kit . The reactions were visualized with 0.02% 3ʹ3-diaminobenzidine tetrachloride in 0.006% HMale WT and NMU KO rats were sacrificed by decapitation under isoflurane anesthesia. Liver and adipose tissues were fixed in 4% paraformaldehyde, dehydrated in an ascending ethanol series, immersed in xylene, embedded in paraffin, and sectioned at a thickness of 10 µm. The sections were dewaxed, washed with DW and stained with hematoxylin and eosin (3 min for each staining). Stained sections were washed in running tap water, dehydrated with alcohol series and with xylene, and then mounted for light microscopic observation and imaging with a digital camera. For adipose tissue analysis, three regions were randomly selected in each section and photographed, and the cell number was counted to calculate the average cell number.45. Digoxigenin-labeled antisense and sense rat Nmu cRNA probes  and rat Nmur2 cRNA probes  were synthesized using a labeling kit  with SP6 or T7 RNA polymerase.Male F344 rats were sacrificed by decapitation under isoflurane anesthesia and brains were quickly removed. ISH was performed on frozen 8-μm-thick frontal sections as described previously46. Briefly, the sections were fixed with ice-cold acetone for 2 min, rehydrated by an ethanol series, and stained with 0.1% toluidine blue. Representative micrographic images of dissections are shown in Supplementary Figure Male F344 rats were sacrificed by decapitation under isoflurane anesthesia and brains were quickly removed. Samples of the rat suprachiasmatic nucleus (SCN), PVN, dorsomedial hypothalamic nucleus (DMH), VMH, arcuate nucleus (ARC), PT, and ependymal cell layer of the third ventricle (3 V) were collected from serial frontal sections of rat brains (30 µm thick) using an LMD system  as described previouslyRpl19 expression was evaluated as an internal control. The amplicon size and specificity were confirmed by a melting curve analysis and 2% agarose gel electrophoresis.RT-qPCR was performed using the LightCycler 96 System (Roche Diagnostics) with SYBR Premix Ex Taq (Takara Bio). The primers used for RT-qPCR are shown in Supplementary TablePlasma glucose (GLU), total cholesterol (T-CHO), and triglycerides (TGs) were analyzed using a Hitachi 7180 automatic analyzer  with Wako commercial kits . The analysis was performed by Oriental Yeast Co., Ltd. Insulin levels were measured using an Rebis Insulin-rat T ELISA kit (FUJIFILM Wako Shibayagi). Leptin levels were measured using a Leptin ELISA kit .P < 0.05 was considered statistically significant.Data are presented as means ± SEM. Comparisons between two groups were performed by Student’s t test. All statistical analyses were performed using GraphPad Prism 6 software . Supplementary Information.


Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.Propagation by somatic embryogenesis in  Theobroma cacao L. (2n = 20) is a preferentially allogamous plant, classified in the Malvaceae family. It has been grown in the lowlands of tropical regions  with social and economic importance1. Cacao beans are used as raw material to produce cocoa powder, liquor and cocoa butter, which are the main ingredients of chocolate, and to produce additives for pharmaceuticals3. Despite its importance, cacao propagation has been done by traditional methods with sometimes low efficiencies, such as grafting and rooted cuttings, or by the seedlings generating genetic depression4. Other propagation technologies, such as somatic embryogenesis, have been developed with the aim to obtain plants with genetic uniformity5. Despite the tremendous efforts to develop a viable somatic embryogenesis protocol, there are still some issues to be solved, including the main one related to the high frequencies of abnormal cacao embryos that cannot be converted into normal plantlets6.7. Zygotic embryos (ZE) go through different stages of development, i.e., globular, heart, torpedo and cotyledonal in dicotyledonous plants versus, globular, scutellar and coleoptilar stages in monocotyledonous plants; and early and late embryogenesis in gymnosperm plants8. Asexual embryogenesis, on the other hand is a complicated process that can be induced naturally (gametophytic or sporophytic) or artificially (somatic embryogenesis), where the stages of development are the same between ZE and somatic embryos (SE)7. Although ZE and SE show similarities in the stages of differentiation, their morphologies can be quite different9. Besides, ZE are protected by endosperm and SE do not have this structure8. Somatic embryos in T. cacao have been developed in tissue culture from different explant sources, with the highest success using flower explants 5. Cacao somatic embryogenesis process is divided in six well defined steps as follows: induction, expression, multiplication, maturation, germination and plant conversion10. All these steps are mediated by a complex regulatory network where different genes are regulated at the transcriptional level by both genetic and epigenetic mechanisms11.Embryogenesis in plants can take place sexually (zygotic embryogenesis) or asexually (apomixis). Zygotic embryogenesis starts with double fertilization of the male and female gametes12. Chemical modification in the DNA can occur mainly when is acetylated or methylated. Acetylation allows gene transcription, whereas methylation inhibits or enhances it depending where the methyl group is positioned in the gene region13. DNA methylation has been widely described as a mechanism implicated in genomic imprinting, repression of gene expression and transposon silencing. DNA methylation in plants occurs when a methyl group is attached to the carbon five of the cytosine. This takes mainly place at cytosines in CpG islands (long repetition of C and G), CHG and CHH  sites14. DNA methylation profiles may change under variation in environmental conditions that can be stressful for the plants15. The position of the methyl group in the gene fulfills important and different functions. If the methyl group is positioned at the regulatory sequences, gene expression is repressed, but methylation also plays an important function when it is placed in the gene body. It has been known that exons are more highly methylated than introns, where methylation in the first exon of the gene blocks gene transcription and methylation in the internal exons does not affect it seriously16. On the other hand, methylation in introns is associated with gene transcription enhancement17.Chromatin structure is affected by epigenetic mechanisms such as histone modifications, small RNAs (sRNAs) and DNA methylationArabidopsis thaliana, the methylation profile in CG context is prevalent and remains high constantly during vegetative growth since embryogenesis is initiated, mainly over transposon element (TE) sequences. In contrasts, the methylation in non-CpG contexts is low in TE, especially in the CHH context in post-embryonic development, but during the embryos maturation it gets saturated. The function of DNA methylation in somatic embryogenesis is still no clear but it is related to the morphogenesis activity in of the embryogenic somatic cells, where it acts switching on and off important specific genes in the different stages of embryogenesis, and also in the hypermethylation of TE regions has been associated with the protection of embryogenic cells to the deleterious consequences of TE activity19.DNA methylation plays an important role in early stages of plant development. During embryo growth in 20. 2,4-D plays a role in cell polarity and asymmetric cell division that influence changes in the cell morphology, physiology, metabolism and gene expression22. 2,4-D is also known as a potent inducer of ethylene production in plant tissues23. Ethylene is an important phytohormone that works together with jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), among others, to regulate several biological processes, including oxidative stress, hypoxia and programmed cell death (PCD)24. Furthermore, it is known to act as an important mediator in aerenchyma formation under hypoxic environments25.In somatic embryogenesis, the tissue is subjected to different environmental stimuli, such as plant grow regulators (PGRs), where the explants and the somatic embryos produced in culture undergo stress. A key PGR used in somatic embryogenesis is 2,4-dichlorophenoxyacetic acid , a synthetic auxin that is known as an important cell stressor and has an important role in cell reprogramming through DNA methylationT. cacao, whole-genome bisulfite sequencing (WGBS) was carried out with the aim to identify differentially methylated genes (DMGs), and their functions, among ZE, normal and abnormal SE. In addition, scanning and transmission electron microscopy analyses were carried out to identify morphological and structural differences between ZE and SE. Understanding the main factors that influence the abnormal SE formation could provide guidance towards somatic embryos development processes to improve the quality and efficiency of the plantlets produced. Additionally, our results provide new knowledge in the somatic embryogenesis regulatory network during ZE and SE differentiation in T. cacao, providing insights in the basic biology of the somatic and zygotic embryogenesis development.To understand the epigenetic basis of abnormal somatic embryo production in We first compared the phenotypic characteristics among ZE, normal and abnormal SE at the late torpedo stage to identify the types of abnormalities present in primary SE and secondary SE from CCN 10  due to their morphology similarities with normal S. In difference, this type of abnormal SE does not have the capacity to germinate when is compare with other abnormal types like AT2 and AT3 that can germinate producing apical shoots or axillar shoots and adventitious roots respectively  analysis of ZE and normal SE showed that their cells have nuclei with normal appearances, whereas abnormal SE showed an average of 194 cells without nucleus and lytic vacuoles presence in an area of 42 mmons Fig. A–C.FigurNormal and abnormal SE exhibited PCD with some differences Fig. A–H. Red 2 in its cotyledons. On the other hand, abnormal SE showed in its cotyledons an average of 195 cells with PCD in the same area. Statistical analysis showed significant differences with p-values < 0.05 in fluorescence intensity in cells of cotyledon, root and shoot regions between normal and abnormal SE. Cotyledons in abnormal SE showed the highest fluorescence intensity, with an average of 2.E+05 arbitrary (arb.) units  is 21.04%, 24.88% and 24.3% in each sample, respectively , For methylation calling we used a total read of 203,071,419 from ZE, 263,500,094 from normal SE and 245,099,294 from anormal SE , the lower sequencing depth cut-off was10. 7,361,253 CpG, 10,679,910 CHG and 65,894,628 CHH methylation sites to be analyzed. In the CpG context we found a binomial distribution in the level of methylation for most of the data  and 23,957  were significantly differentially methylated gene regions in CpG context found in this study. These sequences were functionally annotated as retrotransposons, transcriptional factors involved in autophagy, hypoxia cellular response, DNA repair, auxin response, auxin transport, stress response, disease resistance, cell polarity, PCD, cell wall biogenesis, cell transport, cell division, cellular homeostasis, cell signaling, cellular metabolism, and developmental processes such as embryo morphogenesis  important in defense response in plants are differentially methylated between the different type of embryos. The Thecc1EG010128 is methylated in the exon for ZE with 0.75 level of methylation, whereas the methylation levels for normal and abnormal SEs are 1.0 and 0.9, respectively. The exon and intron of the gene Thecc1EG012913 are methylated with level of 0.30 and 1.0, respectively for ZE, in normal and abnormal SE the intron is 0.60 and 0.1 respectively, but exon in normal SE is unmethylated, while for abnormal SE exon is 0.1 level of methylation. The intronic region of the gene Thecc1EG045556 is highly methylated for ZE (1.0 level of methylation) and for normal and abnormal SE is 0.30 and 0.70 level of methylation, respectively. Those methylation profiles might be associated with the low stress environment ZE are exposed inside the fruit, in difference normal and abnormal SE are more exposed to inappropriate environment in the in vitro culture conditions.The different regions of genes that code for the Nucleic acid-binding proteins superfamily (Thecc1EG021358), Nudix hydrolase (Thecc1EG024914), Chromatin remodeling factor18 (Thecc1EG025136) and Peroxidase superfamily protein (Thecc1EG045425) between others are highly methylated for ZE but low methylated for normal and abnormal SE. These genes code for proteins important in stress response. The exonic region of Thecc1EG021358 and Thecc1EG024914 are more methylated in ZE than the same regions in normal and abnormal SE. In addition, the intronic regions of Thecc1EG024914, Thecc1EG025136 and Thecc1EG045425 are more methylated in ZE than the same regions in those genes for normal and abnormal SE. Methylation in exons is associated with induction or repression of gene expression depending on the position of the exon, but methylation in intronic region is associated with gene expression induction. The low level of methylation in the exon region of those genes in normal and abnormal SE might be associated with their high stress response to the in vitro culture conditions.PIN proteins). Methylation levels in ZE are 0.6 in the intronic region of the gene Thecc1EG039622 and 1.0 level of methylation for both normal and abnormal SE in the same region of the gene. ZE have 0.8 level of methylation in the UTR3 region for Thecc1EG020308 gene and in normal and abnormal SE it reached values of methylation levels 0.0 and 0.25, respectively, in the same regions of the gene. ZE have a methylation level of 0.75 in upstream of the transcriptional start site and 0.8 in the intronic region of the gene Thecc1EG000039. This is quite different from the methylation levels of 0.0 and 0.25 in the upstream region, and the levels of 0.0 and 0.1 in the intronic region in normal and abnormal SE. Auxin efflux carrier proteins fulfill important function in cellular transport where ZE seem to be active differently in this function compared to normal and abnormal SE. Another important gene that codes for an auxin transport protein is Thecc1EG019010 which is located on chromosome 4. In ZE, methylation levels are 0.7 in the exons and 0.75 in introns. Interestingly for normal and abnormal SE both regions have a methylation level of 1.0 which might indicate that probably the auxin trafficking in these cells is blocked  and Thecc1EG005126 (Auxin response factor 11) are differently regulated in ZE, normal and abnormal SE. In ZE the Auxin response factor 6 is methylated in its exonic region and upstream of the transcriptional start site with values of 0.8 and 0.9, respectively. In normal SE, the same regions have methylation levels of 0.1 and 0.2 (upstream and exonic regions). In abnormal SE, the methylation levels are 0.25 for both regions. In the Auxin response factor 11, methylation levels are 0.3 in upstream of the transcriptional start site and 0.7 in exon regions in ZE. In normal SE, methylation levels are less than 0.2 for both of regions. In abnormal SE, the methylation level is 0.2 for upstream transcriptional start site region and 0.4 for the exon region  and 19,783  differentially methylated gene regions in CHG context. In the CHH context, there were 24,658 , 23,820  and 22,893  methylated gene regions. All these differentially methylated gene regions are part of genes that code for proteins with functions in autophagy, hypoxia cellular response, DNA repair, auxin response, auxin transport, stress response, disease resistance, cell polarity, PCD, cell wall biogenesis, cell transport, cell division, cellular homeostasis, cell signaling, cellular metabolism, and developmental processes such as embryo morphogenesis as is the case in the CpG context 28.29. PCD in abnormal SE was notoriously higher than in normal SE in this study. The TUNEL analysis showed that there was DNA fragmentation because the cell nuclei stained fluorescent red in the tissue analyzed, mainly in the embryo body in abnormal SE30. The TUNEL analysis is characterized for the label of free 3ʹ OH ends in the fragmented genomic DNA with a fluorescein-dUTP or TMR-dUTP (Tetramethylrhodamine-dUPT) that bright red fluorescent at fluorescence microscope. Another indication of PCD in abnormal SE was the presence of lytic vacuoles in their cells and the absence of a nucleus. These signals are characteristic of vacuolar cell death in plants, which can be stimulated by recycling part of their cells during normal development or stress response31. In this study we identified genes differentially methylated in ZE, normal and abnormal SE with important functions in PCD, specifically in cell response to hypoxia, calcium signaling and production of peroxidases which is one of the important reactive oxygen species (ROS) key modulator of plant growth and development, and stress adaptation. Those findings can explain the presence of cavities in the embryo body of abnormal SE, a probably product of lysigeny via PCD for lack of oxygen in the in vitro culture environment, inducing the death of the tissue mediated by ROS28.PCD in plants is induced by biological processes essential for embryo development, formation and maturation of many tissues and stress response to environmental conditions for adaptation or reaction to biotic and abiotic stimuliT. cacao was developed for first time with the aim to identify gene regions differentially methylated and methylation profiles among them using WGBS. For cacao, we found a global distribution of methylation in the three contexts with some specific characteristics, where non-CpG sequences in ZE, normal and abnormal SE were manly hypomethylated in the CHH context, but in the CHG context there were high frequencies of hypomethylation, but few sequences were also highly methylated. Whereas CpG sequences displayed similar frequencies of hypo and hypermethylation, this result has been consistency with other studies, where levels of DNA methylation are higher in the CpG context than in the CHG and CHH contexts in many plants species32.The methylome landscape of zygotic and normal and abnormal SE from 33. It has been known that TEs and repetitive DNA sequences are mainly located in intergenic regions of heterochromatin where DNA is highly compacted avoiding the transcription of these regions34. Intergenic region of the DNA is heavily associated with closed heterochromatin state, in which its compaction is mainly regulated by the level of methylation in the CpG and CHG contexts, and less in the CHH context35. In our study we found that the intergenic region is the second most differentially methylated region between ZE, normal and abnormal SE in the three contexts where were identified retrotransposons significantly differentially methylated spread over the chromosomes 3 to 10 in the CpG context and spread over all 10 chromosomes in the CHG and CHH contexts. Mostly of these retrotransposons were hypermethylated in ZE but hypomethylated in normal and abnormal SE. it was reported in many studies that low levels of methylation in retrotransposons can be associate with their activation36, as a result, retrotransposons act as mediator to the stress conditions in the in vitro culture of SE, but retrotransposon activation can also induce abnormal phenotypes SE, as it was identified in other works where the activation of TEs is associated with adaptative response to environmental stress and abnormal phenotypes in plants38.In several studies was reported that methylation in CpG, CHG and CHH contexts is associated with activation or inactivation of transposon elements (TEs) and repetitive DNA sequences33. Our results demonstrated that introns in ZE, normal and abnormal SE are differentially methylated in high frequency followed by intergenic and exonic regions in all the three contexts. Methylation in exons has been linked to splicing where the mature mRNA is formed by removing introns from the transcribed mRNA precursor39. Splicing occurs cotranscriptionally when the transcript is still attached to the DNA and a large fraction of introns are removed from the mRNA precursor by RNA polymerase II40.Methylation in DNA can inhibit or enhance gene expression depending on where the methyl group in placed. Methyl group can be placed in exon or introns in the gene bodies, or in the transcription start sites, which regulated the expression by enhancing or blocking the gene transcription depending where the methyl group position41. The high CpG sequences quantity in exons influence in the amount of the total CpG methylation content in the methylome, being exons recognized by the RNA polymerase II and the spliceosome machinery from introns in the splicing39. In difference of the studies reported above, in our work we identified high frequencies of differentially methylation regions in introns in all of three contexts  when is compared ZE, normal and abnormal SE methylomes, being exons the third gene site more methylated, important finding because those differences have a strong association with the abnormalities in SE and probably influence the abnormal splicing in the abnormal SE phenotypes.There is not information about methylation in the different gene regions and their function in normal SE development, only few information in plants development. Therefore, it is important to understand the epigenetic mechanism in other organism as mammalians and unicellular. Regarding to the proportion of methylation between exon and intros, the analysis of DNA methylome in humans showed twice density of methylation in exon when is compare with introns42 and other genes in yeast43 where genes with methylation in the 5ʹ splice sites (5ʹ SSs) of intron regions revealed highest expression compared with genes without methylation in the same regions44. In plants, introns play an important role in gene regulation as enhancer element45. The mechanism of intron-mediated enhancement occurs when introns are located next to the promotor in the transcribed region, thereby increasing the levels of mature mRNA in the cytosol or when the 5′ SSs (highly methylated) of introns are recognized by U1 Spliceosomal RNA  in the alternative splicing, causing a threefold increase in the transcription products46.Considerable increase of gene expression is associated to intronic regions, such as the enhancement of ubiquitin C gene expression in humans47. In our work, part of the differential methylation of introns among ZE, normal and abnormal SE was probably due to the different phenotypes presented among them. In addition, differences in the methylation profiles of important genes involved in embryo structure development, stress response to the environmental conditions, and the effects of cellular compounds production can be the consequence of abnormalities in SE. A similar observation was mentioned in a study that was made in non-small cell lung cancer, where methylation profiles were different in two types of cell lung cancer and can be divided in two phenotypically distinct subtypes of tumors using methylation profile as a biomarker platform48.In mammals, intronic methylation is an important factor in gene expression regulation of cancer, where hypomethylated motifs in the intron region are associated with upregulation of oncogene expression and hypermethylation is associated with downregulation of tumor suppressive expression49. The principal ROS that are crucial inducer of oxidative stress are mainly singlet oxygen (1O2*), hydroxyl radical (·OH), hydrogen peroxide (H2O2), superoxide radical (O2·–) and nitric oxide (NO)50. Hydrogen peroxide is one of the major ROS compounds produced in the cells during abiotic and biotic stress conditions51. ROS production can increase and accumulate in the cells, creating an imbalance between ROS production and detoxification, thereby generating abnormal chemical signaling50.The main differentially methylated genes in this work were those involved in oxidative stress, auxin transport, auxin responsive genes, disease resistance, senescence, hypoxia, and developmental processes. Oxidative stress can start when homeostasis in the cells is disrupted52. Genome regions coding for peroxidase superfamily proteins were identified in our work as differentially methylated, it may be associated with the oxidative stress presented in ZE and normal and abnormal SE in different ways due to abiotic stress induced by the environmental conditions. Hypermethylation in intergenic regions of genes that code for peroxidases in ZE and low levels of methylation in normal and abnormal SE  confirmed that ZE have different environmental conditions than normal and abnormal SEs. In theory, low methylation in intergenic regions is associated with TEs activation when plants are under stress conditions. At the same time, we can infer that abnormal SE presented a particular methylation profile regarding to genes involved in oxidative stress, auxin transport, and developmental processes. Microscopy analysis of the abnormal SE showed abnormal apical-radical axial cell divisions and PCD in the apical shoot and root meristem. Compared to normal SE, this indicates a relationship between ROS and their effect in the wrong auxin polar transport in cells of the abnormal SE.Another important function of ROS is the direct effect in the biosynthesis, transport, metabolism and signaling of auxin in plants50. The genes that codified the proteins precursor of these compounds were identified in our work as differentially methylated between ZE, normal and abnormal SE. One of the most important is ethylene or its precursor 1-aminocyclopropane-1-carboxylate (ACC) since their production can be associated with the use of 2,4-dichlorophenoxyacetic acid  in the culture medium. Ethylene in high concentration can be accumulated, incrementing respiration rate in the tissue and inducing hypoxia in the culture environment53. Ethylene is necessary to stimulate hypoxic induction and it can also regulate aerenchyma formation in root tips of maize plants exposed to hypoxic conditions by schizogeny or lysigeny mediated by PCD and dissolution of cells54.Oxidative stress induces chemical signals such as abscisic acid, ethylene, brassinosteroids, auxin, gibberellic acid, methyl jasmonate, salicylic acid etc.Arabidopsis thaliana, in both rosette development and hypocotyl growth, and inhibits primary root elongation independently of ethylene perception when it was applied exogenously55. In our research we identified in the methylome of normal and abnormal SE the precursor of ethylene (ACC) differentially methylated downstream of transcription start site of the gene Thecc1EG037684 when is compared with the methylome of ZE. This leads us to speculate that SE are exposed to hypoxic environments in in vitro conditions due to ACC acumulation. Hipoxic environment in the tissue culture can affect some cells in the same tissue generating abnormal development of SE induced by ethylene or ACC.ACC the direct precursor of ethylene has a negative effect in PIN proteins in the chromosomes 1, 4 and 9. As mentioned above, abnormal SE presented a disorganized growing, without polarity between shoot and root meristems, suggesting problems in auxin efflux transportation56. The PIN-formed proteins are transporters acting in the efflux of signal molecules as auxin in plants57. PIN proteins are asymmetrically localized within cells and their polarity determines the direction of intercellular auxin efflux57. PIN proteins are located in the plasma membrane and they have influence in plant development by mediating auxin efflux through cells compartments57. When the gene that encodes the PIN7 protein is mutated in the A. thaliana genome, induced anomalies in zygotic embryos because the auxin efflux and polarity in early embryogenesis is disrupted56. Ethylene is an important regulator in auxin influx and efflux by basipetal auxin transportation. Exogenous application of ethylene inhibits the auxin transportation by proteins as PIN1, PIN2, PIN3 and PIN4 in consequence root formation is disrupted in A. thaliana. Ethylene was also reported as important hormone that downregulates the genes that encodes the lateral auxin movement in Zea mays and it downregulates the genes that encodes root tips and epicotyls formation in Pisum sativum confirming that there is a crosstalk between ethylene and auxin efflux carrier proteins in cell polarity58.As hypothesis, the provascular tissue abnormalities in abnormal SE might be associated with and interruption of the auxin transport from the shoot meristem throughout the embryo axis to the root meristem due to the significative differential of methylation profiles between ZE, normal and abnormal SE of genes that encode the In conclusion, for the first time the methylomes of torpedo ZE and SE in cacao are reported, revealing a unique profile in abnormal SE accompanying the morphologies presented in those embryos. Although abnormalities in SE prevent embryo germination, we identified seven abnormal phenotypes in a SE production of CCN10 genotype where some of them germinated partially but did not convert in normal plants. The methylome landscape of ZE and normal and abnormal SEs are different showing a specific methylation profile for each one. Different methylation levels were found in the three contexts  in ZE, normal and abnormal SE in cacao, CpG and CHG had a bimodal distribution of hypo and hypermethylated regions. We found similar frequencies of hypo and hypermethylation in the CpG context, and higher frequencies of hypomethylation than hypermethylation in the CHG context. In contrast, the CHH context had the highest frequencies of hypomethylation in a right-squeezed distribution.The main differentially methylated regions in the three contexts were intronic and intergenic regions. Methylation in intronic regions the highest region methylated in this study is in theory associated with gene expression enhancing, and probably in the wrong identification of introns in splicing process to form the mRNA, important finding in our work that might explain the abnormal phenotypes in SE. Intergenic regions were the second highest differentially methylated regions when ZE are compared with normal and abnormal SE, being less methylated in the abnormal ones. Intergenic regions are heavily associated with heterochromatin remodeling where generally TEs placed. The TEs are implicated in the regulation of stress in the tissue culture environment. Stress environment in the tissue culture was reveled in this study because genes related to oxidative stress were found differentially methylated between ZE, normal and abnormal SE. Microscopy analysis of Abnormal SE revealed cavity formation in their axes. In addition, cotyledons, shoots, and roots in abnormal SE had the highest area of fluorescence values due to PCD. Cavity formation in abnormal SE can be associated with lysigeny via vacuolar cell death probably as a response of the cell to the lack of oxygen in tissue culture environment. Hypoxia induction in abnormal SE can be associated with genes that codes for ethylene and its precursor ACC, which were differentially methylated in this work when compared with ZE. Ethylene production in in vitro environment reduces oxygen concentration and helps stimulating hypoxia induction. Differentiation and cellular auxin transportation of abnormal SE could also be influenced by ethylene in the cell environment due to the in vitro culture condition. Ethylene production in tissue culture could be mediated by 2,4-D (the main synthetic auxin used in the SE culture in this work) that in high concentration is accumulated in somatic tissue affecting embryos development by ethylene effect. An in-depth study of the gene expression of a core selection of the 873 genes found differentially methylated in this work would be required to confirm our results.Colección Castro Naranjal) with CCN 51 as pollen donor in the field at the Mars Center for Cocoa Science (MCCS) germplasm collection. Cacao pods were harvested 13 weeks after pollination (before maturation), which corresponds to the later torpedo stage when embryos maturation has not started yet as previously described8. A total of 10 g of ZE were extracted from each pod, frozen in liquid nitrogen and stored at − 80 °C for DNA and RNA extraction.Zygotic embryos at the torpedo stage from cacao pods were obtained by controlled hand pollination of CCN 10  and secondary SE generated from primary SE cotyledons (60 primary SE) of CCN 10 were obtained using the Li et al. (1998) and Maximova et al. protocols respectively with some modifications previously described in Ref.59, The analysis was performed in a digital microscope . The percentage of normal and abnormal SE was calculated counting the number of embryos  into the total of embryos present in each sample and multiplying by hundred. In addition, the percentage of conversion of normal and abnormal secondary SE to plantlets was also calculated to assess the viability of embryos and the capacity to generate normal plantlets. Embryo maturation and conversion were performed using the protocol previously described6. Embryos were considered as converted when the shoot apex developed and formed radical extension.Morphological evaluation was done with a representative sample of 118 primary SE  and 530 secondary SE (produced from cotyledons of 20 primary SE as explants) of CCN 10 in late torpedo stage and using as a control zygotic embryos in the same stage of development and following the references ratings previously describedSEM  60. The pictures were captured with CCD and Nav-Cam™ cameras and processed by the Scandium Image software (SIS).Samples of secondary SE  in late torpedo stage  of CCN 10 were collected and fixed in 2.5% glutaraldehyde, in a sodium cacodylate buffer at 0.1 M, pH 6.8, during 12 h. Subsequently, embryos were washed in the same buffer and were dehydrated in an increasing acetone series . Immediately after dehydration, the material was taken to a critical point dryer , which allowed the full elimination of water in the plant tissue. Afterward, the samples were placed in a metal stub with double-sided tape on it for subsequent metallization in a sputter COATER, , followed by subsequent observation in TEM MORGAGNI and wide angle Olympus MegaView III camera and the iTEM software  61.Samples of ZE and secondary SE  in late torpedo stage  of CCN 10 were collected and fixed in 3% glutaraldehyde, in a sodium cacodylate buffer at 0.1 M, pH 6.8, during 12 h. The samples were subjected to a series of washes (3 times for 15 min) in a sodium cacodylate buffer at 0.1 M, pH 7.2, and post-fixed in 1% osmium tetroxide, prepared in the same buffer, during 1 h at 4 °C. Next, the samples were washed 3 times in the same sodium cacodylate buffer for 15 min each one and were then dehydrated in an increasing ethanol series , followed by two washes in 100% ethanol (each one for 30 min). Shortly after, the samples were soaked in a mixture of 100% ethanol and LR White resin in the proportions of 3:1 (2 h), 1:1 (2 h), 1:3 (overnight), followed by two changes of pure LR White resin every 4 h, always under slow agitation. Afterwards, the samples were placed in gelatin capsules and covered with pure LR White resin. Resin polymerization was completed after 24 h at 60 °C. The ultrathin section (70 nm) were made with a diamond knife, using a Leica ultramicrotome . The cut sections were deposited on copper grids (300 mesh), contrasted with uranyl acetate in aqueous solution for 25 min, and then with lead citrate for 30 min. Subsequently, they were observed in 2O, and 5 parts acetic acid) during 12 h. The samples were submerged in paraffin and sectioned in slices of 5 µm using Leica ultramicrotome . The tissue sections were pretreated with proteinase K diluted in Tris–HCl 10 mM (20 mg/mL) pH 7.4 during 20 min. After that we followed the manufacturer recommendations. Tissue sections were analyzed in LEICA fluorescence microscope model DM2500 identifying red fluorescence with the Leica Application Suit (LAS X) software (Cells-stained fluorescent red are positive for cell death). The negative control was prepared using normal SE tissue incubate sections with label solution only, instead of TUNEL reaction mixture. The positive control was prepared using the abnormal SE tissue. Sections of abnormal SE were incubated with DNase I ) for 10 min at 15–25 °C to induce DNA strand breaks, prior to labeling procedure.Cell death was detected using the “In situ” cell death detection fluorescein kit provided by ROCHE. Samples of normal SE and abnormal SE in late torpedo stage of CCN 10 were collected and fixed in FAA solution , and the statistical analysis was performed with the software R 4.0.2.Pictures were captured with the QImaging Retiga 4000R monochrome camera, using the Texas Red fluorescent filter (Leica # TX2)2 to a fine powder using a mortar and pestle, transferred to a 2 µL Eppendorf microtube and DNA immediately extracted, following the kit protocol . The extraction procedure was repeated two times to ensure the DNA quality and quantity. After the second extraction, DNA was precipitated with 1:1 volume of isopropanol and 2 µL of glycogen from a stock of 20 mg/mL for 20 min at − 20 °C. The samples were centrifuged in a bench-top centrifuge  at 8000×g for 30 min at 20 °C. The supernatant was discarded, the resulting pellet was left to dry for 30 min in environmental conditions. Afterward, the pellet was re-suspended in 50 µL of 100% ethanol. The DNA quality was checked on a 1% agarose gel in 1× TBE  using a molecular marker  as reference. DNA quantification was performed using a spectrophotometer Multiskan GO, Thermo Scientific brand by absorbance at a wavelength of 260/230 and 260/280 nm.Genomic DNA from a population of 200 normal SE, 200 abnormal SE and 200 ZE at the late torpedo stage was extracted using the DNeasy extraction kit Plant Mini Kit (250) from QIAGEN for future methylation analysis. A tissue sample of 100 mg was disrupted in liquid NThe Whole-Genome Bisulfite Sequencing was performed on the total DNA extracted from two replicates of a set of mixed embryos from each of the three representative samples , using the protocol to prepare samples for Illumina sequencing platform ). For a few minutes, genomic DNA (5 μg) was normalized with unmethylated lambda (λ) DNA 5% . DNA was fragmented by sonication using a Covaris S220/E220 sonicator, and fragment selection (150–300 bp) was performed with AMPure XP beads (Agencourt Bioscience). Genomic DNA libraries were constructed using the Illumina TruSeq Sample Preparation kit, considering the Illumina’s standard protocol. Repair was performed at the ends of the DNA; at the 3ʹ end of each fragment an adenine was added, and the Illumina TruSeq adapters were also ligated to both ends. After ligation of the adapters, DNA was treated with sodium bisulfite using the EpiTect Bisulfite kit (QIAGEN). The bisulfite conversion was performed two times to ensure a conversion rate over 99%. Seven PCR cycles were carried out to ensure the enrichment of the adaptor-ligated DNA using PfuTurboCx Hot-Start DNA polymerase (Stratagene). Library quality and concentration were checked using the Agilent 2100 Bioanalyzer. Paired-end DNA sequencing (2 × 100 bp) with 30× coverage was then performed using the Illumina HiSeq 2000 platform.https://sourceforge.net/projects/fastqc.mirror/) with Phred quality score over 20 at each cycle. Also the adapter sequences were trimmed off the raw sequence reads using Trimmomatic program (http://www.usadellab.org/cms/index.php?page=trimmomatic)62. Read mapping was carried out using the BSMAP (http://code.google.com/p/bsmap/) which is based on the SOAP (Short Oligo Alignment Program) against to the Cacao_Matina1-6 (Tcacao_genome_v1.1) genome as a reference (https://www.cacaogenomedb.org/Tcacao_genome_v1/). The only uniquely mapped reads were selected to sort; index and PCR duplicates were removed with SAMBAMBA (v0.5.9). The evaluation of the quality of the alignment data (a BAM file) and the basic statistics of the alignment  was performed with Qualimap 2.2. The methylation ratio of every single cytosine location was called from the mapping results using ‘methylatio.py’ script in BSMAP. The results of the coverage profiles were calculated as the number # of C/effective CT counts for each cytosine in CpG, CHH and CHG contexts63.Before mapping and estimation of cytosine methylation levels, the raw sequence reads were filtered to make the quality check using the FASTQC bioinformatics tool . The methylation ratio values of all samples were normalized using median scaling normalization to compare the methylation level between samples. During data preprocessing, low coverage and high coverage bases were filtered. Lower read cut-off of 10 means that bases with coverage below 10× were discarded because a high enough read coverage will increase the power of the statistical tests. Higher read cut-off of 500 means that bases with more than 500× in each sample were also discarded.64. Differential methylation was obtained from the regions significantly different using Logistic regression (p < 0.001). The analyzes were performed with the aid of packages methylKit, complexHeatmap, circlize and annotate using software R 4.0.2.Statistical analysis was performed using the difference of two groups (delta_mean) per comparison pair. The significant results were selected on conditions of |delta_mean|≥ 0.2 with p-value of 0.05. Level of methylation was displayed as hypermethylation when the delta mean ≥ 0.2; and hypomethylation when the delta mean ≤  − 0.2. For significant CpG list, hierarchical clustering analysis was performed to group the similar samples grouped in CpGs, CHGs and CHHs. A cluster analysis was performed considering the correlations between the samples and grouping with Ward’s method65. The gene function was identified by making the alignments with expectation values  < 0.001 with the aim to infer gene homology comparing with others genomes such as A. thaliana using the cacao genome database in the Phytozome V13 (https://phytozome-next.jgi.doe.gov/info/Tcacao_v2_1) and the translated-DNA: protein alignments by BLASTX similarity search tool in the National Center for Biotechnology Information (NCBI) databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The protein metabolic function was identified using the collection databases from Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/).Each cytosine locus in CpG, CHH and CHG contexts was annotated using the Table Browser tool of the University of California Santa Cruz (UCSC) Genome Browser. Annotation included functional location of each gene , transcripts identification (ID), gene ID, strand, and sequence contextThis research was carried out in Brazil and the plant material used in it was donated from the germplasm collection of the Mars Center for Cacao Science (MCCS), which complies with the relevant national guidelines and the legislation of the Conselho de Gestão do Patrimônio Genético—CGEN permit for access to genetic resources number 02000.000070/2015-05.Supplementary Information.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.Supplementary Table S9.Supplementary Table S10.Supplementary Table S11.Supplementary Table S12.Supplementary Table S13.Supplementary Table S14.


The epidermis and cuticle play an important role in reducing dehydration and protecting the cashew pseudofruit in both the production environment and the postharvest stage. This study analyzes the alterations on the epidermis and cuticle of CCP 76 cashew pseudofruits harvested in five developmental and maturation stages . The epidermis and cuticle of the samples were analyzed under light microscopy (LM) , scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The epidermal cells at S3 reached maximum outer periclinal wall thickness, which reduced during ripening (S4 and S5), while the cuticle increased in thickness during the same period. These changes coincided with the rapid initial growth of the cashew pseudofruit when the epidermis and cuticle need to accompany the expansion of internal tissues. At the ultrastructural level, lipid material is transported via vesicles through the cell wall to the cuticle, increasing its thickness. Epicuticular waxes, previously deposited as plates and globules, began to develop an amorphous shape during maturation. This process possibly occurs due to changes in wax composition that can be related to the development of greasiness on the fruit skin. These findings provide a better understanding of cashew pseudofruit skin, which will aid future studies and strategies to preserve quality during the postharvest stage. Anacardium occidentale L.) belongs to the Anacardiaceae family [The cashew tree  evaluation of the pseudofruit’s rind surface revealed notable differences among fruit cuticles at several maturation stages. In S1, a very high density of unicellular non-glandular trichomes occurred a–c. The In S2, fewer trichomes were seen because of their breakage, with visible scars on the surface. Irregularly shaped wax platelets were seen d–f. In SIn S4, some fragments of trichomes were seen. At the broken trichome sites (scars), apertures reaching deeper layers, devoid of cuticles, were visible. Globular wax was maintained, the topographic ornamentation became less evident, and some regions displayed small whitish wax crystals. Some shallow surface microcracks were observed j–l. In S5, the epicuticular wax looked amorphous, suggesting structural and biochemical changes. Stomata became sparser due to the maximum growth of the mature pseudofruit, scars were left by the ablation of trichomes, and some microcracks were clearly observed on the cuticle m–o. Transmission electron micrographs revealed that the epidermal cells in S1 showed an evident nucleus, many vacuoles were partially filled with an electron-dense content, and the periclinal cell walls were thin a. In theIn S3, the periclinal walls became evident and two overlapping cells were observed that formed a biseriate epidermis c. In thiIn stages S4 d–f and SThe CCP 76 cashew pseudofruit grew slowly in the initial stages (S1 and S2); however, its growth increased quickly after 30 days until its cycle was completed in about 50 days, confirming the descriptions for this species [The epidermis was uniseriate with palisade-like cells until S3. From this stage, it became biseriate, with shorter and wider cells. The literature data show two differentiation possibilities for the epidermis throughout the development of fleshy fruits, that is, fruits with a uniseriate epidermis and fruits in which epidermal cells divide into more advanced development stages and become bi- or multiseriate. ‘Cascada’ tomato ,31 and ‘During the maturation period of the pseudofruit (S4 and S5), the epidermis’s thickness reduced due to increased cell width and narrow external cell wall thickness. In pear cultivars ‘Clapp’s Favorite’ and ‘Conference’, an epidermis formation pattern similar to that of the cashew pseudofruit occurred, that is, with periclinal divisions and stratification, and subsequent reduction in cell height up to 40% during maturation . Epiderm−1 of anthocyanins in cashew pseudofruit skin harvested with 70% orange-red skin, which was responsible for the increased color. The same authors also identified 0.22 g kg−1 of hydrolyzable polyphenols and 20–24 g kg−1 of proanthocyanidins in the skin and 0.7–1.0 g kg−1 in the pulp, indicating that the skin was highly astringent. Epidermal and subepidermal cells showed many vacuoles of different sizes with phenolic content in all the stages analyzed. The vacuolar content, along with pigments in chloroplasts (chlorophylls and carotenoids), caused changes in the pseudofruit’s color index. Tezotto-Uliana et al. (2018)  found 0.In the cashew pseudofruit’s epidermis, non-glandular trichomes and stomata were distributed very densely. The removal of trichomes left scars that remained on the epidermis throughout development until the fruit’s maturation. The presence of trichomes in the early developmental stages and during scar formation has also been recorded in some apple cultivars, such as ‘Gala’, ‘Galaxy’, ‘Jonagold’, and ‘Szampion’ . TrichomStomata were already formed in S1 with different degrees of opening. The coating and its natural openings determine the diffusion of gases (respiration) and transpiration in fruit . TherefoThe cuticle has particularities in fruit of all species; however, Lara et al. (2015)  report tSEM observations showed few superficial microcracks in the cuticle, possibly due to increased wax biosynthesis during maturation. Microcracks occur on the cuticle because of its inability to accompany fruit growth ,25, as wGreasiness has been recently investigated as a disorder identified on apple skin. Greasiness is known to be related to the accumulation of liquid cuticle constituents, such as esters of oleate and linoleate of -Farnesol and short-chain alcohols (C3–C5) ,38,39. SIn the early stages S1 and S2, epidermal cells have thin anticlinal walls, evident nuclei, and small vacuoles with an electron-dense content. In S3, cells of both epidermal layers exhibit a thickening of the anticlinal and periclinal walls, along with the presence of numerous plasmodesmata. The role of plasmodesmata in mediating macromolecule traffic is well-known in the vegetative organs of plants ,42, but The mechanism of biosynthesis and wax deposition is not fully understood in fruit ,17,24. ADuring the cashew pseudofruit maturation process, many vesicles cross the outer wall of epidermal cells, and the contents of these vesicles are released onto the cuticular surface. Similar electron micrographs have been recorded on citrus petal epidermis by Marques et al. (2016) , who relAnacardium occidentale L.) were obtained in 2017 from an orchard in the municipality of Artur Nogueira, São Paulo State, Brazil . The complete development of cashew apples on the plant requires 50 days on average [−1 of StartClor®) for 10 min and dried at room temperature. Early dwarf cashew CCP 76 clones  were used to determine both morphometry and skin color. Moreover, three cashew pseudofruits from different plants (four replicates per sample) were used for the analysis of skin structure under light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM).The length and diameter of the median region of the cashew pseudofruits were determined with a digital caliper . Weight (cashew pseudofruit and nut) was established on a precision scale .The cashew pseudofruits’ skin color was evaluated by a digital colorimeter  using the CIELAB color space system and calibration at D65: the three-dimensional components were L* (lightness) (dark to light), with variation between −100 and +100, and a* (green to red) and b* (blue to yellow), with variation between −60 and +60. The readings were performed in four regions of the cashew pseudofruit.®, Heraeus Kulzer, Hanau, Germany) to make blocks. The blocks were sectioned in a rotary microtome  at 5–7 μm thickness. Sections on slides were stained with ruthenium red for pectins and polysaccharides [®, Merck, Darmstadt, Germany) to analyze the epidermis and its outer cell walls. Sudan IV was used to detect lipids to delimit the cuticle [Transversal skin samples from the equatorial regions of the three cashew pseudofruits were fixed in Karnovsky’s solution , subjectcharides  and moun cuticle . Digital® software . Four images were taken per sample, with six measurements on each image to measure the thickness, width, and height of the outer wall of epidermal cells and cuticle thickness, totaling 72 measurements per stage. Measurements were taken using ImageJ2 [Skin samples from the equatorial regions of the three cashew pseudofruits were fixed in Karnovsky’s solution  and dehy2 , mounted2 in sodium cacodylate buffer ) [Skin transversal samples from the equatorial regions of the three cashew pseudofruits were fixed in Karnovsky’s solution with modifications )  for 48 hpH 7.2)) . Observap < 0.05). The statistical software used was SASM-Agri [Data were submitted to analysis of variance (ANOVA) and the means were compared using Tukey’s test (ASM-Agri .In conclusion, this is the first comprehensive study of the epidermis and cuticle changes that take place during the development and maturation of cashew pseudofruits. The ultrastructural analyses demonstrated that lipid material is transported via vesicles through the cell wall to the cuticle, increasing its thickness. Epicuticular waxes, previously deposited as plates and globules, start to develop an amorphous shape during maturation. This process possibly occurs due to changes in wax composition and leads to the development of greasiness on the fruit skin, which is related to the preservation of cashew quality during the postharvest process.


The histological diagnosis of sarcoma can be difficult as it sometimes requires the combination of morphological and immunophenotypic analyses with molecular tests. A total of 2705 tissue samples of sarcoma consecutively collected from 2006 until 2020 that had undergone molecular analysis were assessed to evaluate their diagnostic utility compared with histological assessments. A total of 3051 molecular analyses were performed, including 1484 gene fusions tested by c/qRT–PCR, 992 gene rearrangements analysed by FISH, 433 analyses of the gene status of MDM2, 126 mutational analyses and 16 NGS analysis. Of the samples analysed, 68% were from formalin-fixed, paraffin-embedded tissue and 32% were from frozen tissue. C/qRT–PCR and FISH analyses were conclusive on formalin-fixed, paraffin-embedded tissue in 74% and 76% of samples, respectively, but the combination of the two methods gave us conclusive results in 96% and 89% of frozen and formalin-fixed, paraffin-embedded tissues, respectively. We demonstrate the utility of c/qRT–PCR and FISH for sarcoma diagnosis and that each has advantages in specific contexts. We conclude that it is possible to accurately predict the sarcoma subtype using a panel of different subtype-specific FISH probes and c/qRT–PCR assays, thereby greatly facilitating the differential diagnosis of these tumours. Sarcomas are a heterogeneous group of mesenchymal neoplasms for which histological assessment is challenging. The morphology and immunoprofile of different tumour types frequently overlap and can mimic other tumour types. Nevertheless, an accurate diagnosis of many of these tumours remains a challenge, since they often share similar morphological features and similar cellular differentiation ,2,3. Of The diagnosis of BSTSs is challenging as their morphology and immunophenotype often overlap with each other and with tumours of other lineages, such as carcinomas and melanomas. Many BSTSs are associated with characteristic, reproducible genetic aberrations , which can be used for diagnostic purposes. As most genetic aberrations are tumour-specific, their detection by cytogenetic or molecular genetic techniques is diagnostically useful. Translocations can be detected by fluorescence in situ hybridization (FISH), and novel fusion transcripts can be detected by conventional or quantitative reverse-transcriptase polymerase chain reaction (c/qRT–PCR). The ability to achieve reliable molecular test results from formalin-fixed, paraffin-embedded (FFPE) tissue has been well documented ,8,9. FreMDM2 gene amplification tests. In the last 5 years, routine molecular testing for giant cell tumour (GCT) has been performed to assess the mutation status of H3F3A, and molecular testing for chondroblastoma using the H3F3B gene status has also been introduced. Experience with these techniques has also increased within the department as more molecular tests are routinely performed. In recent years, the use of a novel technique, anchored multiplex PCR (AMP) for next-generation sequencing (NGS) using the Archer Fusion Plex Sarcoma kit, has been implemented. Recently, assays have been established for fusion gene transcript detection that combine multiplex anchored PCR amplification of specific targets with NGS [The aim of this study, therefore, was to evaluate the impact and feasibility of the analysis of chromosomal alterations using interphase FISH and c/qRT–PCR in BSTSs, without a known diagnosis at the time of investigation. To this end, we reassessed 15 years of activity by comparing the use of FFPE tissue vs. the frozen tissue to see whether there were discernible improvements in the efficacy of rearrangement and with NGS ,11. ThisMDM2 amplification and gene variations were included in this study. In the case of molecular investigation for fusion transcript, the workflow followed the algorithm, as shown in In the present study, we collected the results of molecular analyses that were performed over 15 years using different c/qRT–PCR and FISH assays. All cases with a request of molecular investigation for fusion transcripts, gene rearrangement analysis, MDM2 status) (396 patients), synovial sarcoma (SS) (393 patients), myxoid/round cell liposarcoma (MLPS) (388 patients) and extraskeletal myxoid chondrosarcoma (EMC) (125 patients). Less frequent tests were performed for desmoplastic small round cell tumours (nine patients), low-grade fibro myxoid sarcoma (LGFMS) (nine patients), sclerosing epithelioid fibrosarcoma (nine patients), angiomatoid fibrous histiocytoma (eight patients) and pseudomagnetic haemangioendothelioma (three patients). The summaries of the findings for each tumour type tested are shown in The tumours most frequently tested for molecular confirmation were Ewing sarcoma (ES) (761 patients), well/dedifferentiated liposarcoma  consisted of FFPE tissue samples (590 were from blocks prepared elsewhere for a second opinion or for diagnostic review) and 32% (884) were from processed frozen tissue from our institution A. In tote status B. In 14%e status C.Of the c/qRT–PCR analyses performed on frozen tissue samples, 91% were evaluable, 1% were not evaluable and 8% were unsuitable material. Of the c/qRT–PCR analyses performed on FFPE tissue samples, 74% of cases were evaluable, 10% were not evaluable and in 16% the material was unsuitable for analysis A. SimilaMDM2 gene with the FISH method, the samples transported in formalin solution were compared with the samples transported under vacuum conditions. In the latter case, the samples were sealed in plastic bags in a vacuum apparatus immediately after surgical removal  [p < 0.0001).To evaluate the analytical quality of the samples tested for the amplification of the Bergamo) . SamplesEWSR1 analysis in FISH had a very doubtful setting, with split signals observed in 28 of 200 (14%) nuclei. A repetition of FISH on the same paraffin block showed similar results. Targeted NGS showed a translocation involving EWSR1 and FEV; the presence of a translocation was confirmed by cRT-PCR. For Case 5, FISH indicated that EWSR1 was rearranged, and NGS subsequently revealed a fusion between EWSR1 and PBX3 , but targeted NGS revealed a fusion between EWSR1 and ATF1. This result was confirmed by qRT–PCR. Case 12 tested negative in the MLPS assay by c/qRT–PCR and FISH. Subsequently, NGS revealed a fusion between SS18 and SSX1, which was confirmed by a qRT–PCR assay (NGS was performed for 16 cases that were determined to be negative by the first molecular screening (c/qRT–PCR and FISH); they were received for diagnostics (in the period 2019–2020). Of the samples analysed, nine cases (57%) did not have fusions, and six (37%) cases had fusions. One sample (6%) failed due to poor material quality and was determined to be unsuitable by failing the minimum quality control for parallel massive sequence analysis . Regardiand PBX3 A,B. Genequencing C. Case 1CR assay .Clinically relevant molecular analysis requires results to be reported in a clinically useful timeframe. We tracked the laboratory turnaround time (TAT) by retrieving specimen status data from the laboratory information system. The detection of tumour-specific genetic aberrations is becoming an integral part of the diagnostic investigation of bone and soft tissue tumours ,21,22,23MDM2 analysis or when the material was limited . In our experience, the c/qRT–PCR was the more frequently used ancillary test for gene rearrangement because it can quickly provide an answer, as demonstrated by the evaluation of TAT. Another reason for the prevalence of c/qRT–PCR tests is related to the availability, in our laboratory, of a large panel of assays  of suspected tumours of the musculoskeletal system that were collected over 15 years were analysed. Specific molecular abnormalities were identified in 1669 of 2705 cases (62%) of sarcomas, including 693 of 986 bone tissue sarcomas (70%). A total of 1610 investigations were carried out by c/qRT–PCR using a panel of 63 different c/qRT–PCR assays developed for fusion transcript analysis of FFPE and frozen tissues from 22 different tumour types. To the best of our knowledge, this is the largest analysis of sarcomas based on molecular studies. Based on the availability of the material, the first analysis would usually first involve the c/qRT–PCR and subsequent FISH analysis. Only the FISH method was needed for f assays  for geneIn determining the sensitivity and specificity of the molecular detection, here we report that c/qRT–PCR tests have greater sensitivity and specificity than FISH analysis in the tumours most frequently tested (ES and SS). These results are in agreement with a previous paper reporting that FISH and RT-PCR show similar sensitivities, while RT-PCR showed a higher specificity . WhereasIn this study, we evaluated the impact of preanalytical factors and the quality control measures required to improve the reliability of RNA extraction from FFPE material. RNA expression analysis from FFPE tissues has been broadly used in biomedical research ,27. It iMDM2 gene amplification test. It is probably optimal for the fixation process to be controlled by the pathology laboratory. This may involve the transport of fresh specimens to the pathology laboratory, preferably under vacuum for transport times of more than an hour.This study shows a failure rate of 26% in the c/qRT–PCR performed on FFPE material, including unsuitable/not amplifiable material (16%) and uninterpretable results (10%). The RNA is prone to degradation, and the efficiency of extracting amplifiable nucleic acids from FFPE tissue is dependent on the calcification, ischemia and fixation time ,31. DespThis study we also report our experience with recently implemented NGS technology used for all cases that have not been diagnosed with c/qRT–PCR and FISH. A total of 100% and 90% of samples were evaluable from fresh and FFPE tissues, respectively. Of the samples tested, 57% did not have gene fusions, while we found the presence of gene fusions in 37%. Only 6% (one case from FFPE tissue) did not pass the PreSeqRNA quality control; these results are in agreement with those reported before . RegardiAt present, in our experience, the methodological approach to evaluate gene fusions, which first involve c/qRT–PCR analysis and then FISH, has proven to be rapid, economical and reliable. TAT is a key aspect of clinical feasibility but has not previously been well documented for a clinical NGS cancer test. The combination of PCR and FISH allowed us to diagnose 54% of the samples in 3 days and 95% in 8 working days. However, a realistic increase in TAT, was observed for the samples that required an in-depth diagnosis by NGS. Some samples required multiple attempts at tissue and RNA preparation. Some samples required FISH and/or RT–PCR for orthogonal confirmation. Some samples showed fusion that required laborious interpretation and preparation and development of a specific c/qRT–PCR assay and performing Sanger sequencing for confirmation.EWSR1 present in different tumours [In conclusion, using the two methods ensures a higher success rate as both have limitations. RT–PCR is a rapid test designed to detect specific fusion transcripts, but this also means that rare fusion transcripts, or unusual translocation breakpoints, cannot be detected. Conversely, FISH assays utilize ‘break-apart’ probes that only identify a breakpoint in one of the common genes  with tumours . Therefo tumours ,37,38. TA series of 2705 tissue samples consecutively collected from January 2006 until December 2020 that had undergone FISH, c/qRT–PCR, gene mutational analysis or next-generation sequencing (NGS) were performed. The study was approved by the ethical institutional committee (code: TRASLOCASARC CE AVEC: 729/2020/Oss/IOR).The material assessed was that of tumours biopsied or excised at the Rizzoli Institute and processed locally, as well as tumours in paraffin blocks from other institutions or sent for a second opinion on a diagnosis. Since January 2017, a new method was implemented to improve the transport of samples in our institution. Briefly, the tissue samples were vacuum sealed in plastic bags using a semi-professional machine  inside the surgical theatre immediately after surgery and kept at 4 °C until transfer to the pathology laboratory. Once in pathology laboratory, the surgical specimens were processed without delay.Tests were requested to confirm or exclude a histological diagnosis. Tumours with an obvious histological diagnosis  were not analysed by molecular tests, and most cases tested were those with a degree of diagnostic uncertainty. All small round cell sarcomas with molecular tests performed on FFPE tissue and fresh material were included. Tests were requested to confirm or exclude a histological diagnosis. Tumours and tests for which routine molecular diagnostic assays are available in the laboratory are listed and described in MDM2 amplification and gene variations were included in this study. In the case of molecular investigation for fusion transcript, the workflow followed the algorithm, as shown in All cases with a request of molecular investigation of fusion transcripts, gene rearrangement analysis, For analysis of fusion transcripts, total RNA was isolated from frozen or FFPE tissue using a modified method, including TRIzol reagent  and RNeasy Mini Kit spin columns , as previously described . To extrWith some modifications the same QIAamp FFPE Tissue kit (Qiagen) was used for DNA purification of frozen samples. A fragment of less than 10 mg tumour tissue was selected using scalpel blades. Haematoxylin–eosin cryostat sections were performed to evaluate if the area of tissue selected was representative of the tumour. For PCR reactions, >50 ng of DNA was amplified using AmpliTaq Gold 360 Master Mix , according to described methods, with 0.5 μM of primers . ReactioMDM2 gene, cases were scored by counting a minimum of 100 tumour cell nuclei at X100 magnification with a DAPI/green/red triple band pass filter. The number of MDM2 and CEP12 signals was determined and a MDM2/CEP12 ratio was calculated for each nucleus. A ratio >2.0 in at least 10% of nuclei was considered amplified for the MDM2 gene. A Color View III CCD camera soft imaging system (Olympus) was used to capture images, then analysed with a CytoVision imaging software version 7.5 .For FISH, 2–4 mm thick FFPE sections were treated, as previously described. FISH was performed using specific Dual Color (Break-apart) DNA probes, as described in ® Sarcoma Panel kit  following the manufacturer’s protocol. Archer Library preparation reagents included FusionPlex Reagent and unidirectional gene-specific primer (GSP1 and GSP2) targeting 26 (FusionPlex® Sarcoma) or 63 (FusionPlex® Sarcoma v2) genes involved in bone and soft tissue tumours and Archer MBC adapters for Ion Torrent . This test relies on Anchored Multiplex PCR (AMP™) technology to generate target-enriched libraries. To assess the initial RNA quality, the Archer PreSeqRNA quality control (QC) assay was performed. If satisfactory for testing (PreSeq qPCR crossing point 30), cDNA libraries were produced. During library preparation, the cDNA was purified using Agencourt AMPure XP beads . Final libraries were quantified with the Ion Library TaqMan Quantitation Kit  and pooled to equimolar concentration. The prepared libraries were loaded on an Ion 520 or 510 chip using the Ion Chef system  and sequenced using the Ion Torrent Personal Genomic Machine (PGM) or S5 Machine. With the Archer analysis software version 6.0.4 (ArcherDX), the produced libraries were analysed for presence of relevant fusions. The sequence quality was assessed by the following criteria: a minimal total read number of 1.5 million with >7% unique fragments, and >40% RNA reads. If all criteria were met, the specimen was considered negative for fusions in the selectively captured genes. Archer Analysis Software version 6.0.4 at default parameters was used for reads alignment, fusion genes identification, data visualization and annotation. Human reference genome hg19/GRCh37 was used for comparison to obtained sequences.The NGS technology has been introduced in diagnostics since 2019. The measurement of RNA quantity for both FFPE and frozen material was measured by Qubit RNA HS Assay Kit , in accordance with Archer FusionPlex Protocol for Ion Torrent. This test relies on Anchored Multiplex PCR (AMP™) technology to generate target-enriched libraries. The methodological details are described elsewhere ,14,15. A