Ficus carica L.), the differentiation between fig and breba is increasingly common in the bibliography. In this regard, keep in mind that the fig tree generally produces two crops a year, the parthenocarpic breba, also called as early fig, and the main non-parthenocarpic crop, the fig proper. In this study, four brebas varieties  were selected in order to identify compositional, nutritional, and chemical diversity. These varieties were chosen for their commercial relevance in Spain. Color , fruit and peel weight, size, pH, total soluble solids (TSS), titratable acidity (TA), maturity index (MI), sugar, and organic content were determined for all the breba fruits samples. In addition, polyphenolic profile, amino acids, and volatile aromatic compounds were also identified. The varieties ‘Colar’ and ‘SuperFig1’ showed the highest fruit weight and size, while ‘Cuello de Dama Negro’ presented the higher pulp yield. The higher organic acid and sugar contents were determined for ‘SuperFig1’ and ‘Cuello de Dama Negro’, respectively. Although in low concentrations, the phenolic compound quercetin 3-(6-O-acetyl-beta-glucoside) and the amino acid tyrosine were only detected in the ‘’Cuello de Dama Negra’ and ‘SuperFig1’ fruits, respectively. Of the eighty volatile aromatic compounds identified, only eight were common in four varieties. An important knowledge gap was identified in relation to the characterization of the two Ficus carica L. crops, that is, the differentiation and specification in the literature when working with brebas and/or figs.Although most of the published articles generalize with the fruit of the fig tree ( Ficus carica L.), considered one of the oldest fruit trees cultivated in the Mediterranean region, continues to present an important economic and nutritional role today due to the high consumption around the world of its fruit, the fig and breba (early fig) [The fig tree (rly fig) ,2,3.Although its origin is indicated in Asia Minor, its cultivation is widespread throughout the world, mainly in the Mediterranean region due to its mild winters and hot and dry summers . Among tFicus carica L. fruits, are called brebas and/or figs. The figs appear from the bloom of the year, while the brebas, in those varieties that have this aptitude, are dormant figs that do not begin their development until the following spring. The “biferous” varieties are those that produce a crop of brebas and another of figs, while the “uniferous” produce a single crop of figs. The breba are larger and juicier and are normally eaten fresh and figs are smaller and can be eaten both fresh and dry [In general, and due to their maturity date and bloom, the  and dry ,8.Early fig fruits or brebas are popular for their sweet, smooth taste, and fluffy, fresh texture. Its consumption is mainly fresh and dry but also processed as a jam. On a nutritional level, breba fruits are a valuable source of minerals, vitamins, sugars, antioxidants, and phenolic compounds among other important metabolites. Due to its nutraceutical characteristics, records have been found that indicate its use since ancient times by different systems of traditional medicine, such as Siddha, Unani, and Ayurveda, to treat different cardiovascular, gastrointestinal, and respiratory diseases ,4.Ficus carica L.) is the most important species at a commercial level, it consists of more than 600 varieties with significant genetic diversity and verified by numerous studies [However, it should be noted that, although the fig tree , both for their national and international importance, that is to say, their commercial representation mainly destined for fresh consumption ,8,13. Mosenes L. .Ficus carica L.) with consolidated commercial interest were selected for fresh consumption, these being [‘Cuello de Dama Negro’: Biferous variety with an important breba production and very well adapted to rainfed conditions. Breba fruits present a good aptitude for manipulation and transport, fulfilling the demands of the market for their fresh consumption.‘San Antonio’: Biferous variety perfectly adapted to rainfed cultivation conditions, it is interesting for the early ripening of its fruits.‘Colar’: The most widespread variety in the southeast of Spain, due to its productivity and earliness, as well as its size, color, and flavor.SuperFig1’ (‘SF1’): Little-known parthenocarpic biferous variety, presents a productive and precocious tree. It has resistant skin and its taste quality is high.To the knowledge of the authors, few works differentiate between brebas and figs. Being the breba fruits, generally poor studied, characterized and almont no compared between the different varieties. Based on this, for the present study, four biferous varieties of fig trees  of nitrogen, 80 UF of P2O5, and 150 UF of K2O. At the time of fruit collection, all the trees presented a good phytosanitary status.The 15-year-old trees are located on a commercial plot in Ojós (Murcia), in southeastern Spain. The cultivation and management conditions remained homogeneous for the four varieties studied in order to reduce the environmental impact on the parameters studied. The trees are disposed of in a 6 × 4 frame and irrigated by a drip irrigation system. Each fig tree has available 4 drippers of 4 L hn = 20) was determined according to the minimum sample size, guaranteeing the representativeness of the population with an interval of 95%, calculated by Equation (1) [n represents the minimum representative sample size of the population; S is the standard deviation of the sample; For each variety, a total of 20 brebas were collected from four different trees (5 fruits per tree). The number of figs per variety (tion (1) .(1)n ≥1All the figs were collected manually in their commercial maturity cap, which was determined by the softening of the fruit, typical coloration according to the variety, and longitudinal cracking of the skin. Once collected, the figs were immediately transferred to the laboratory and processed and analyzed on the same day.The morphological characterization of the figs was carried out using the parameters of weight, size, and color. The weight of the figs was determined using an analytical balance  with a precision of ±0.01 g, for all the fruit samples the total fruit weight and the peel weight was determined, both weights in grams. The breba size was determined with the measurements relative to the maximum equatorial diameter of the fruit , the diameter of the free ostiole , the longitudinal height of the breba with peduncle , and longitudinal height of the peduncle on its shortest side , as shown in n = 20) and their standard deviation.The external and internal color of the brebas was established using a spectrophotometer  according to the Commission Internationale de l’Éclairage (CIE) and was expressed as color values L *, a *, and b *; where L * indicates the lightness of the color , a * it is position between green and red  and b * its position between blue and yellow . The target color n = 4) per variety. The juice samples were obtained from the processing of sub-samples composed of five breba fruits each, using a domestic press blender (Orbegozo LI model 5060). The juices were placed in sterile polyethene containers until their use to ensure their preservation, and TSS, TA, and pH analyses were determined the same day.The biochemical analyzes of each breba variety were carried out on four different juice samples (−1) were determined using an automatic potentiometric titrator . In addition, the maturity index (MI) was also calculated as the TSS/TA ratio. The results represent the mean values (n = 4) with their standard deviation.Total soluble solids  were measured using a refractometer . The pH and titratable acidity  were previously centrifuged at 15,000 rpm for 10 min at 4 °C, then the supernatant was removed carefully and filtered with a 0.45 µm nylon membrane (Filter-Lab NY 0.45 µm). The determination of sugars and organic acids was performed by Agilent 1100 high-performance liquid chromatography (HPLC) with ChemStation software using a Supelcogel C610H column, 30 cm × 7.8 mm, and a Supelguard guard, 5 cm × 4.6 mm . To measure the absorbance of fatty acids, a diode array detector (DAD) (Diode Array DAD G1315A) set at 210 nm and a refractive index detector (RID) (G-1362-A) were used for the sugar content. In both cases, reference standards of organic acids and sugars were used supplied by Sigma-Aldrich  with calibration curves with R2O:formic acid (75:24:1) using a vortex for 5 min. Subsequently, they were centrifuged for 10 min at 12000 rpm and filtered by 0.45 µm. The relative quantification of the phenolic compounds present in the samples was carried out through chromatography comparison with pure standards  and their maximum absorbance spectrum at an emitted wavelength. at 290 nm, 320 nm, and 520 nm, respectively, through a diode UV detector (DAD) integrated into the HPLC and connected online to the mass spectrometer.The polyphenolic profile of figs was identified by HPLC-DAD-ESI-MSn (Agilent 1200 series). For extraction, 50 mg of lyophilizate pulp were weighed and dissolved in 1 mL of MeOH:Hv/v) were used. The extract was vortexed for 1 min, sonicated for 3 min and finally centrifuged at 5000 rpm for 20 min at 4°C The supernatant was collected and dried in a vacuum oven at 27 °C and resuspended in 800 μL of D2O phosphate buffer (100 mM KH2PO4 pH = 6.0) containing 0.58 mM TSP . The mixture was centrifuged at 4000 rpm for 5 min at 4 °C and the supernatant (600 µL) was placed in a 5 mm nuclear magnetic resonance (NMR) tube for analysis. All spectra were recorded on a Bruker AV-HD NMR operating at a proton NMR frequency of 500.16 MHz. Each 1H NMR spectrum consisted of 64 scans with the following parameters: 0.191 Hz/point, pulse width = 4, 0 μs (90°) and relaxation delay = 2.0 s. The free induction decay was Fourier transform with line broadening = 1 Hz, Gaussian broadening = 0, and sensitivity to peak selection = 1.0. 1H-NMR spectra were analyzed with Chenomx Profiler  to obtain amino acid concentrations in figs.For amino acids identification and quantification, aliquots of 50 mg of lyophilized fig pulp in a methanol/water solution . The GC-MS system was equipped with a 30 m × 0.25 mm Supelco  SLB-5 MS (fused silica) column with a 0.25 µm film thickness. The carrier gas used for this analysis was helium maintained at a column flow rate of 0.6 mL minp < 0.05. In addition, for all results, principal component analysis (PCA) and cluster analysis (CiA) were also performed. Cluster analysis was applied to the standardized data for hierarchical associations employing Ward’s method for agglomeration and the squared Euclidean distance as the dissimilarity measure. Statistical analyzes were performed with the Statgraphics Centurion 18 Software data analysis software.The significant differences of the experimental data obtained were evaluated using a one-way analysis of variance (ANOVA) followed by a contrast of means separation using the Tukey test for The morphological characterization results of the four fig varieties studied are presented in The mean weight of the brebas studied ranged between 62.58–134.63 for the whole fruit and between 15.02–35.77 g for the peel. While the varieties ‘Colar’ and ‘SF1’ presented the highest weights, both of breba (133.92 g ‘SF1’) and peel (34.49 g ‘Colar’), the weights of the varieties ‘Cuello de Dama Negro’ and ‘San Antonio’ were significantly different, with values between 62.58–75.68 g and 15.02–16.07 g for whole fruit and peel, respectively. The weight results for the ‘Colar’ and ‘SF1’ varieties were only different when compared to the weights of the ‘Cuello de Dama Negro’ and ‘San Antonio’ varieties, but, in both cases, without statistical differences between them. On the other hand, the highest pulp yield was presented by the ‘Cuello de Dama Negro’ variety with an average yield of 80%. This pulp yield was significantly higher than that of the other varieties, which ranged between 73 and 74% (‘Colar’> ‘San Antonio’ = ‘SF1’).p > 0.05). The luminosity parameter (L *) did not present significant differences both for the external color , and for the internal color (range between 51.29 and 55.65), in both cases, the results indicated luminous colors .The external and internal color of the brebas showed little variability between the studied varieties (The Hue angle (H°), or objective color, presented values between 31.93 and 40.52, without significant differences between the varieties for the external color, while for the internal color, the highest tone angle (81.00) was determined for the ‘San Antonio’ variety. This result is significantly higher when compared to the other varieties, which ranged from 54.45 to 58.40.Regarding the external color, the variety ‘Cuello de Dama Negro’ exhibited values for the parameters a* (2.51) and b* (1.39) well below the other varieties, which remained without significant differences in the ranges of 7.32–8.69 and 5.68–7.44 for parameters a* and b*, respectively. For the internal color, the varieties ordered from highest to lowest a* were established as: ‘Cuello de Dama Negro’ > ‘Colar’ > ‘SF1’ > ‘San Antonio’, highlighting the difference between the maximum result (14.36 for ‘Cuello de Dama Negro’) and the lowest value obtained (3.41) for ‘San Antonio’. Based on the results obtained for the internal color parameter b*, the varieties can be divided into two groups, on the one hand, ‘Cuello de Dama Negro’ and ‘San Antonio’, which presented the highest values , and on the other, the varieties’ SF1’ and ‘Colar’, with values > 17 and >18.−1) significantly different when compared to the TA of the varieties that presented the lowest values, these being ‘Cuello de Dama Negro’ and ‘San Antonio’ with 1.14 and 1.18 g citric acid L−1, respectively. The MI for all varieties was between 129.62 and 153.94, but without statistically significant differences. The results are presented in In relation to the chemical characterization of breba fruits, the four varieties presented pH values between 8.35 and 8.59, without significant differences between them. The ‘Cuello de Dama Negro’ variety (17.25 °Brix) showed the highest levels of total soluble solids, while the lowest amount was determined for the ‘Colar’ variety (12.37 ° Brix). On the other hand, the variety ‘Colar’ was the one that presented the highest TA value . Succinic acid was detected between 0.222–0.267 g kg−1 in all varieties (‘San Antonio’ > ‘Colar’ > ‘Cuello de Dama Negro’) except for the breba ‘SF1’, in which it was not detected.The sugars and organic acids present in the breba varieties studied are shown in −1, while, for the other three varieties, the acid content was less than 0.1 g kg−1, without differences significant among them. Lactic acid was also detected in the four varieties, where the ‘Colar’ variety presented the highest amounts (0.292 g kg−1) and ‘Cuello de Dama Negro’ the lowest values (0.153 g kg−1), the results for all the varieties showed significant differences.For the ‘San Antonio’ breba was identified the highest content of formic acid, with a concentration of 0.119 g kg−1), followed by ‘Colar’ (10.444 g kg−1) > ‘San Antonio’ (9.506 g kg−1) > ‘Cuello de Dama Negro’ (8.630 g kg−1).In general, the variety ‘SF1’ presented the highest content of organic acids (11.584 g kg−1), and ‘San Antonio’ of fructose (122.66 g kg−1), while the lowest results, for both sugars, were determined for the variety ‘SF1’, with 100.07 g kg−1 of fructose and 99.59 g kg−1 of glucose. Sucrose was also detected in all samples, with maximum concentrations between 4.6 and 4.8 g kg−1 for the ‘Cuello de Dama Negro’ and ‘San Antonio’ varieties, respectively. On the contrary, the lowest values of sucrose were determined for ‘Strain’ (3.1 g kg−1) and ‘SF1’ (3.5 g kg−1).In relation to the sugars contained in the breba juices, fructose and glucose were the major sugars in all the juices studied. In this sense, the variety ‘Cuello de Dama Negro’ presented the highest concentrations of glucose (131.187 g kg−1) followed by the varieties ‘San Antonio’ (248.30 g kg−1) > ‘Colar’ (208.43 g kg−1) > ‘SF1’ (203.24 g kg−1).‘Cuello de Dama Negro’ variety presented the highest total sugar content  and ‘SF1’ (17.5) the lower.Due to the important influence on the fruit quality, mainly caused by its impact on parameters such as flavor and odor, the phenolic profile of breba fruits was determined by identifying and quantifying the polyphenolic compounds as shown in −1, 1.231 mg g−1 and 0.278 mg g−1 respectively, in the other varieties, the concentrations detected were at least 30% lower.‘Cuello de Dama Negro’ variety stood out for showing the highest contents for all compounds compared to the other varieties. In this sense, while the concentrations for the cyanidin 3-rutinoside, caffeic acid 4-O, and 5-CQA compounds in ‘Cuello de Dama Negro’ breba’s were 1.897 mg g−1 for Caffeic acid 4-O; 0.05 mg g−1 for 5-CQA; and 0.210 mg g−1 for cyanidin 3-rutinoside. Additionally, ‘Cuello de Dama Negro’ was the only variety in which quercetin 3-(6-O-acetyl-beta-glucoside) was detected with a concentration of 0.153 mg g−1. The heterogeneity of the results, with distant maximums and minimums, is confirmed with the statistical study since all the results showed significant differences between the varieties.In all cases, ‘Colar’ variety presented the lowest values, with contents of 0.444 mg gIn order to study and identify in detail the breba fruits and their possible compositional variations related to the variety, a metabolomic characterization was carried out due to their metabolic and nutritional importance .A total of eleven amino acids were identified, among them, only aspartate and valine did not show significant differences between the varieties. The brebas of the varieties ‘Cuello de Dama Negro’ and ‘San Antonio’ showed the highest glutamate and glutamine contents, with concentrations between 0.539–0.604 mM and 0.813–0.856 mM, respectively, while the varieties ‘SF1’ and ‘Colar’ showed values around 0.3 mM of glutamate and between 0.652 mM and 0.455 mM of glutamine, respectively.In the ‘San Antonio’ brebas, the highest concentrations of alanine (0.759 mM) and proline (1.44 mM) were detected; meanwhile, the variety ‘SF1’ presented the highest contents of isoleucine (0.153 mM) and leucine (0.062 mM), the latter together with the variety ‘Colar’ (0.068 mM), and the lowest contents of proline (0.439 mM). On the other hand, the ‘Cuello de Dama Negro’ brebas showed the lowest concentrations of alanine (0.998 mM), isoleucine (0.052 mM), and leucine (0.041 mM). The highest GABA values were identified in the ‘San Antonio’ fruits (0.528 mM) and ‘Colar’ (0.461 mM) while ‘Cuello de Dama Negro’ (0.358 mM) and ‘SF1’ (0.378 mM) presented the lower values, without significant differences between both groups. The variety ‘Colar’ showed the highest concentrations of asparagine  while the other varieties showed ≤ 8704 mM (‘Cuello de Dama Negro’) but without differences between them. The only variety in which tyrosine was detected, with a concentration of 0.018 mM.On the other hand, two additional metabolites were identified, choline and trigonelline. Regarding choline, ‘Cuello de Dama Negro’ (0.169 mM) showed the lowest concentration compared to the other varieties, which was around 0.2 mM. Already for trigonelline, the results showed significant differences between the varieties, where for ‘Colar’ it was not detected and for the others ‘San Antonio’ > ‘Cuello de Dama Negro’ > ‘SF1’.Among the four breba varieties studied, a total of 80 different volatile compounds were identified . For theOnly seven of the eighty volatile compounds were detected in the four varieties, these being: benzyl alcohol; benzaldehyde; cycloheptasilo1ane, tetradecamethyl-; 3-ethyl-4-methylpentan-1-ol; -2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol; cyclopentasilo1ane, decamethyl-; and cyclotetrasilo1ane, octamethyl-.Aiming to better understand the trends and relationships between the many variables studied (52) for the different samples of brebas (four varieties), principal component analysis (PCA) was applied. The first three principal components (PC) explained 100% of the total variation. The first two PCs explained 91.04% of the observed variability. The first component (PC1), which represents 50.61% of the total variance, was related to the analyzed variables trigonelline, sucrose, fructose, glucose, fruit weight, peel weight, D1, D2, L1. L* external, L* internal, b* internal, AT, pH, SST, glutamate, glutamine, isoleucine, and leucine. These variables are related to the most important sugars, fruit size, exterior and interior brightness of the fruit, acidity, and TSS, as well as various amino acids.PC2 represented 40.42% of the total variance. It was correlated with the content of Choline, Chlorogenate, myo-Inositol, L2 a* external, C* external, a* internal, H° internal, C* internal, MI (TSS/TA), quercetin acetyl-glucoside, cyanidin 3-rutinosido_1, GABA, alanine, aspartate, phenylalanine, pyroglutamate, tryptophan, and tyrosine .PC3 represented 8.96% of the observed variability and was correlated with succinate, b* external, H° external, 5-CQA (chlorogenic acid), quercetin acetyl-glucoside, and proline. .The PCA results showed that the PC1 axis allows clearly discriminating the ‘San Antonio’ and ‘Cuello de Dama Negro’ varieties from the ‘Colar’ and ‘SF1’ cultivars. . PC2 allPC3 helps to discriminate the ‘Colar’ cultivar from the other three and the SF1 cultivar from the other three. The morphological and chemical parameters dendrogram showed and confirmed the differences between the brebas varieties studied but indicated similarities between ‘Colar’ and ‘SF1’ varieties and ‘Cuello de Dama Negro’ and ‘San Antonio’ cultivars .Regarding the morphological characteristics, it should be noted that, for all the varieties considered in the present work, the fruit weight and size were higher than that indicated by other authors. In this sense, while the lowest and highest fruit weight of the studied varieties was 62.58 g (‘San Antonio’) and 134.63 g (‘Colar’), respectively, in a study carried out by Çalişkan and Polat  for two Considering the color diversity presented by the different fig varieties worldwide, and which can vary from green/yellowish tones to intense blacks, in general, consumers common trend, showing a preference for figs that have dark colors and intense for fresh consumption . In thisThe analysis of TSS, TA, and MI (TSS/TA) parameters are important in the evaluation of the quality of the fruit, and are used to define its levels of maturity, and, therefore, its quality attributes. This means that high TSS and MI (TSS/TA) values will imply greater acceptance by the consumer . In thisBrebas, such as figs, represent a role in the Mediterranean diet, with frequent consumption in its various forms . Their high appreciation and consumption are due to their fluffy, fresh, and sweet texture . For theSince both the amount and the composition of the sugars have a direct influence on the sweetness of the fruit and its taste appreciation, the results indicate differences between the varieties, since for ‘Cuello de Dama Negro’ and ‘SF1’ their sweetness is mainly due to fructose, while for ‘San Antonio’ and ‘Colar’ it would be glucose. The values obtained are higher than those obtained in other studies ,26,32. T−1), while the Crna petrovka varieties (3.95 g kg−1) and Bela petrovka (2.7 g kg−1) for ‘Colar’ and Santo Antonio, respectively [−1) compared to those determined in the present work. The higher content of organic acids can result in a less sweet appreciation in the flavor of the fruit, counteracting the sweetness provided by the sugars. As far as the authors are aware, no work has been reported where the presence of lactic acid in brebas is reported.However, the sweetness of the fruit is not only related to sugars, but also to organic acids. Most studies indicate malic and citric acid as the main organic acids of figs , which cectively . Howeverectively  determin−1 and 0.028 mg g−1 for the Portuguese varieties ‘Pingo de Mel’ and ‘Branca Tradicional’. It should be noted that, in the same study, the authors did not detect the flavonoid glycoside, quercetin 3-glucoside, in the fruits, only in the peel and leaves, while, in the present study, it was identified in ‘Cuello de Dama Negro’. This fact is not unusual, since protective effects of cell structures against UV radiation are attributed to this compound, as well as a high influence on the coloring of the fruits, where ‘Cuello de Dama Negro’ presented a pulp with a much more intense coloration [On the other hand, with their use and consumption increasingly widespread throughout the world, brebas have shown to be an excellent source of phenolic compounds . All theloration ,37. Cyanloration ,39, so iloration  for the Amino acids are building blocks of proteins and polypeptides. In addition to the nutritional and clinical properties, some amino acids can influence the perception of taste, since they can present a sweet taste . To the On the other hand, in addition to the mentioned characteristics, the quality of the fruits is also affected by other parameters, such as aroma. Several studies indicate that volatile compounds significantly affect flavor and aroma quality . DespiteBased on the experimental results, the following points can be concluded:‘Cuello de Dama Negro’ was the variety with the highest amount of TSS (17.25 °Brix), with significant differences over ‘San Antonio’ and ‘SF1’, and these in turn also present significant differences over ‘Colar’ (12.37 °Brix). However, there are no differences between the four varieties regarding MI.Malic and citric acids were the predominant organic acids in breba fruits. Malic acid predominates significantly in ‘San Antonio’, ‘SF1’ and ‘Colar’ while ‘Cuello de Dama Negro’ has a significantly lower content. Citric acid is presented in higher quantity in ‘Cuello de Dama Negro’ compared to ‘San Antonio’.The variety with the greatest diversity and content of phenolic compounds was ‘Cuello de Dama Negro’. The phenolic compound cyanidin 3-rutinoside was only detected in the ‘Cuello de Dama Negro’ fruits.The amino acid tyrosine was only detected in ‘SF1’, while the metabolite trigonelline was presented in all varieties except ‘Colar’.The varieties presented a great diversity of volatile compounds, where only seven of the eighty compounds were detected in all the varieties. Further studies need to be carried out to verify the potential of establishing a form of varietal identification based on the presence and/or absence of volatile compounds.This is the first study that compares and characterizes the chemo-nutritional and pomological properties of the four varieties of figs that are most relevant from the commercial point of view of southeastern Spain.Due to its content in bioactive compounds, the consumption of this fruit can contribute to the prevention of certain diseases, however, more specific studies are needed to corroborate this statement.An important knowledge gap was identified in relation to the characterization of the two crops of Ficus carica L., that is, the differentiation and specification in the literature when working with brebas and/or figs.


We evaluated the impact of the genotype on hepatic, pancreatic and myocardial iron content, and on hepatic, cardiac and endocrine complications in children with transfusion-dependent β-thalassemia (β-TDT). We considered 68 β-TDT patients  consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia network. Iron overload was quantified by T2* technique and biventricular function by cine images. Replacement myocardial fibrosis was evaluated by late gadolinium enhancement technique. Three groups of patients were identified: homozygous β+ (N = 19), compound heterozygous β0β+ (N = 24), and homozygous β0 (N = 25). The homozygous β0 group showed significantly lower global heart and pancreas T2* values than the homozygous β+ group. Compared to patients with homozygous β+ genotype, β0β+ as well as β0β0 patients were more likely to have pancreatic iron overload . No difference was detected in biventricular function parameters and frequency of replacement fibrosis. No patient had cirrhosis/fibrosis, diabetes or heart failure, and the frequency of endocrinopathies was comparable among the groups. In pediatric β-TDT patients, there is an association between genotype and cardiac and pancreatic iron overload. The knowledge of patients’ genotype can be valuable in predicting some patients’ phenotypic features and in helping the clinical management of β-TDT patients. Beta-thalassemia is a genetic blood disease with a high incidence in the Mediterranean basin, Middle East, Indian subcontinent, Central Asia, and Far East . HoweverBased on the clinical severity and transfusion requirement, thalassemia can be classified into two main groups: transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT). TDT is the most severe clinical form of β-thalassemia, and its current treatment consists of regular transfusions every 2–5 weeks) to maintain pre-transfusion hemoglobin levels above 9–10.5 g/dL  weeks to,7. Iron A superior understanding of organ damage can be achieved by a comprehensive assessment of the children with TDT, representing the ideal population to study the initial stage of iron loading and the onset of its complications.The aim of the present study was to evaluate the impact of the genotype on hepatic, pancreatic and myocardial iron content, and on hepatic, cardiac and endocrine complications in children with transfusion-dependent β-thalassemia.From the 1727 patients with hemoglobinopathies enrolled in the E-MIOT  Network, we retrospectively selected 68 pediatric (age < 18 years) TDT patients who had undergone at least one MRI scan. We excluded the patients with Hb Lepore either in heterozygosis or in homozygosis and those with alfa abnormalities.The E-MIOT Network is constituted by 66 thalassemia centres and 11 MRI sites where MR exams are performed using standardized and validated procedures ,18. All All patients were regularly transfused to maintain a pre-transfusion hemoglobin concentration above 9–10 g/dL. MRI scanning was performed within one week before regular scheduled blood transfusion. This study complied with the Declaration of Helsinki and was approved by the institutional ethic committees. Parents gave their informed consent for all patients.MRI exams were performed on conventional clinical 1.5 T scanners of three main vendors , equipped with a phased-array receiver surface coil. ®, V2.0, Consiglio Nazionale delle Ricerche and Fondazione Toscana Gabriele Monasterio, Pisa, Italy) [For iron overload assessment, a mid-transverse hepatic slice , five or, Italy) . Hepatic, Italy)  and were, Italy) ,27. Thre, Italy) . Global , Italy) . The gloSteady-state free precession (SSFP) cine images were acquired in sequential 8-mm short-axis slices (gap 0 mm) from the atrio-ventricular ring to the apex to quantify biventricular function parameters in a standard way . The int®; Bayer; Berlin, Germany) intravenous administration at the standard dose of 0.2 mmol/kg using a fast gradient-echo inversion recovery sequence. The use of Gadobutrol has been demonstrated to be safe in patients with hemoglobinopathies [To detect the presence of replacement myocardial fibrosis, late gadolinium enhancement (LGE) short-axis and vertical, horizontal, and oblique long-axis images were acquired 10–18 min after Gadobutrol , good (60–80%) or insufficient (<60%).An MRI LIC ≥3 mg/g/dw was considered indicative of a significant iron load . It was Liver fibrosis was diagnosed if the liver stiffness assessed by transient elastography was >7.0 kPa, while a liver stiffness >12.5 kPa was indicative of cirrhosis. Diabetes mellitus (DM) was defined by fasting plasma glucose ≥126 mg/dL or 2-h plasma glucose ≥200 mg/dL during an OGTT, or a random plasma glucose ≥200 mg/dL with classic symptoms of hyperglycemia or hyperglycemic crisis .Hypogonadotropic hypogonadism was defined as luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels below 2 IU/L, with an estradiol concentration of below 20 pg/mL in girls or a testosterone concentration of below 3 ng/mL in boys. Hypogonadism was detected in females by the absence of breast development and in males by the absence of testicular enlargement (<4 mL) by the age of 16 years .Hypothyroidism was defined as a high serum TSH concentration with normal or reduced free thyroxine levels (primary form) or normal or low serum TSH concentration with reduced free thyroxine levels  .Hypoparathyroidism was defined as low serum calcium concentration, increased serum phosphate, low serum parathyroid hormone or, if normal, inappropriate for the calcium level .Diagnosis of growth hormone (GH) deficiency required integration of growth criteria, medical history, laboratory tests (measurements of insulin-like growth factor 1 and insulin-like growth factor binding protein type 3 levels and provocative testing), and imaging studies . Heart failure (HF) was identified based on symptoms and signs, according to the AHA/ACC guidelines . ArrhythAll data were analyzed using the SPSS v27.0 statistical package. Continuous variables were described as mean ± standard deviation (SD) and categorical variables were expressed as frequencies and percentages.The normality of distribution of the parameters was assessed by using the Kolmogorov–Smirnov test.For continuous values with normal distribution, comparisons among groups were made by one-way ANOVA. Kruskal–Wallis test was applied for continuous values with no normal distribution. The χ2 test was used for the comparison of non-continuous variables. Bonferroni post-hoc test was used for multiple comparisons between pairs of groups.Correlation analysis was performed using Pearson’s test or Spearman’s test where appropriate. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by using logistic regression.In all tests, a two-tailed probability value of 0.05 was considered statistically significantAll patients were white and 35 (51.5%) were females. Mean age was 11.98 ± 3.67 years (range: 4–18 years). Thirty-three different genotypes were recorded and the commonest were homozygous CD39, CD39/IVS-1,110, and homozygous IVS-1,110 . Each allele belonging to genotype was classified according to the corresponding phenotypic expression (β+ or β0) and patients were divided into three groups: homozygous β+ , compound heterozygous β0β+ , and homozygous β0 .The clinically relevant findings in the three groups are summarized in p = 0.258).The number of transfusional units in the 12 months before the MRI scan was available for 14 patients with homozygous β+ genotype , 18 patients with heterozygous β0β+ genotype , and 17 patients with homozygous β0 genotype  and, besides the trend, no association with the genotype was detected (No difference was found in the distribution of the different chelation regimens or in the compliance.Mean MRI LIC was 7.19 ± 8.79 mg/g dw and hepatic iron overload was detected in 44 (64.7%) patients. In the 71 patients in whom pancreatic T2* images were available, mean global pancreas T2* value was 19.82 ± 11.54 ms, and pancreatic iron overload had an incidence of 73.1%. Mean global heart T2* value was 33.75 ± 10.49 ms. All seven (10.3%) patients with myocardial iron overload had both hepatic and pancreatic iron overload. p < 0.0001) and inversely correlated with global pancreas T2* values  and global heart T2* values . No association between iron overload in the different organs and age at start of regular transfusions or chelation was detected. Mean serum ferritin levels were directly correlated with MRI LIC values , as well as global heart T2* values , and a positive correlation was detected between global pancreas and heart T2* values . MRI LIC values were inversely correlated with global pancreas T2* values   (p = 0.009) for patients with the heterozygous β0β+ genotype and 10.08  for patients with the homozygous β0 genotype versus patients with the homozygous β+ genotype.Global pancreas T2* values were significantly lower in the homozygous β0 group than in the homozygous β+ group (= 0.024) A. The pectively) B. The ORp = 0.048)  . In homoBiventricular function parameters were assessed in 63 patients, because for five patients a short MRI protocol was chosen to avoid sedation. There were not significant differences among groups in bi-atrial areas, biventricular volume indexes and ejection fractions, and LV mass index.Eleven patients completed the MRI protocol with acquisition of the LGE images and only one of them showed replacement myocardial fibrosis.No patient had liver fibrosis or cirrhosis.Diabetes mellitus was not diagnosed in any patient. Hypogonadism, hypothyroidism, and GH deficit developed in 5.9% of patients and prevalence of hypoparathyroidism was 4.4%. At least one endocrine complication was detected in 13 (19.1%) patients. Frequency of hypogonadism, hypothyroidism, hypoparathyroidism, GH deficit, and endocrinopathies globally considered was comparable among the three groups .No patient had heart failure. One patient had an ectopic atrial tachycardia which resulted in left ventricular dysfunction and required hospitalization and treatment with anti-arrhythmic drugs. This patient was a nine-year-old male with a homozygous β0 genotype and elevated iron levels .The knowledge of the molecular background of β-thalassemia can play a key role in the understanding of the factors affecting the diverse clinical manifestations. In the present study, we evaluated the impact of an underlying genetic defect on the development of disease complications in children with TDT. To the best of our knowledge, few studies, involving mainly patients from a limited area of Egypt, have explored this issue ,16. The We analyzed a representative sample of the Italian pediatric population with TDT, in which 33 different genotypes were identified. In line with previous studies on adult Italian patients ,47, the Patients were divided into three groups according to their genotype: homozygous β0, with two severe mutations and consequently a high alpha-non alpha globin chain imbalance, heterozygous β0β+, characterized by a combination of mild/severe mutations, and homozygous β+, with two mild mutations and a lower imbalance between alpha and beta globin chains.The three groups were homogeneous for age, sex, and hemato-chemical parameters. The transfusion demand tended to be lower in the homozygous β+ group than in the other two groups, but the difference was not statistically significant, likely because the datum was not available for all patients and the transfusion burden may change from center to center, depending also on red blood cell preparation and concentration. Moreover, it has been suggested that other factors  could contribute, more accurately than the genotype, to provide a basal evaluation of residual erythropoietic activity, and therefore, of the blood consumption . Other sMRI LIC values tended to be higher in the homozygous β0 group, but the difference among the group was not significant. Conversely, global heart T2* values were significantly lower in the homozygous β° group than in the homozygous β+ group. Liver and heart have not only different iron uptake mechanisms, resulting in a “delay” in cardiac iron overload, but also different rates of iron clearance . The capTo the best of our knowledge, this is the first study to show an association between genotype and levels of pancreatic iron overload. Likely, this link was not masked by the effects of the iron chelation therapy, since it seems extremely hard to remove iron from the pancreas . We founWe failed to detect a correlation between genotype and biventricular volumes and ejection fractions and LV mass index, likely because all patients had normal or near-normal values of these parameters.In our study population, no patient had liver fibrosis or cirrhosis and only one patient had hepatitis C virus (HCV) infection, confirming that in Italy the implemented measures to improve blood transfusion screening have significantly reduced the risk of getting an infection. Indeed, in an Italian study involving 1079 TDT patients with a mean age of 37.79 ± 10.11 years, only 37.1% had never contracted the HCV infection . In the The prevalence of the other endocrine complications in our pediatric population was hypogonadism, hypothyroidism and GH deficit—5.9%, and hypoparathyroidism—4.4%. This prevalence was consistent with some studies ,67, whilWe were not able to explore the association between genotype and cardiac complications, since no patient had heart failure and only a single patient had arrythmias. Of note, this patient had a homozygous β0 genotype, which, in a prospective multicenter study, emerged as a risk factor for the development of cardiac arrhythmias and complications globally considered .The main limitation of this study is the small sample size. Moreover, the previous complete transfusion history and chelation therapy, as potential determinants of the described pattern of iron organ distribution, were not available.In pediatric TDT patients there is an association between genotype and cardiac and pancreatic iron overload. Patients with homozygous β+ genotype had significantly lower myocardial iron levels than β0β0 patients and significantly lower pancreatic iron levels than β0β+ and β0β0 patients. So, the knowledge of the genotype can be valuable in predicting some patients’ phenotypic features and in helping the clinical and instrumental management of TDT patients.


More than 100 grapevine varieties are registered as suitable for wine production in “Douro” and “Trás-os-Montes” Protected Designations of Origin regions; however, only a few are actually used for winemaking. The identification of varieties cultivated in past times can be an important step to take advantage of all the potential of these regions grape biodiversity. The conservation of the vanishing genetic resources boosts greater product diversification, and it can be considered strategic in the valorisation of these wine regions. Hence, one goal of the present study was to prospect and characterise, through molecular markers, 310 plants of 11 old vineyards that constitute a broad representation of the grape genetic patrimony of “Douro” and “Trás-os-Montes” wine regions; 280 samples, grouped into 52 distinct known varieties, were identified through comparison of their genetic profiles generated via 6 nuclear SSR and 43 informative SNP loci amplification; the remaining 30 samples, accounting for 13 different genotypes, did not match with any profile in the consulted databases and were considered as new genotypes. This study also aimed at evaluating the population structure among the 65 non-redundant genotypes identified, which were grouped into two ancestral genetic groups. The mean probability of identity values of 0.072 and 0.510  were determined. Minor differences were observed between frequencies of chlorotypes A and D within the non-redundant genotypes studied. Twenty-seven pedigrees were confirmed and nine new trios were established. Ancestors of eight genotypes remain unknown. V. vinifera samples is the first prerequisite for the correct management of germplasm collections, selection of suitable parents for breeding programs, and clarification of synonyms, homonyms, and naming inaccuracies among grape varieties [The wine denominations “Douro”, the oldest demarcated and regulated winemaking region in the world, and “Trás-os-Montes”, represent approximately 29% of the Portuguese vineyard area for wine production  . These Parieties ,4,5,6.Identification of grape varieties has been generally based on the phenotypic traits of vegetative and reproductive structures, but morphological features are not sufficiently reliable for the classification of closely related varieties due to genotype–environment interactions . MoreoveV. vinifera [V. vinifera spp. sativa (cultivated grapevine), by analysing their distribution in local V. vinifera spp. sylvestris and sativa [SSR markers, because of their high degree of polymorphism, reproducibility, and co-dominant nature, have become the markers of choice for exchange of information concerning grapevine genetic resources . Severalvinifera . Chlorotd sativa , and to At the present time, SNP markers are considered one of the most powerful tools recently developed ,13,20. AThe sequence of the grapevine reference genome is available ,24 and cV. vinifera populations, to infer genetic structure, and to identify kinship relationships [V. vinifera subsp. vinifera and sylvestris) germplasm were also been undertaken, using the ICVV sets of 48 [Vitis International Variety Catalogue  includes SNP data of 112 markers for many varieties [These SNP markers are used to investigate genetic variability, discriminating among wild and cultivated ionships ,36,37,38ts of 48 . Currentarieties .VIVC database and the ICVV-SNP database, respectively; (ii) evaluation of genetic diversity and relationships among the grape genotypes detected, through SSR and SNP (240 SNP) markers; (iii) determination of chlorotypes and their frequencies through cpSSR and SNP loci amplification; and (iv) determination of first-order kinship relationships among grape varieties using the 240-SNP set.As a result of changes in worldwide wine consumption and European Union incentives , radicalA total of 310 grapevines were sampled in traditional vineyards throughout “Douro” and “Trás-os-Montes” PDO regions . These vSamples were characterised using molecular markers, namely Simple Sequence Repeats (SSR) and Single Nucleotide Polymorphisms (SNP), starting by varietal identification and followed by population structure, genetic diversity, and pedigree analyses with non-redundant genotypes.All samples were initially analysed with six nuclear microsatellites  and thrAn SNP array was first proposed as an alternative to SSR for varietal identification by Cabezas et al. . These aNon-redundant genotypes for the 48-SNP set were then genotyped with 192 additional SNP loci for subsequent analyses of population structure, genetic diversity, and parentage relationships. However, only 226 out of 240 SNP markers were informative; 14 SNPs were discarded, because data produced by 11 SNP were missing in at least 61% of the samples, and three SNP were monomorphic in the genotypes analysed.VIVC and ICVV-SNP databases, respectively. The VIVC database includes 5424 genetic profiles [VIVC data, the varieties ‘Hebén’, ‘Mouratón’, ‘Montua’, ‘Tempranillo’, ‘Palomino‘, ’Parraleta’, ‘Grec Rouge’, and ‘Trousseau Noir’ are synonyms of the Portuguese varieties ‘Mourisco Branco’, ‘Tinta Gorda’, ‘Diagalves’, ‘Aragonez’, ‘Malvasia Rei’, ’Tinta Caiada’, ‘Rabigato Francês’, and ‘Bastardo’, respectively , ‘Mouratón’ and ‘Síria’ (17), ‘Roseira’ (16), ‘Marufo’ (13), ‘Tempranillo’ (13), ‘Camarate Tinto’, ‘Carrega Branco’, and ‘Malvasia Preta’ (11), and ‘Trousseau Noir’ (10). These most commonly found genotypes across the sampled old vineyards may reflect their relevance in the past century.Sixteen identified genotypes  and eight non-identified genotypes  were found only once in the study . The selection of plants was based on the difficulty of their morphological identification by ampelographers, where only 31 out of 310 samples were previously named but mainly with local names . MicrosaMoreover, ‘Mourisco’ and ‘Bastardinho’ samples share the same nSSR and SNP profiles, but were revealed to be the ‘Mouratón’ (syn. ‘Tinta Gorda’) genotype described by Cunha et al. . ‘MourisVIVC and ICVV-SNP databases. This fact led to the conclusion that the two first samples were incorrectly named, but further analysis of the other two samples is needed to clarify if they are a situation of naming inaccuracy. Nevertheless, all four samples were considered as new genotypes , one to a table variety (1.9%), 14 to varieties with a double wine/table use (26.9%), and one to a variety with double table/raisin use 1.9%; .There are 343 authorised varieties for wine production in Portugal . All varAnother fact to take into account was that seven red varieties  and five white  listed as suitable for production of PDO wines are described as late-maturing ones ,52. HencV. vinifera germplasm diversity studies assessed with SSR markers. An analysis of 51 varieties using the six OIV loci described a range of 7 (VVMD27) to 11 (VVS2) alleles, with an average of 8.2 [SNP and SSR profiles were compared to assess the genetic diversity of the 65 non-redundant genotypes and these results are summarised in e of 8.2 . The nuce of 8.2  showed ae of 8.2 , also usThe allele size varied between 132 (VVS2) and 263 bp (VVMD7). VVMD7-239 and ssrVrZAG62-191 alleles were the most frequent, with over 43% of frequency. On the other hand, 25 alleles (43.86%) showed a frequency lower than 5%, eight of them being unique alleles . SpecifiV. vinifera spp. sativa germplasm studied by Emanuelli et al. [Allele frequencies and genetic parameters were also determined for the 226 SNP set . The mini et al.  (MAF = 0Ne) differed from 3.928 (VVMD7) to 6.995 (VVMD5), and an average number of 4.962 effective alleles was obtained for nSSR markers, similar to the mean Ne value (4.658) attained by Castro et al. [Ne ranged from 1.016 (SNP625_278) to 1.999 (SNP1327_56), with an average of 1.604, which is consistent with other reports on the genetic diversity of cultivated grape varieties .Ho) varied between 0.785 (VVMD7 and VrZAG62) and 0.954 (VVMD5); the lowest expected heterozygosity (He) was detected at VVMD7 locus with 0.745 and the highest one at VVS2 locus with 0.820 (Ho = 0.859 and He = 0.790) was similar to that observed for other sets of grape varieties analysed with SSR markers (Ho = 0.833 and He = 0.767 in [Ho = 0.833 and He = 0.769 in [p < 0.05) from HWE at the six SSR loci analysed. Differences between SNPs and SSRs were also observed with respect to heterozygosity. The observed heterozygosity  and expected (He = 0.351) heterozygosity values than SSRs (p < 0.05) between Ho and He values was found. However, deviation from the HWE (p < 0.05) was observed for 13.27% of the SNP markers; for 23 SNPs (10.18%), Ho was significantly higher than He, whereas Ho was significantly lower than He in the remaining seven SNPs (3.09%).As expected, SSR loci, due to the SSR multiallelic nature and high level of polymorphism, exhibited a significantly higher heterozygosity than biallelic SNP loci. This trend was observed by Emanuelli et al. . On averhan SSRs . For 196PIC values for each of the six nSSR and 226 SNP loci. VVMD7 and VVMD5 markers displayed the minimum (0.736) and maximum (0.844) PIC values, respectively  and 0.492 . PIC vaNP locus  or by co−7) was considerably higher than that determined for the 226 SNP set  used for the varietal identification would give a similar identification power as 14 microsatellites. The global probability of identity (PI) obtained for the six SSR set (1.0 × 10chl) of the 65 non-redundant genotypes was determined using ccmp3, ccmp5, and ccmp10 loci, which were considered the three most polymorphic chloroplastidial microsatellite (cpSSR) loci in grapevine [The chlorotype , but a high percentage of chl D was also detected  could be a consequence of crosses between non-Iberian introduced varieties and Portuguese native germplasm. Chlorotypes of ‘Carrega Branco’, ‘Nevoeira’, ‘Roseira’, and the 13 new genotypes identified were determined for the first time in this work, as no previous references were found in the literature or databases used. Most of these genotypes bear chl D, except ‘Nevoeira’ and ‘NG013’  [The genetic stratification in the set of 65 non-redundant grape genotypes was tested through a STRUCTURE analysis, using 226 SNP profiles. The delta ion (ΔK)  suggesteq-value) threshold of 0.7 for genetic group assignment was considered. Twenty (30.77%) and 18 (27.69%) genotypes were assigned to SNP-group 1 and SNP-group 2, respectively. The percentage of admixed genotypes was 41.54%  was performed to infer the distribution of genetic relationships among structure groups as revealed by 226 SNP loci A. The fiAn Unweighted Pair Group Method with Arithmetic Mean (UPGMA) distance tree was constructed to investigate the genetic relationship among the 65 non-redundant genotypes from genetic distance matrices  and duos (parent—offspring pairs) involving genotypes analysed and the corresponding LOD values and the number of mismatching loci are presented in Pedigree results revealed 36 compatible trios, with high LOD values, ranging from 52.20 to 101.40, using a maximum of two mismatching loci as threshold, with the confirmation of 27 already reported trios see .Several grape varieties have been previously reported to have an important role in the establishment of local genetic networks, such as ‘Hebén’, ‘Alfrocheiro’, and ‘Marufo’ in the Iberian Peninsula ,59,60. Ichl D) and ‘Hebén’ (chl A) need to cross-pollinate to produce descendants, a process that increases genetic diversity and increases hybrid plant vigour, which could have favoured their selection as seed donors by early farmers to ensure grape production [Unlike hermaphrodite grape varieties, female progenitors, such as ‘Marufo’ (oduction .chl A), a variety classified by Lacerda Lobo [chl D as the female progenitor ‘Marufo’ and obtained from hybridisation events between the referred variety and seven different Portuguese (six) and French (one) varieties (chl A), an old variety from Douro wine region. ‘NG006’, ‘NG007’, ‘NG008’, and ‘NG003’ resulted from the cross between ‘Marufo’ and, respectively, ‘Tinto Cão’, ‘Camarate Tinto’, ‘Folha de Figueira’, and ‘Trousseau Noir’ . Moreover, two new pedigrees were proposed as a result of crosses between ‘Marufo’ and ‘Trincadeira’ (‘NG009’ and ‘NG011’ as progenies). The participation of ‘Marufo’ and ‘Vinhão’ (chl A) as genitors of ‘Tinta Mesquita’ and ‘NG012’ was also observed  was found to be a parent in eight trios previously described, two of them together with ‘Hebén’ (chl A), and six with ‘Cayetana Blanca’  [‘Alfrocheiro’ (h chl A) ,59,60.q-value > 0.7 for group assignment) could be explained by parentage analysis. For example, both ‘Touriga Fêmea’ and ‘NG007’ were determined as admixture genotypes; ‘Touriga Fêmea’ is a progeny derived from ‘Malvasia Fina’ and ‘Touriga Nacional’ , whereas ‘NG007’ is a reliable result of a cross between ‘Marufo’ and ‘Camarate Tinto’  were considered. Five reliable duos were detected and summarised in However, no reliable trios or duos within the ICVV-SNP database were found for other genotypes, mainly the Iberian ones: ‘Tamarez’, ‘Tinta Francisca’, ‘NG005’, and ‘NG013’. Since the ICVV-SNP database includes a high number of Iberian profiles and even so no parentage relationships were established, most likely progenitors of the referred genotypes are extinct or close to. Their extinction, as proposed by Cunha et al. , may be V. vinifera in “Douro” and “Trás-os-Montes” PDO regions, 310 plants were sampled across 11 different old mixed variety vineyards, all predating the 1970s  agarose gel electrophoresis containing Gel-GreenTM Nucleic Acid Gel Stain 0.5x  and stored at −20 °C until use. The final concentration was confirmed using a NanoDrop® ND-1000 UV-Vis spectrophotometer .Genomic DNA was extracted according to Doyle & Doyle , with soVitis characterisation—VVS2, VVMD5, VVMD7, VVMD27, ssrVrZAG62, and ssrVrZAG79. The forward primer of each pair was fluorescently labelled with 6-FAM (VVMD5 and VVMD27), VIC (VVMD7 and ssrVrZAG62), or NED (VVS2 and ssrVrZAG79). Two multiplex PCRs (A and B) were carried out as previously described by Castro et al. [Samples were analysed with the set of six nuclear microsatellite (nSSR) loci recommended by OIV  for Vitio et al. , with 0.The chlorotype was determined using the three chloroplastidial microsatellite (cpSSR) loci designed by Weising & Gardner : ccmp3, w/v) containing Gel-GreenTM Nucleic Acid Gel Stain 0.5x (Biotium). Fluorescently labelled cp and nSSR products were separated by capillary electrophoresis using the ABI PRISM® 3130 automated sequencer  and GeneScanTM 500 LIZ®  as the internal lane size standard. Data produced were analysed by Peak Scanner v1.0 software . The sizes of the amplicons were scored in base pairs (bp) based on the relative migration of the internal size standard.All PCR products were visualised by electrophoresis on 2.0% agarose gels , and samples that produced nSSR profiles that did not match with those of the VIVC database were also genotyped for a set of 240 SNP markers previously identified by Lijavetzky et al. [DNA samples from non-redundant genetic profiles  of alleles. For SNP data, numerical values were assigned to each nucleotide .Non-redundant grapevine genotypes with genetic profiles for 6 nSSR and 48 SNP loci were used for grape variety identification. Genetic profiles for 240 SNP loci were used for population structure and genetic diversity analyses.Na), the average number of effective alleles (Ne), observed heterozygosity (Ho) and gene diversity or expected heterozygosity (He) were calculated through the GenAlEx software (version 6.5) [To compare SSR and SNP results, genetic parameters of polymorphism, such as the average number of different alleles per locus (ion 6.5) . They wePIC was calculated as follows: PIC = He − (2 × MAF2 × (1-MAF)2), where MAF was the minor allele frequency.Polymorphism information content (PIC)  of each q-value). Structure analyses were performed using the STRUCTURE software (version 2.3.4) ,73,74,75To assess the relationship among the non-redundant genotypes, the pairwise genetic distance matrix was computed based on SNP data, through the ‘Genetic Distance’ procedure in the GenAlEx software (version 6.5) , for subData from the 240 SNP set were also used to identify possible first-order kinship relationships—trios (mother–father–offspring) and duos (possible parent–offspring pairs)—among the non-redundant grape genotypes in the study, since their SNP profiles merged with those of the ICVV-SNP database. The likelihood-based method in the CERVUS software was used (version 3.0) . The likSSR and SNP analyses were very useful in the identification and characterisation of the plants analysed and overcame the ampelography difficulties in grapevine prospections to contribute to the ultimate goal of conserving grape varietal legacy in Northeast Portugal. Further morphological, agronomical, and oenological analyses are being undertaken to complement the molecular data reported in this study.In the present work, 280 plants in the “Douro” and “Trás-os-Montes” PDO regions were identified and grouped into 52 different grape varieties. Thirteen additional unique genotypes were also detected from the study of other 30 vines, which were clustered, along with cv. Marufo (mother of the majority of these new genotypes), in an exclusive independent cluster, accordingly to UPGMA data. Some of these 13 new genotypes could be considered minor neglected Portuguese grape varieties, while others, found only in plants belonging to a single local vine grower still using traditional techniques for grapevine propagation, are probably individual grapevines that emerged as seedlings and can be the initial steps of the establishing of local grape varieties as it occurred in the past. Altogether, their discovery highlights once more the huge patrimony of the Portuguese grape germplasm. The aforementioned observations emphasise the constant demand for prospection and identification of old grape material in traditional vineyards, to enlarge the knowledge about the still existing varietal diversity for its conservation, characterisation, and eventual exploitation. The broader the knowledge, the greater the chances to overcome biotic and abiotic stresses affecting the vineyards today.


Altogether, these results will be useful for uncovering the molecular mechanisms of flavonoid biosynthesis and subsequently accelerating quality pecan breeding.Flavonoids influence the flavor and nutritional value of pecan nuts. However, limited information is available regarding the molecular mechanisms underlying pecan flavonoid biosynthesis. Here, we used a high (“YLC28”) and a low (“Oconee”) flavonoid content cultivar as the research objects. The changes in flavonoid content and the gene transcription patterns during kernel development were identified. Different accumulation patterns of total flavonoids (TF) and condensed tannins (CT) were observed between the two cultivars. The contents of TF and CT in “YLC28” were 1.76- and 2.67-fold higher levels than that of “Oconee” on 150 days after full bloom of female flowers, respectively. In total, 30 RNA-Seq libraries were constructed and sequenced. The upregulated genes in “YLC28” were highly enriched in flavonoid-related pathways. Thirty-three structural genes were identified, and the expression of two  Carya illinoinensis (Wangenh.) K. Koch] is native to North America, belongs to the Juglandaceae family and is an important nut crop throughout the world   . To datees (C4H) . Howeveres (C4H) .Camellia sinensis (Zanthoxylum bungeanum (Carya illinoinensis CHSs (CiCHSs) were isolated, and two of them exhibited significant correlations with the total phenolic content of pecan  and a high flavonoid content cultivar (“YLC28”). The content variation of total flavonoids, condensed tannins, quercetin, and apigenin along with embryo development were determined. The gene expression profiles during kernel maturation of the two cultivars were identified by using RNA-Seq technology. Structural genes and transcription factors associated with flavonoid biosynthesis were obtained. The investigation of molecular mechanisms behind phytochemical accumulation based on comparative transcriptome was rarely performed in pecan. We believe that the findings will deepen our understanding of flavonoid accumulation in pecan kernels and provide ideas for the study of molecular mechanisms underlying other phytochemical biosynthesis in this plant. Subsequently, the present study will facilitate breeding research in pecan.The pecan kernels were sampled at approximately 100, 107, 114, 121, 128, 135, 142, and 150 days after the full bloom of female flowers  from “YLC28”  and “Oconee”  trees in Jiande , China. The trees were 11-years old. The kernels were frozen in liquid nitrogen and stored at −80°C for further analysis.The plant materials were dried in a freeze dryer  for 96 h. Then, the kernels were finely ground in a tissue blender . The powder of each sample was used to detect the contents of major classes of flavonoids, such as total flavonoids, condensed tannins, quercetin, and apigenin.2. Then, 0.3 mL 0.1 g/mL Al(NO3)3 was added, incubated for six min before mixture with 3 mL 0.04 g/mL NaOH, and added distilled water to make the total volume up to 10 mL. After incubating for 15 min, the absorbance was detected at 510 nm using a spectrophotometer. The content of TF was expressed as rutin equivalent (RE).The total flavonoids were extracted using an ethanol reflux method . 0.5 g pThe CT content was detected using a vanillin assay . 1.0 g pThe standards of quercetin (Qu) and apigenin (Ap) were obtained from Sigma-Aldrich . The pecan kernel powder (0.5 g) was weighed, mixed with 1.5 mL of extracting solution and incubated at 80°C for 90 min, then extracted in an ultrasonic bath for 60 min. The mixtures were centrifuged (4°) at 10,000 r/min for 10 min and filtered through a 0.45 μm filter membrane. Finally, the extractives were dried under nitrogen flow, dissolved in 1000 μL methanol, and filtered (0.22 μm). HPLC was performed on a Rigol L3000 HPLC system  with a Kromasil C18 column  at 35°C. The mobile phases were methanol/water/phosphoric acid (60/40/0.08). The flow rate was 0.8 mL/min. The detection wavelength was 360 nm. The contents of quercetin and apigenin were calculated using regression equations.To identify the molecular mechanism that regulates the different flavonoid contents between “YLC28” and “Oconee,” RNA-Seq technology was employed. Based on the trends of TF and CT content variation, five different developmental stages  of kernels from the two cultivars were used as plant materials. The number of “YLC28”  samples ranged from H100 (100 DAFB) to H150 (150 DAFB). Similarly, the number of “Oconee” samples  ranged from L100 to L150. Total RNA was extracted using a TRIzol reagent kit  according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer  and assessed using RNase-free agarose gel electrophoresis. Library construction was performed as described previously. Next, the 30 separately constructed libraries (five stages with three bioreplicates of two cultivars) were sequenced using Illumina HiSeq 2500 by Gene Denovo Biotechnology Co. .Q-value ≤ 20) higher than 50% were removed. An index of the reference genome was built (1) term annotation for each gene was performed by Blast2GO software2 using default parameters, taking FDR ≤ 0.05 as a threshold. All the sequences were aligned to the KEGG database,3 and PlantTFdb database4 by BlastX .Data quality control, clean read assembly and gene abundance calculation were performed as described in a previous study . The rawas built , and paias built . The mapas built . To idenas built  was empl5 The DEGs associated with “flavonoid biosynthesis” and “phenylalanine metabolism” pathways were selected and mainly analyzed.A differential expression gene (DEG) analysis was performed by using DESeq2 software . DEGs we6 A co-expression analysis was displayed by Cytoscape 3.9.0 . Here, 18s RNA served as the reference gene , and then 223.34 Gb clean data were retained after removing low-quality sequences and adaptors . The aveDifferential gene expression analysis was performed between the two cultivars, and along with the kernel development of each genotype. A gene temporal expression pattern analysis was carried out using a STEM software. All the DEGs in ten comparison groups, including L114 vs. L100, L121 vs. L100, L135 vs. L100, L150 vs. L100, L121 vs. L114, L135 vs. L114, L150 vs. L114, L135 vs. L121, L150 vs. L121, and L150 vs. L135, were used to generate temporal expression profiles in “Oconee” . Similar11 , four CHSs, two CHIs, one flavanone 3-hydroxylase (F3H), one flavonoid 3’-hydroxylase (F3’H), three flavonoid 3’,5’-hydroxylases (F3’5’Hs), two DFRs, three leucoanthocyanidin reductases (LARs), one anthocyanidin synthase (ANS), one anthocyanidin reductase (ANR), and one flavonol synthase (FLS). The expression trends of these genes were dramatically different between the two genotypes. In “Oconee,” most of these genes exhibited relatively high expression levels at the first (100 DAFB) and fifth (150 DAFB) developmental stages. In contrast, these genes were upregulated from the first to the third stages in “YLC28.” The transcription levels of three PALs , four C4Hs , three 4CLs , one CHS (CIL0006S0011), two F3’5’Hs (MSTRG.25379 and MSTRG.27951), and one FLS (CIL1060S0040) in the second stage (114 DAFB) of “YLC28” were significantly increased compared with those in “Oconee” at the same stage. One 4CL (CIL1192S0044), two CHSs (CIL1399S0001 and CIL1549S0032), one CHI (CIL0301S0006), one F3H (CIL1020S0081), one F3’H (CIL0909S0093), and one DFR (CIL0203S0007) gene were upregulated at stage three (121 DAFB) in “YLC28” compared with “Oconee.” The contents of CT and TF in “YLC28” were significantly increased compared with those in “Oconee” at 114 and 121 DAFB, respectively. The high expression of these structural genes might be important for abundant flavonoid accumulation in “YLC28” kernels. In addition, one 4CL (CIL0937S0156), one CHI (CIL1546S0028), one LAR (CIL1032S0007) and one ANR (CIL1099S0044) gene exhibited increased transcription levels in the third and fourth (135 DAFB) stages in “YLC28” compared with “Oconee.” Similarly, the high expression of structural genes in organs rich in flavonoids has also been reported in potato  one F3’H (CIL0909S0093) and one FLS (CIL1060S0040) exhibited a significant positive correlation  with the contents of TF, CT, Ap, and Qu. In “YLC28,” one, ten and one genes  exhibited positive correlations with the TF, CT, and Ap, respectively. Among them, two PALs (CIL0945S0098 and CIL0945S0099), one CHS (CIL0006S0011), one F3’5’H (MSTRG.27951), and one FLS (CIL1060S0040) exhibited high correlation  with the CT content in “YLC28.” They might play crucial roles in the CT biosynthesis in “YLC28.” The content of CT can affect the flavor and palatability of pecan nut . Similar results were reported in strawberry  with each other , ERF (74), MYB (70), NAC (54), and MYB_related (45) were the main categories . R2R3-MYbidopsis . AtMYB3,bidopsis . As showMYBs was illustrated in PtrMYB57 led to reduced or increased anthocyanin and CT contents, respectively  was observed, indicating that the transcriptome data were reliable.To verify the RNA-Seq data, the expression of fourteen randomly selected genes was analyzed using qRT-PCR. The qRT-PCR results of most genes were consistent with the RNA-Seq data . Then, aPALs, one CHS, one F3’5’H, and one FLS were significantly correlated with the CT content changes in “YLC28.” A putative subgroup 4 MYB transcription factor (CIL1093S0100) was identified, which might act as a flavonoid biosynthesis repressor during kernel development. The present study will be useful for accelerating the study of the molecular basis of flavonoid biosynthesis in pecan kernels.Flavonoids are key nutrition components for pecan nuts. However, the molecular mechanisms of flavonoid biosynthesis in pecan kernels have not been fully elucidated. Here, the flavonoid content changes and gene expression patterns during embryo development in “YLC28” and “Oconee” were detected. The contents changing patterns of TF and CT were significantly different between the two cultivars during kernel development. RNA-Seq results indicated that the upregulated genes in “YLC28” were significantly enriched in flavonoid biosynthesis pathways. Thirty-three differentially expressed flavonoid-related structural genes were obtained. The expression profiles of two PRJNA792564.The raw data have been deposited in the NCBI database with the bioproject accession number CZ, XY, and HR conceptualized the project. CZ performed the experiments, did the data analysis, and wrote the manuscript. KW and JC helped CZ to did the data analysis. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Desmophyllum dianthus occurs in high densities, in spite of low pH and aragonite saturation. If and how these conditions affect the energy demand of the corals is so far unknown. In a laboratory experiment, we investigated the carbon and nitrogen  budget of D. dianthus from Comau Fjord under three feeding scenarios: (1) live fjord zooplankton , (2) live fjord zooplankton plus krill (>7 mm), and (3) four-day food deprivation. In closed incubations, C and N budgets were derived from the difference between C and N uptake during feeding and subsequent C and N loss through respiration, ammonium excretion, release of particulate organic carbon and nitrogen . Additional feeding with krill significantly increased coral respiration (35%), excretion (131%), and POC release (67%) compared to feeding on zooplankton only. Nevertheless, the higher C and N losses were overcompensated by the threefold higher C and N uptake, indicating a high assimilation and growth efficiency for the krill plus zooplankton diet. In contrast, short food deprivation caused a substantial reduction in respiration (59%), excretion (54%), release of POC (73%) and PON (87%) compared to feeding on zooplankton, suggesting a high potential to acclimatize to food scarcity . Notwithstanding, unfed corals ‘lost’ 2% of their tissue-C and 1.2% of their tissue-N per day in terms of metabolism and released particulate organic matter (likely mucus). To balance the C (N) losses, each D. dianthus polyp has to consume around 700 (400) zooplankters per day. The capture of a single, large krill individual, however, provides enough C and N to compensate daily C and N losses and grow tissue reserves, suggesting that krill plays an important nutritional role for the fjord corals. Efficient krill and zooplankton capture, as well as dietary and metabolic flexibility, may enable D. dianthus to thrive under adverse environmental conditions in its fjord habitat; however, it is not known how combined anthropogenic warming, acidification and eutrophication jeopardize the energy balance of this important habitat-building species.In the North Patagonian fjord region, the cold-water coral (CWC)  Corals are ecosystem engineers , formingDesmophyllum dianthus thrives under low pH (7.4–8.4), near and below aragonite saturation  form one of the most structurally-complex habitats of the deep sea , but occ to 1.6; ). The lo to 1.6;  and terr to 1.6; . The nat to 1.6; .Desmophyllum dianthus is a solitary, ahermatypic (not reef-building) CWC species with a cosmopolitan distribution , while the basal end is bare and provides a settlement substrate (habitat) for various epibiontic and endolithic organisms, e.g., foraminiferans, bio-eroding sponges, and photoautotrophic microorganisms , such as copepods, but also larger micronekton, such as euphausiids (krill)  per day  . To fuel per day . Howevern budget . In the n budget . In expen budget , but it D. dianthus under present-day low-pH conditions in Comau Fjord, Chile, i.e., the difference between C and N uptake and C and N loss  budget of the CWC d N loss . CWCs red N loss  as partid N loss . The remd N loss . Since Cd N loss , we deted N loss : (1) livD. dianthus specimens . The corals were chiseled off the substrate by SCUBA divers and immediately placed into water-tight, sealed plastic containers, ensuring no contact with either the brackish surface water layer or air during transport to the laboratory. To remove epibiontic and endolithic organisms . Before the start of the experiment, corals were fed for two hours per day with 1/4 of a haul of live, freshly-collected fjord zooplankton (see next section). This corresponds to a zooplankton concentration of 571 ± 203 zooplankton individuals L−1 (mean ± standard deviation), equivalent to 85 ± 30 µmol C L−1 and 16 ± 6 µmol N L−1, as analyzed in four additional aliquots of 1/4 of a zooplankton haul . During feeding, water exchange was interrupted.For maintenance before and during the experiment, corals were kept in three 28 L-maintenance tanks (−3) with an analytical balance equipped with an underfloor weighing basket (Sartorius CPA225DOCE). Polyp dry mass was derived from buoyant weight according to −3  were collected at night in Comau Fjord between 20 and 80 m water depth, in a 45-min horizontal trawl at 2 knots with a ring trawl net . Krill were drained on paper tissue, measured , weighed (wet mass) and stored frozen (−13 °C) until utilization in the experiment (see below). Six euphausiids were dried , weighed again  and used for C and N analysis (see below).Krill  was subsequently offered a surplus of food, i.e., zooplankton plus krill , while batch II  remained unfed, mimicking food (zooplankton) shortage, e.g., in winter . Within each batch, at least two C and N budgets were determined separately for each individual coral, by repeated measurements on the same specimens  in separate experimental bottles for 2 h . Zoopla2 h  in separate experimental bottles with one coral each . The bottles were filled with 10 µm-filtered fjord water, pumped from 25 m depth off Huinay. The coral was fixed in its natural ‘upside-down’ position . The flow was high enough to kept live zooplankton in suspension, but low enough not to bend the coral tentacles  and the number of zooplankton individuals (n = 2) added to each experimental bottle (n = 3), but not for zooplankton. Start POC and PON concentrations in the feeding scenario ‘zooplankton’ were 164 ± 36 µmol C L−1 and 30 ± 7 µmol N L−1, start POC and PON concentrations in the feeding scenario ‘zooplankton+krill’ were 126 ± 0.3 µmol C L−1 and 24 ± 0.1 µmol N L−1. The start zooplankton concentration was 1,025 ± 332 zooplankters L−1 in the feeding scenario ‘zooplankton’ ; the start zooplankton concentration in the feeding scenario ‘zooplankton+krill’ was presumably similar. Per coral subset , due to logistical limitations (handling time). Due to the low activity in the seawater-only controls, we expect no significant bias related to the longer duration. End water samples for the amount of POC and PON (290–370 mL) and for the number of zooplankton individuals  were taken from each experimental bottle after thorough mixing , C and N fluxes on our results. At the start of the incubation, the start O2 concentration was measured with an optode  in a separate experimental bottle Bo (‘start O2’). Start water samples were taken by syringe from Bo for analysis of dissolved inorganic nitrogen , DOC, and DON . The remaining water in Bo (300–600 mL) was used for POC and PON analysis. During the incubation, coral polyps remained protruded with extended tentacles. At the end of the incubation, the corals were removed and returned to their maintenance tanks. In each experimental bottle, the end O2 concentration was first measured, before taking end water samples for DIN, DOC, DON, POC and PON. The end O2 concentration was never below 80% of the start O2 concentration, a conservative threshold to avoid effects of low-oxygen concentration on coral physiology (−1) to prevent microbial activity. Samples for DOC and DON were filtered through GF/F-filters into glass vials , acidified to pH = 2 with 32% hydrochloric acid to avoid microbial activity and stored dark at 4 °C. Sample processing for POC and PON was described in the previous section (‘Feeding’).For the incubations (without food), the corals were placed into the experimental (SCHOTT) bottles, that were filled with 10 µm-filtered fjord water and closed air bubble-free with a lid . Per corysiology . DIN samn = 6 aliquots per coral) were collected on pre-combusted , pre-weighed GF/F-filters and dried up to constant mass at 40 °C. Coral samples were transported to Germany under CITES permit E-00427/12.At the end of the experiment, the coral tissue was removed from the skeleton with an airbrush filled with 0.7 µm-filtered seawater and homogenized with an ultra turrax . The voli.e., <1% relative standard deviation for triplicate measurement of acetanilide). To measure the OC content of coral tissue (tissue-C), subsamples on GF/F filters  were vapor-acidified with 12 N-hydrochloric acid prior to analysis, to remove remainders of skeletal inorganic carbon (n = 3) without acidification. The total tissue-C and -N content of each coral was calculated by multiplying the OC and ON content measured in the subsamples (one mL) with the volume of the tissue-seawater suspension (see previous section). Samples of krill, POC and PON (from feeding and incubations) were not acidified, as previous tests revealed negligible amounts of inorganic carbon. POC concentration was determined as −1] = 3.9 wet mass [mg] + 48.3; R2 = 0.86) and the relation between wet mass and N content . Zooplankton samples were counted in a Bogorov chamber under a stereo microscope. Only undamaged, non-gelatinous zooplankters were counted.Samples of coral tissue, krill, POC and PON were weighed for dry mass and subsequently analyzed for organic carbon (OC) and organic nitrogen (ON) content on an elemental analyzer  with acetanilide calibration , nitrate and nitrite concentration was analyzed spectrophotometrically at the ICBM-Terramare Wilhelmshaven according to −1). DOC and total dissolved nitrogen (TDN) concentration was quantified via high-temperature catalytic oxidation  and seawater-control (‘control’) trials. The  C uptake of the corals from zooplankton  was determined as i.e., µmol C and N d−1), because the corals were fed once per day only. In treatment ‘zooplankton’, we additionally determined the zooplankton capture of the corals, as All C, N, and O2 h−1) were derived as 2] is the O2 concentration. The C respiration rate was derived from the O2 respiration rate, assuming a respiratory quotient CO2:O2 = 1; this quotient was measured for CWCs in situ  were obtained as i.e., by bacterioplankton passing the 10 µm-filter and/or by the coral microbiome . The term ‘scope for growth’  models were fitted, separately for coral batch I and II  as fixed effect ). In batch I, C and N fluxes directly  after feeding on zooplankton (‘zoopl’) were compared with C and N fluxes directly after feeding on zooplankton+krill (‘krill.d1’); and C and N fluxes directly after feeding on zooplankton+krill (‘krill.d1’) were compared with C and N fluxes one day later (‘krill.d2’). In batch II, C and N fluxes after feeding on zooplankton (‘zoopl’) were compared with C and N fluxes after four days of food deprivation (‘unfed’). Detailed results of the LME models are available as i.e., ‘zoopl’, ‘krill.d1’, ‘krill.d2’, ‘unfed’). The lines ‘C loss = C uptake’ and ‘N loss = N uptake’, i.e., ‘SfG = 0’, were interpreted as the minimum C and N demand of the corals to balance their C and N loss, associated with each feeding treatment.Graphical and statistical analysis was performed with R . Values I and II , with thI and II . The LMEEx situ experiments, as presented here, allow the measurement of the total C and N fluxes of a CWC, i.e., its C and N budget. This measurement proves difficult in situ, as it typically requires a closed-off water volume. Nevertheless, ex situ experiments only provide specific simulations of the dynamic fjord environment −1 d−1 −1 d−1).When fed with zooplankton (treatment ‘zooplankton’), C)−1 d−1  and a N  (−1 d−1 . One eupD. dianthus (D. dianthus respired on average 27.5 ± 5.8 µmol O2 (mmol tissue-C)−1 d−1 and excreted 3.2 ± 1.3 µmol NH4+ (mmol tissue-C)−1 d−1. When the corals received krill in addition to zooplankton, they showed an on average 35% higher respiration rate than when they were fed with zooplankton only −1 d−1 −1 d−1). Four days of food-deprivation reduced POC and PON release by 73% and 87% relative to the POC and PON release after feeding on zooplankton −1 d−1 in corals fed with zooplankton plus krill (‘krill.d1’), corresponding to 52.7 to 108.4 µmol C polyp−1 d−1. Accordingly, the minimum N demand of D. dianthus −1 d−1 in corals fed with zooplankton plus krill (krill.d1), corresponding to 3.6 to 23.8 µmol N polyp−1 d−1. This means that unfed corals lose 2% of their tissue-C per day to respiration and POC release and 1.2% of their tissue-N per day to ammonium excretion and PON release  than indicated here.Corals fed with zooplankton plus krill showed a positive scope for growth (SfG) for C and N within the first day after feeding . We first discuss the metabolic flexibility  of the corals in response to varying food availability. Then, we evaluate how the corals could sustain their C and N demand under the low-pH conditions in the fjord and speculate how their C and N balance might change in the future.In this study, we report C and N budgets of the CWC D. dianthus shows a high metabolic flexibility in response to varying food availability, indicated by increased respiration and ammonium excretion with increasing meal size −1 d−1) compared to the respiration rate of Mediterranean D. dianthus −1 d−1;  and temperature (12 °C).e energy . This farivation . Overall)−1 d−1; ), in spiD. dianthus could allow colonization of different habitats, from shallow areas of fjords with a high variability of food, temperature, salinity and pH, to the environmentally more stable, deep CWC habitats , due to the lower fecal loss in relation to the high C and N uptake , as feces egested from their gastrovascular cavity  and as c feeding , 5F suggN uptake .L. pertusa rapidly dissolved in seawater, hence, the DOC release of this CWC species was >30 times higher than its POC release. This high DOC:POC ratio was not confirmed for D. dianthus (present study); instead, rates of DOC release/uptake were highly variable −1 d−1; POC plus DOC) than its Mediterranean conspecifics −1 d−1, −1 d−1, ). Higher)−1 d−1, . In the d−1, −3, , and hen −3, −3; .e.g., from the extensive salmon farming in the Chilean fjords . Anthropogenically increased sedimentation, n fjords , could fD. dianthus often co-occurs with other suspension feeders, such as the bivalve Acesta patagonica and the sponge Mycale thielei  release, unfed corals have to expend 2% of their tissue-C and 1.2% of their tissue-N per day. With growing meal size, feeding-related costs and losses increase the total C and N loss to 4.4% of the tissue-C and 6.4% of the tissue-N. To grow somatic and reproductive tissue, the corals need additional resources. Conspecifics from the Mediterranean show an even higher C demand, due to their higher respiration rate , less-acidic conditions, and due to the lower POC release  high temperatures and low pH  zooplankton individuals (>100 µm) per day. As a voracious zooplankton predator, D. dianthus is capable to exploit high concentrations of zooplankton  losses, a medium-sized plankton , which mkton . We observed dense krill swarms directly next to the corals during dives by remotely-operated vehicle between 160 and 200 m depth (D. dianthus captured live krill at a similar rate (18% h−1) as small zooplankton  resilience to experimentally acidified conditions when their energy supply was high , a high food quality increases assimilation and growth efficiency of CWCs (present study) and hence their reserves to actively counteract adverse conditions  budget, the solitary coral needs to capture a minimum of 700 (or 400) zooplankton individuals per polyp and day, or one larger prey item such as one euphausiid. Under experimental food deprivation, the corals swiftly reduced all C and N loss terms, likely to conserve energy. We argue that the exploitation of zooplankton swarms and/or the consumption of krill, combined with a high metabolic flexibility, are important in sustaining the energetic requirements of D. dianthus in Comau Fjord under naturally low pH. The bulk of the population, however, thrives in deeper waters under even lower pH, near or below aragonite saturation (in situ, along the pH depth gradient. Climate change, ocean acidification and the intense salmon aquaculture in the Patagonian fjord region likely impact the energetic balance of D. dianthus. The species appears particularly sensitive to a combination of stressors, such as high temperatures and acidification (The CWC turation , which mturation ; this mafication  or hypoxfication . A disru10.7717/peerj.12609/supp-1Supplemental Information 1Click here for additional data file.


Murine Double Minute Clone 2, located at 12q15, is an oncogene that codes for an oncoprotein of which the association with p53 was discovered 30 years ago. The most important function of MDM2 is to control p53 activity; it is in fact the best documented negative regulator of p53. Mutations of the tumor suppressor gene p53 represent the most frequent genetic change in human cancers. By overexpressing MDM2, cancer cells have another means to block p53. The sarcomas in which MDM2 amplification is a hallmark are well-differentiated liposarcoma/atypical lipomatous tumor, dedifferentiated liposarcoma, intimal sarcoma, and low-grade osteosarcoma. The purpose of this review is to summarize the typical clinical, histopathological, immunohistochemical, and genetic features of these tumors. Murine Double Minute Clone 2 is an oncogene, the function of which was first described in DNA associated with paired acentric chromatin bodies, termed double minutes, harbored in spontaneously transformed mouse 3T3 fibroblasts .,16.MDM2.DNA FISH . An impofeatures .MDM2 amplification does not entirely preclude the diagnosis of ALT/WDL. In Li–Fraumeni syndrome, the occurrence of p53+/MDM2− ALT/WDL can be an early expression of the disease . Th. ThMDM2 MDM2 in sarcomas gives us insight into ‘M’olecular mechanisms. It has created a ‘D’iagnostic tool, FISH being more sensitive than immunohistochemistry. MDM2-amplified sarcomas ‘M’ay be druggable in the future. Finally, it helps us ‘TO’ sort out part of the numerous and complex sarcomas.In conclusion, the story of


Under ambient conditions, the only known valence state of calcium ions is +2, and the corresponding crystals with calcium ions are insulating and nonferromagnetic. Here, using cryo-electron microscopy, we report direct observation of two-dimensional (2D) CaCl crystals on reduced graphene oxide (rGO) membranes, in which the calcium ions are only monovalent (i.e. +1). Remarkably, metallic rather than insulating properties are displayed by those CaCl crystals. More interestingly, room-temperature ferromagnetism, graphene–CaCl heterojunction, coexistence of piezoelectricity-like property and metallicity, as well as the distinct hydrogen storage and release capability of the CaCl crystals in rGO membranes are experimentally demonstrated. We note that such CaCl crystals are obtained by simply incubating rGO membranes in salt solutions below the saturated concentration, under ambient conditions. Theoretical studies suggest that the formation of those abnormal crystals is attributed to the strong cation-π interactions of the Ca cations with the aromatic rings in the graphene surfaces. The findings highlight the realistic potential applications of such abnormal CaCl material with unusual electronic properties in designing novel transistors and magnetic devices, hydrogen storage, catalyzers, high-performance conducting electrodes and sensors, with a size down to atomic scale. Counter to conventional wisdom, two-dimensional CaCl crystals with +1 calcium ions have been observed, which show unexpected metallicity, room-temperature ferromagnetism, heterojunction, piezoelectricity-like property, and distinct hydrogen storage and release capability. Calcium ions are present in rocks, bones, shells, biominerals, geological deposits, ocean sediments and many other important materials . Calcium60 [Since the discovery of C60 , carbon 60 , carbon 60 . The cry60 . Here, u2 solution below the saturated concentration under ambient conditions. This is quite similar to preparation of 2D Na–Cl crystals with Na2Cl and Na3Cl crystals in the rGO membranes in NaCl solution below saturated concentration [2 solution under protection of argon atmosphere conditions, and were incubated in 5.0 mol/L (M) CaCl2 solution overnight under ambient conditions. Preparation of Ca–Cl@rGO membranes of such thinness, corresponding to only several layers of the graphene sheets, allows clear characterization of the 2D Ca–Cl crystal structures and minimization of the unfavorable effects of inhomogeneous frozen rates on the membranes during the freezing process.2D Ca–Cl crystals were obtained by soaking ultra-thin rGO membranes (thickness <10 nm) in CaClntration , but thein situ formation [−1. Further, double-orientated crystals with the same lattice structure of the single Ca–Cl crystal were also observed both on TEM diffraction patterns and high-resolution images . In Figs −1, too, which correspond to the (1−100) reflections of the graphene. Notably, the (2−200) reflections of the Ca–Cl crystals are coincident with these (1−100) reflections of graphene . The hexagonal (2−200) reflections of the Ca–Cl crystals are also consistent with the (1−100) reflections of the graphene sheets, demonstrating that the structure of the Ca–Cl crystals in the dried Ca–Cl@rGO membranes is consistent with the structure of the Ca–Cl crystals observed above in the ultra-thin Ca–Cl@rGO membranes.Then, we measured the atomic ratio of Ca to Cl and the properties of this Ca–Cl crystal. The ultra-thin Ca–Cl@rGO membranes, which include only several layers of graphene sheets, are too thin to perform elemental analysis and it is too difficult to perform measurements of the electrical, magnetic and hydrogen storage properties. We fabricated thick freestanding rGO membranes (thickness >10 μm) ,16,17 fr2 crystals, there are some stable structures with a Ca : Cl ratio of ∼1 : 1, which we denoted as CaCl. To further investigate the stoichiometries of Ca to Cl in the membranes, we performed XPS elemental analysis by etching the membrane with argon ions. The depth profile shows that the Ca : Cl ratios vary from a value below 0.6 : 1 at the membrane top surface to a stable ∼1.2 : 1 in the inner sheets  TEM energy-dispersive X-ray spectroscopy (EDS) analysis and X-ray photoelectron spectroscopy (XPS) . The dark-field high-angle annular dark field scanning TEM (HADDF-STEM) image shows typical distributions of the Ca–Cl crystals in the membranes . Interestingly, the corresponding TEM EDS analysis  shows that, in addition to the formation of regular CaCl2, Ca3Cl4, Ca3Cl2 , on the graphene surface or confined between graphene sheets.To discuss the possible structures of the crystals, we used density-functional theory (DFT) ,19 combiThese CaCl crystals with unusual electronic structure of +1 calcium ions exhibit revolutionary new properties for the crystals with calcium ions, such as metallicity, ferromagnetism, heterojunction, coexistence of piezoelectricity-like property and metallicity, and adsorption capacity with hydrogen storage. The projected electronic band structure and density of states (DOS) of model I shows that CaCl crystals have distinct metallicity in which Ca plays a dominant role Fig. a. The co2,3-edge peaks for regular CaCl2 at 347.82 and 351.12 eV, there are new Ca L2,3-edge peaks at 348.02 and 351.22 eV  membrane is larger than both that of pure calcium metal (∼347.92 eV) and of regular CaCl2 crystals (∼347.82 eV) . To further verify this phenomenon of valence state change, we performed NEXAFS experiments on the dried Cu–Cl@rGO membrane, which was prepared by incubating the rGO membranes with CuCl2 solutions. The L2,3-edge peaks for Cu in the dried Cu–Cl@rGO membrane are consistent with the L2,3-edge peaks in the regular CuCl crystal, demonstrating that the valence state of Cu has been changed from +2 to +1 in the dried Cu–Cl@rGO membrane .Abnormal valence state change of the CaCl crystals was analyzed and confirmed by soft X-ray absorption spectroscopy . In addition to the L−2 MΩ·cm, indicating their good conductivity. We note that our result on rGO membranes is consistent with metallic properties reported in the literature [Metallic properties of CaCl crystals were confirmed experimentally by measuring the electrical resistivity . Figure terature . In contsM) of the dried CaCl@rGO membrane is ∼2.1 emu/g at 300 K, whereas sM = ∼0.4 emu/g for the rGO membrane at the same temperature, showing a ∼400% enhancement of magnetism by introducing only ∼5%  calcium to the membrane. For a system with periodic model I configuration, Stoner instability analysis and polarized DFT calculations [+, and the saturation magnetic moment computed is consistent with the experimental observations .Room-temperature ferromagnetism of the CaCl crystals in rGO membrane was also experimentally observed. Figure ulations  do not sA graphene–CaCl heterojunction was experimentally demonstrated. The current–voltage curve of the dried Ca–Cl@rGO junction membrane under positive and negative gate voltages is obviously asymmetric, indicating typical rectification behavior Fig. g. FurtheThe metallic CaCl crystals in rGO membrane possess unexpected piezoelectricity-like property. DFT calculations show that, for 5% geometric elongation along the x direction, the charge distribution of the Ca, Cl and C atoms in model I has significant relocation . Our experiment, where a dried Ca–Cl@rGO membrane was connected by two Cu foil electrodes Figs h. AlthouNature continuously produces surprises, and our results demonstrate another one. 2D Na–Cl crystals with unconventional stoichiometries on graphite or graphene  and at e2+, Fe2+, Co2+, Cu2+, Cd2+, Cr2+ and Pb2+) and graphitic surfaces [Such abnormal CaCl crystals are attributed to the strong cation-π interactions between Ca cations and aromatic rings in the graphene surfaces. As strong cation-π interactions also exist between other metal cations (such as Mgsurfaces ,24,28–32surfaces . We noteSee Supplementary section PS1 for details.nwaa274_Supplemental_FileClick here for additional data file.