Lupinus angustifolius L.) as a grain legume crop, exploiting discovered alleles conferring low-alkaloid content (iucundus), vernalization independence (Ku and Julius), and reduced pod shattering (lentus and tardus). In this study, a L. angustifolius mapping population was subjected to massive analysis of cDNA ends (MACE). The MACE yielded 4185 single nucleotide polymorphism (SNP) markers for linkage map improvement and 30,595 transcriptomic profiles for expression quantitative trait loci (eQTL) mapping. The eQTL highlighted a high number of cis- and trans-regulated alkaloid biosynthesis genes with gene expression orchestrated by a regulatory agent localized at iucundus locus, supporting the concept that ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7 may control low-alkaloid phenotype. The analysis of Ku shed light on the vernalization response via FLOWERING LOCUS T and FD regulon in L. angustifolius, providing transcriptomic evidence for the contribution of several genes acting in C-repeat binding factor (CBF) cold responsiveness and in UDP-glycosyltransferases pathways. Research on lentus selected a DUF1218 domain protein as a candidate gene controlling the orientation of the sclerified endocarp and a homolog of DETOXIFICATION14 for purplish hue of young pods. An ABCG transporter was identified as a hypothetical contributor to sclerenchyma fortification underlying tardus phenotype.The last century has witnessed rapid domestication of the narrow-leafed lupin ( Lupinus angustifolius L., is a grain legume crop, appreciated as an organic fertilizer that improves soil structure and productivity, as well as providing a source of protein for human and animals. This species has witnessed rapid domestication during the last century. Several important agronomic traits have been identified and transferred into improved germplasm [Ku and Julius), low-alkaloid content (iucundus), reduced pod shattering (tardus and lentus), soft seededness (mollis), white flower color (leucospermus) and anthracnose resistance (Lanr1).The narrow-leafed lupin, ermplasm . These tKu or Julius loci diminished the need of vernalization and enabled temperature-independent sowing of L. angustifolius [Vernalization responsiveness is the natural adaptation to climatic conditions, based on the requirement of a prolonged low temperature period during germination to induce flowering ,3. Naturtifolius .L. angustifolius, and one of them, iucundus, was extensively implemented in breeding [A high level of quinolizidine alkaloids is a typical feature of primitive populations in many lupin species, as these chemical compounds protect plants from pests and fungi , howeverbreeding ,9. Some breeding ,11.L. angustifolius, namely tardus, affecting sclerenchyma strips of the dorsal and ventral pod seams, and lentus, modifying the orientation of the sclerified endocarp of the pod [Shattering of dry pods is natural process of seed dispersal, however, it is a very undesired trait in modern agriculture because it dramatically decreases harvested yield. Two unlinked recessive alleles contribute to reduced pod shattering in  the pod ,13.L. angustifolius is natively adapted to the Mediterranean climate which has hot dry summers, because of one of its survival strategies which is impermeability of seed coat to water. Hard-seeded germplasm has a long dormancy period and irregular germination. Recessive soft-seediness allele mollis confers water permeability and efficient seed germination [L. angustifolius allele for breeding because the desired phenotype is maternally determined [mination . It is ttermined . L. angustifolius has been reduced by high susceptibility to anthracnose, caused by the pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler and Hagedorn [L. angustifolius was revealed to be controlled by several single dominant genes that were discovered in different germplasm resources, namely, Lanr1 in cultivar Tanjil, AnMan in cv. Mandelup, and LanrBo in the breeding line Bo7212 [The agronomic potential of Hagedorn . The rese Bo7212 ,18,19.L. angustifolius seed and flower color. The most widely exploited is the recessive allele leucospermus, affecting anthocyanin synthesis and resulting in bright seeds and white flowers [Several genes contribute to  flowers . iucundus (marker iucLi) [Ku (KuHM1) [mollis (MoLi) [lentus  [tardus  [To generate numerous molecular markers for agronomic trait selection in narrow-leafed lupin breeding programs, microsatellite-anchored fragment length polymorphisms (MFLP) fingerprinting has been exploited ,21. Trair iucLi) , Ku (KuH (KuHM1) , mollis s (MoLi) , lentus nd LeLi) ,24, and nd TaM2) ,26. Narrnd TaM2) ,28,29,30nd TaM2) ,32, and nd TaM2) ,34.L. angustifolius. These new data were exploited for construction of a linkage map and for determination of expression quantitative trait loci (eQTLs) related to selected domestication traits.Recently, a new method of transcriptome-based genotyping-by-sequencing, called massive analysis of cDNA ends (MACE), has been developed . The MACL. angustifolius mapping population (83A:476 × P27255), yielding 11,864 markers. A total of 9304 markers were localized within gene sequences whereas 2560 markers were found in loci lacking any annotation. There were 4185 MACE markers retained after application of total missing data threshold (counting heterozygotes and no data scores), followed by inference of consensus segregation for genes represented by several single nucleotide polymorphism, SNPs. There were 3532 genes represented by single markers, four genes were represented by pairs of markers with heterogeneous segregation patterns, and 645 markers were localized in unannotated loci. The annotation of markers is provided in the The MACE protocol was applied for 89 RILs and for parental lines of L. angustifolius genome physical and linkage mapping. Because the L. angustifolius karyotype carries numerous small and very uniform chromosomes, the BAC-derived markers have been frequently used as chromosome-specific landmarks to validate physical linkage of particular genome regions, as well as to facilitate assignment of particular chromosomes to linkage groups [Ku locus, as well as for comparative mapping of genes from isoflavonoid and fatty acid synthesis pathways [The MACE is a method providing sequences anchored in the 3′-ends of mRNA and can be used to develop sequence-defined markers, as well as to quantify gene expression ,36. In te groups ,45,46,47pathways ,49,50,51Ku, tardus, lentus, mollis, leucospermus, iucundus and Lanr1) [2p-values of distortion from expected 1:1 segregation are provided in Markers used for linkage map development included 4,185 MACE and 10 BAC-end PCR markers developed in this study as well as previously published data including seven trait loci (d Lanr1) , eight td Lanr1) ,25,52,53d Lanr1) ,50,51,54L. angustifolius draft genome sequence [The segregation pattern of 59.7% of the markers was redundant, and therefore the map contains 1735 loci, namely from 60 to 120 loci per linkage group. The lengths of linkage groups vary from 78.5 to 156.38 cM, reaching 2163.63 cM in total. The results of linkage mapping are provided in sequence  and thisL. angustifolius linkage maps [L. luteus [L. albus [L. angustifolius genome mapping study, namely 87 lines with only 78 lines overlapping with previous mapping studies, was too low to provide the high resolution required for significant improvement of genome assembly, and resulted in high marker redundancy, reaching 89.9% [Mapping data from the most recent age maps ,34 were . luteus , as wellL. albus . Neverthng 89.9% . iucundus, 50 for Ku, 35 for leucospermus, 29 for lentus, 17 for tardus, 11 for Lanr1 and five for mollis. The values of the t-Student test association between domestication trait segregation and gene expression patterns, including FDR correction and statistical significance analysis, are provided in iucundus trait segregation highlighted lysine biosynthesis and lysine metabolism, as well as cofactor binding and coenzyme binding, as the most overrepresented processes and functions, respectively  are provided in ectively . This wamollis) to 61 (iucundus) genes had eQTL peaks localized  was cis-regulated, whereas the vast majority (74%) was trans-regulated. Furthermore, from 34 genes for iucundus that had association values between their expression patterns and trait segregation above 0.5 or below −0.5, as many as 30 revealed a positive association with wild, high alkaloid phenotype. Moreover, as many as 16 genes highly associated with iucundus revealed to have their major eQTL locus explaining more than 50% of their observed expression variance localized directly at iucundus, or very close to it , for example, affects nicotine metabolism, as well as regulates the ETHYLENE RESPONSE FACTOR 189, a key transcription factor in nicotine biosynthesis pathway [L. angustifolius quinolizidine alkaloid biosynthetic gene because it revealed similar expression pattern to the LDC gene [MAJOR LATEX PROTEIN 423 , which is hypothesized to be involved in stress responsive activation of biosynthetic pathway of coumestrol, a coumestan isoflavone in soybean [L. angustifolius due to highly elevated expression in bitter P27255 accession [ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7, a candidate locus for iucundus, evidenced by a gene expression study involving transcriptome sequencing of four accessions and quantitative RT-PCR profiling of 14 accessions differing in alkaloid content, as well as by molecular marker development and linkage mapping [RAP2.7 at iucundus locus, another cis-regulated component, Fe SUPEROXIDE DISMUTASE 2 , revealed similarly high LOD and explained eQTL variance values, but opposite direction of association. Other iucundus-associated genes have included Lup005321 (OIW13431.1) and Lup005322 (OIW13432.1) encoding homologs of DIHYDROFLAVONOL 4-REDUCTASE which is one of the key genes from anthocyanin biosynthesis pathway [iucundus region also includes a Lup021586 (OIV95196.1) gene encoding HXXXD-type ACYL-TRANSFERASE . The expression profile of LaAT has been highly associated with alkaloid content in L. angustifolius [pauper locus in L. albus, as evidenced by linkage mapping and validation survey in a set of 127 bitter and 23 sweet accessions [Among the genes with expression positively associated with high alkaloid phenotypes in the RIL population, the highest LOD values of eQTLs were revealed for Lup009726 , Lup028431 , Lup015923 , and Lup007628  a. The Lu studies ,59,60. L pathway ,62. IndeLDC gene . Lup0159 soybean . Lup0159ccession . Lup0076tifolius . Moreovecessions .cis-regulated genes revealed eQTLs localized at iucundus locus, including three hypothetical components of alkaloid biosynthesis pathways, Lup007706 (OIW07664.1), Lup017658 (OIW07643.1), and Lup032669 (OIW21355.1). Lup007706 encodes a representative of a S-adenosyl-l-methionine-dependent methyltransferases superfamily protein. N-methylation of quinolizidine alkaloids was confirmed to occur in crude protein extracts from Laburnum anagyroides carrying S-adenosyl-l-methionine: cytisine N-methyltransferase [S-adenosyl-l-methionine-dependent N methyltransferase catalyzes a nitrogen methylation involved in vindoline alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus) [4-HYDROXY-TETRAHYDRODIPICOLINATE SYNTHASE gene which is generally involved in biosynthesis of l-lysine, a precursor of quinolizidine alkaloids. This gene revealed considerably elevated expression in bitter accessions of L. angustifolius, indicating its hypothetical involvement in alkaloid biosynthesis pathway [CARBOXYLESTERASE 1 gene. CARBOXYLESTERASE 1 was evidenced to be involved in one of the final three steps of noscapine alkaloid biosynthesis [iucundus locus. This study provided novel evidences supporting the concept that RAP2.7 may control low-alkaloid iucundus phenotype, however, further evidence would require cis-trans tests. Nonetheless, such studies are hampered by very low transformation efficiency in narrow-leafed lupin [In addition to Lup007628 and Lup007632, nine other nsferase . Moreove roseus) . Lup0176 pathway . Lup0326Ku locus . Genes showing the highest gene expression association and eQTL peak co-localization (≤2 cM) with Ku included Lup011808  and Lup015372 . Lup015372 encodes hypothetical uncharacterized protein, whereas Lup011808 a homolog of A. thaliana CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1, CRLK1. CRLK1 confers cold responsiveness in plants via C-repeat binding factors (CBF) pathway [CBF in Arabidopsis delays flowering by promoting the expression of FLOWERING LOCUS C (FLC), indicating a link between cold signaling and flowering time regulation [INDUCER OF CBF EXPRESSION 1 (ICE1) which integrates cold signals into FLC-mediated flowering pathway [SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene was also identified [CBF pathway affects flowering time but does not affect vernalization response in Arabidopsis [NtCBK1) from N. tabacum functions as a negative regulator of flowering; high levels of NtCBK1 in the shoot apical meristem extended the vegetative phase of growth [CRLK1, in this study, was revealed to be positively associated with late flowering phenotype. These observations bring attention to the hypothetical involvement of calcium and calmodulin link with FLC pathway in flowering time regulation in L. angustifolius. Legume genomes do not contain FLC homologs but other genes from this pathway, including activators and repressors of FLC, are present [The P27255 parent is late flowering and requires vernalization for flowering induction, whereas the 83A:476 parent is early flowering and vernalization independent. In this study, seeds were subjected to vernalization procedure to ensure transition from vegetative to generative phase in all analyzed RILs. Such an approach could result in diminishing of some differences in expression profiles of vernalization-responsive genes between early and late flowering RILs. However, despite this partial pre-sowing vernalization, relatively a large number of genes revealed to have their eQTL peaks closely localized to Ku locus b. Contra pathway . Anotherentified . A recenbidopsis . Moreove present .Ku eQTLs include Lup011781 (OIW03144.1), Lup011739 (OIV96743.1), Lup011836 (OIW03199.1), and Lup002110 (OIW20134.1) sequences. Lup011781 has been identified as a homolog of MHM17-10 (AT5G56980) gene. It is pathogen-associated molecular pattern-induced gene with unknown function, putatively participating in jasmonic acid pathway [GALACTURONOSYLTRANSFERASE-LIKE 10, which is involved in cell wall organization and its expression is regulated by FLAVIN-BINDING KELCH REPEAT, F-BOX 1 (FKF1), blue light receptor, and well-known photoperiodic flowering time regulator [GTF2H2), performing basic functions in transcription and nucleotide excision repair of damaged DNA. Lup002110 has been annotated as a representative of the UDP-glycosyltransferases protein family. One of the A. thaliana UDP-glycosyltransferases, UGT87A2, was revealed to be involved in the regulation of flowering in vernalization and gibberellin pathways via the flowering repressor FLC [Other egulator . Lup01180 AT5G569 gene. ItL. angustifolius gene underlying vernalization responsiveness, a homolog of A. thaliana FLOWERING LOCUS T (FT), LanFTc1  [LanFTc1 revealed negative association with late flowering phenotype. Similar LOD values were also revealed for Lup018024 (OIV92673.1), Lup020573 (OIW03269.1), and Lup018485 (OIW19675.1) genes. Lup018024 encodes a homolog of bZIP transcription factor, FD, which mediates signals from the FT gene at the shoot apex and promotes plant flowering in general [FD expression revealed a positive association with late flowering phenotype. In Arabidopsis, the FT-FD complex induces the transcription of several floral-promoting genes, such as SOC1 and FRUITFULL (FUL), which accelerate flowering, as well as APETALA1 (AP1) and LEAFY (LFY), which control floral meristem identity [AGAMOUS-LIKE 8 (AGL8)/ FUL and AP1 MADS-box transcription factors and was found to be negatively associated with late flowering phenotype. Lup020573 was annotated as MYB60 transcription factor. MYB transcription factors perform various regulatory functions in plants in responses to biotic and abiotic stresses, development, differentiation, metabolism, defense, etc. [As plants in this study were subjected to a moderate vernalization procedure, differences in gene expression resulting from variation in vernalization responsiveness should be reduced as compared with those expected for nonvernalized plants. This reduction might be highlighted by a relatively low LOD value (as compared with other eQTLs from this region) of the major LOD 7.0) ,81. As e general . Howeveridentity . Indeed,Ku eQTLs shed light on vernalization pathway in L. angustifolius, providing transcriptomic evidence for the contribution of several genes acting upstream of FLC in CBF and UDP-glycosyltransferases pathways. The study also revealed transcriptomic contribution of conserved mechanism of FT-FD regulon on transition from vegetative to generative development phase in L. angustifolius.To summarize, analysis of lentus (le) allele changes the orientation of the sclerified endocarp in the pod, substantially reducing torsional forces after drying [lentus were identified to have major eQTL peaks localized in the proximity of this locus: Lup018336 , Lup018348 , and Lup018228  , MODIFYING WALL LIGNIN-1, and MODIFYING WALL LIGNIN-2 (At1g31720/MWL-1 and At4g19370/MWL-2) [DETOXIFICATION14, a member of the multidrug and toxic compound extrusion (MATE efflux) family [le allele affects a pod pigmentation, resulting in a purplish hue of young pods and a bright yellowish-brown color on the internal surface of mature pods. Lup018348 may be responsible for this pigmentation, whereas Lup018336 for pod shattering in L. angustifolius.The recessive r drying . In thisOD 13.0) c. All tha margin . Another0/MWL-2) ,87,88. L) family . MATE tr) family . Some MA) family ,92. Anthtardus (ta) allele affects the sclerenchyma strips of the dorsal and ventral pod seams, greatly increasing the fusion of two pod halves and moderately hampering their separation when drying [tardus, namely Lup002465 (OIW17837.1) and Lup002448 (OIW17820.1) (RCN1) is required for hypodermal suberization of roots [ABCG1 confers suberin formation in potato tuber periderm [tardus trait.The recessive n drying . Two gen17820.1) d. Lup002of roots . Similarperiderm . Some ABperiderm ,97. Moreperiderm . ABCG trmollis provides water permeable testa at maturity [A. thaliana DWARF5 gene)  e. Becausfluidity . Mutatiofluidity . Recentlthaliana . Mollis leucospermus allele confers white flower and bright seed pigmentation in L. angustifolius. A similar trait in pea was conferred by a basic-helix-loop-helix (bHLH) transcription factor [leucospermus locus. Two genes with expression positively associated with recessive allele revealed eQTL peaks close to leucospermus, namely Lup008087 (OIW21684.1) and (Lup017573) OIV97389.1  annotated as F-BOX/WD repeat-containing protein. Interestingly, a single mutation in an F-BOX domain-containing protein, OsFBX310, confers brown hull phenotype in rice resulting from a high content of total flavonoids and anthocyanins [Recessive n factor  but eQTLV97389.1 f. Lup008ocyanins , howeverL. angustifolius genome and transcriptome analysis. As a method for gene expression analysis, the MACE was first used in chronic kidney disease survey [Calliphora vicina pupae [Lolium perenne), highlighting a candidate LpPg1 resistance gene and yielding numerous SNPs which were further transformed into PCR-based molecular markers [Pisum sativum) providing single nucleotide variants subsequently converted into CAPS markers [Sym33 [Phaseolus vulgaris seeds and Solanum lycopersicum pollen [Triticum aestivum [This study is the first report on exploitation of the MACE for e survey  and in dna pupae . The MAC markers . The MAC markers . Furthers [Sym33 . The MACm pollen ,40. The aestivum .L. angustifolius genotyping approaches were based on diversity arrays technology (DArT) profiling. DArT studies have highlighted low genetic diversity in narrow-leafed lupin breeding material as compared with primitive and wild germplasm [Several previous ermplasm . This doermplasm ,110. Theermplasm ,112.L. angustifolius can serve as a reference model for such studies across the whole genus. Moreover, information about candidate genes identified in L. angustifolius can be translated to other legume species as these processes are generally conserved.In this study, the MACE was revealed to be an advantageous technique for marker development and gene expression profiling. The eQTL mapping highlighted numerous genes involved in the vernalization response and alkaloid biosynthesis, providing a valuable contribution for further advancement of knowledge on the complexity of molecular networks controlling these two biological processes. Taking into consideration the recent improvements in deciphering the molecular basis underlying early flowering and low-alkaloid phenotypes, as well as addressing results reported here, L. angustifolius domestication several agronomic traits were identified and transferred into improved germplasm by classical selection approaches. Current breeding materials and cultivars usually carry desired alleles of all major domestication traits in homozygous state . However, domestication process was highly focused on these traits and resulted in approximately threefold reduction in genome-wide diversity across domesticated accessions as compared with their wild relatives [LanFTc1, CRLK1, FD, UGT85A2, GAUT10, and MYB60. Moreover, a common issue related to dry and warm weather patterns, which are expected to occur more frequently due to changing climate, is pod dehiscence. Identified candidate genes for lentus (a homolog carrying DUF1218 domain) and tardus (an ABCG5 transporter) await further genotypic and phenotypic exploration in wide genetic background because mapping population represents only a small fraction of diversity existing in L. angustifolius germplasm.During the process of elatives . Furtherelatives . Novel on = 89, F8) of L. angustifolius [ku), pod shattering , hard seeded (Mollis), blue flower and dark seed color Leucospermus), high alkaloid (Iucundus) and anthracnose susceptible (lanr1). 83A:476 has an opposite allele combination . Both parental lines are homozygous in relation to these alleles.The reference 83A:476 × P27255 recombinant inbred line (RIL) population  were vernalized for 16 days at 4 °C in darkness on Petri dishes with moist filter paper. Filter paper  was changed every four days to maintain phytosanitary conditions. Following vernalization, plants were transferred to pots  and grown in controlled conditions  at the Wielkopolska Center of Advanced Technologies in Poznań, Poland. Tissue was sampled from young leaves two times a day, 4 h after beginning of photoperiod and 1 h before the end of photoperiod on the 28th, 36th, and 44th day from sowing. Five biological replicates were collected.L. angustifolius genome assembly [http://www.lupinexpress.org). Normalization procedure was as follows [Frozen plant tissue  was homogenized using TissueLyser II  and two stainless steel beads (ø 5 mm) placed in a 2 mL tube . RNA isolation was performed using SV Total RNA Isolation System  according to the protocol. The concentration of RNA was measured using NanoDrop 2000  and A260/A280 ratio. RNA quality was visualized by 1% agarose gel electrophoresis (1X TAE) of denaturated samples. RNA concentration was equalized to 400 ng/µL in nuclease-free water. Samples from particular line  were bulked together in equal aliquots. 10 µL of mixture (4 µg of RNA) was subjected to the MACE protocol. The MACE profiling and SNP calling was outsourced . The MACE reads were aligned to the assembly  (http:// follows : The ave2) values for Mendelian segregation were estimated using the expected 1:1 ratio. The calculation of probability was based on χ2 and 2 degrees of freedom. Based on the segregation distortion observed in recently published L. angustifolius linkage map versions [2p-value threshold of 1 × 10−7 was applied. The 83A:476-like scores were assigned as “a”, the P27255-like scores as “b”, and the heterozygotes as “h”. If several cosegregating MACE markers in particular gene were identified, the marker with the lowest percentage of missing data was chosen to infer consensus segregation representing each cluster. To provide a mapping file, heterozygote scores were removed. Accepted missing data threshold was 11%. Chi-square  and sequenced using ABI PRISM 3130 Genetic Analyzer XL . Allele-specific PCR (AS-PCR) polymorphisms were visualized by 1% agarose gel electrophoresis, whereas nucleotide substitution polymorphisms were revealed by the cleaved amplified polymorphic sequence (CAPS) and dCAPS approaches [Segregation data for domestication traits and tightly linked SSR-derived markers were included in the study ,25,52,53proaches ,116. Resproaches ,118. Digproaches .Ku, tardus, lentus, mollis, leucospermus, iucundus, and Lanr1 segregation data (binary trait) by t-Student test in two classes of polymorphism. Obtained p-values were false discovery rate (FDR) corrected [p-value ≤ 0.01 were subjected to composite interval mapping performed in Windows QTL Cartographer V2.5  using 5 background control markers, window size 10 cM, walk speed 0.5 cM, and backward regression method. LOD threshold for QTL calling was established by permutation test (N = 1000) using the same parameters. Linkage groups and LOD graphs were drawn in MapChart [p-value ≤ 0.01 were analyzed for gene ontology (GO) term enrichment by hypergeometric test with FDR correction in Bingo [L. angustifolius genes obtained from Ensembl Plants Genes database . Whole-genome annotation was used as reference set. Results were provided as −log10 .Normalized gene expression values (continuous traits) obtained for RILs and mapping population parental lines were associated with orrected . Genes wMapChart . Moreovein Bingo  using GOThe massive analysis of cDNA ends was revealed to be applicable for molecular marker development and linkage map construction, as well as for gene expression evaluation and expression quantitative trait loci mapping.Ku locus shed light on vernalization response via FLOWERING LOCUS T and FD regulon, providing transcriptomic evidence for contribution of several genes acting in C-repeat binding factor (CBF) cold responsiveness and in UDP-glycosyltransferases pathways. This information can be relevant to decipher vernalization pathway in legumes, because legume genomes do not contain a major vernalization-responsive gene FLOWERING LOCUS C (FLC) but other genes from this pathway, including activators and repressors of FLC, are present.The analysis of vernalization independence iucundus locus highlighted a high number of cis- and trans-regulated alkaloid biosynthesis genes with gene expression orchestrated by a regulatory agent localized at iucundus locus, supporting the concept that the ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7 gene may control low-alkaloid phenotype in narrow-leafed lupin.The study of low-alkaloid lentus locus selected a DUF1218 domain homolog as a candidate gene controlling the orientation of the sclerified endocarp and a DETOXIFICATION14 homolog for purplish hue of young pods.Research on reduced pod shattering ABCG transporter gene was identified as a hypothetical contributor to sclerenchyma fortification underlying reduced pod shattering tardus locus.An


SD = 1.9) receiving FFT to investigate whether outcomes in FFT are predicted by CU traits and to what extent reliable changes in CU traits can be observed. The results showed that although CU traits are related to increased problem severity at baseline, they predicted neither treatment dropout nor post-treatment externalizing behavior and family functioning. CU traits were related to diminished improvement ratings, in particular with respect to parental supervision. Reductions in CU traits were observed across the time of treatment, and these were most profound among adolescents with elevated levels of CU traits at baseline. Further research should investigate whether certain evidence-based treatment components are more suited for adolescents with CU, and if the addition of specific intervention elements for reducing CU-traits could further improve outcomes for this high-risk population.Despite the availability of evidence-based treatment models for adolescent behavior problems, little is known about the effectiveness of these programs for adolescents with callous-unemotional (CU) traits. Defined by lack of empathy, lack of guilt, flattened affect and lack of caring, CU traits have been linked to long-term anti-social behavior and unfavorable treatment outcomes and might be negatively related to outcomes in evidence-based programs such as Functional Family Therapy (FFT). This study used a single-group pre-post evaluation design with a sample of 407 adolescents (49.1% female, mean age = 14.4 years,  Problem behaviors in adolescence can range in severity from heated verbal arguments and breaking curfew to aggression, violence, criminal behavior and drug use . These bEngagement to ensure family members participation in sessions; (2) Motivation to decrease the level of blame and negativity in the family and increase hope and motivation for change; (3) Relational Assessment to assess how risk and protective factors and family relational dynamics relate to the behavior problems; (4) Behavior Change to improve family skills such as communication, conflict management and problem solving to reduce problem behavior; and (5) Generalization to maintain and extend skill use inside and outside the family, prevent relapse and refer the family to additional support and services, if needed. FFT has a primary focus on family level risk factors as the reduction of these have been linked to diminished behavior problems  and age (older scoring lower than younger) .Secondly, we sought to evaluate if CU traits predict completion status and treatment outcomes in the context of FFT treatment. We assessed for this both related to adolescent behavior problems and family level problems. In line with previous research by Lastly, we wanted to determine the level of change in CU traits over time in the context of FFT-treatment. Previous research has shown mixed and limited evidence for the therapeutic malleability of CU traits in adolescents, so we hypothesized that for the majority of the participants in our study, CU traits would not reliably change and that any observed effect-size in relation to changes in CU traits would be small. We also examined the relationship between change in CU traits and change in adolescent behavior problems and family level problems.SD = 1.9). See This study builds upon data from a single-group pre-post design evaluation of FFT in eleven municipalities and two private treatment agencies in Denmark . Inclusir(405) = 0.689, p < 0.001, and the factor structure of the ICU12 might mostly be due to method variance effects  is a 6-item questionnaire completed at the end of FFT-treatment. Adolescents, parent(s) and therapist rate the family’s change on a general level (one item) and on five specific areas (one item per area): family communication skills, adolescent behavior, parenting skills, parental ability to supervise and monitor adolescent and family conflict levels. Each item is rated on a 6-point scale ranging from 0 (Things are worse) to 5 (Very much better), with a no change anchor at scale point 1 (Things are no different). Reliability across rater groups was good with αs ranging from 0.90 to 0.95.The FFT quality assurance database provided data on the number of days between each of the first three sessions , the number of sessions (as a measure of treatment intensity) and treatment duration. In addition, we collected the therapist assigned closing category, defined as either completion , drop out (family discontinued treatment prior to treatment completion), or non-completion .The adolescents and their parents completed the study questionnaires prior to or during their first FFT sessions for the baseline assessment, and during their last FFT session or shortly thereafter for the post-treatment assessment. The time between baseline assessment and post-assessment ranged from 17 to 473 days, with a median of 169 days and interquartile range of 88 days. Therapists entered data for each case in the FFT quality assurance database in relation to session dates and treatment completion status.There is no formal agency for ethical approval of survey and register-based studies in Denmark. The present study was, however, presented to the regional research scientific ethical committee in Southern Denmark in February of 2017 and deemed not required to obtain formal ethical approval as the study only used questionnaire-based data, according to governing law in Denmark. Information about the survey was provided to potential participants in an invitation letter, and the participants’ voluntary completion and return of the survey questionnaires constituted implied consent. The University of Southern Denmark (SDU) is the controller for processing of personal data in connection with the project, which has been included in SDU’s internal record of processing activities under file number [18/28527], cf. GDPR Article 30.All statistical analyses were conducted in SPSS Version 26. The relationships of CU traits to pre-treatment variables were assessed by partial bivariate correlations controlling for pre-treatment within-rater scores on the SDQ externalizing problems scale. This allowed for an assessment of the unique contribution of CU traits, as co-occurring levels of conduct problems and hyperactivity/impulsivity constitute the central aspects of externalizing problems .The association between CU traits and treatment completion was assessed by binary logistic regression of the two non-completion categories combined against the completed category. The conditional predictive value of CU traits on treatment service parameters, e.g., treatment length, were assessed by bivariate partial correlation controlling for baseline parent-rated externalizing problems. The prediction of CU traits on the continuous outcome measures was assessed by multiple regression analyses. As we were interested in the additional predictive value of CU traits, we controlled for baseline externalizing behavior as they might predict treatment gains to a large extent . In addiGiven that CU traits are meant to identify a more severe subgroup amongst adolescents with CD , a lack dRM) was calculated as the average-based change statistics over the time of treatment  and handled by pairwise deletion in the correlation and regression analyses. There were 3 outliers in relation to treatment pacing with more than 60 days between sessions, and these values were treated as missing in our analyses.The baseline zero-order correlations between CU traits and the measures of adolescent and family functioning and covariates are presented in p = 0.067 in the full sample. In the clinically elevated subsample, CU traits did statistically significant predict treatment non-completion, OR: 1.053, 95% CI , p = 0.037. However, the sensitivity of the model in its ability to correctly predict treatment non-completion, was relatively poor, with only 12.2% correct identification of non-completers in the clinically elevated subsample.Among all cases, 268 (65.8%) completed, 47 (11.5%) dropped out, 75 (18.4%) were non-completers and 17 (4.2%) were missing therapist-reported completion data. The results of the binary logistic regression model including both baseline externalizing and baseline CU traits as predictors, showed that CU traits were not significantly related to treatment non-completion, OR: 1.036, 95% CI , SD = 8.2, the total number of sessions averaged 11.8, SD = 5.6, and treatment duration averaged 160.2 days, SD = 72.3. The partial correlations of CU traits to treatment parameters controlling for baseline parent-ratings on the SDQ-externalizing scale are presented in r(206) = −0.166, p = 0.016.Treatment parameters showed that initial treatment pacing between the first two sessions averaged 9.46 days, SD = 0.92, for adolescent-report, 3.50, SD = 0.89, for parent-report and 3.06, SD = 1.08 for therapist-report. The post-treatment improvement ratings averaged 3.39, To further explore the significant predictions of CU traits on post-treatment improvement ratings, dRM) for the group average reduction was 0.36, 95%CI . The threshold value of elevated CU traits generated as 0.5 SD above the mean of the ICU12 was 16 scale points, which coincided well with the mathematically derived cut-off value of 15 scale points. In the subgroup of 60 adolescents with elevated pre-treatment ICU12 scores and an available post-treatment score, 26 (43.3%) were reliably declining, 32 (53.3%) had no reliable change and 2 (3.3%) were reliably increasing, with a corresponding group average based dRM of 0.91, 95%CI . The computation of a reliable change index for CU traits from baseline to post-treatment showed that for the 216 adolescents with available post-treatment CU ratings, 37 (17.1%) were reliably declining, 166 (76.9%) had no reliable change and 13 (6.0%) were reliably increasing. The corresponding repeated measures effect size  of the study participants had no reliable change, as hypothesized, while 17.1% had a reliable decline and 6.0% had a reliable increase. The theoretical values of standardized reliable change scores beyond −1.96 and +1.96 within a normal distribution is 2.5% in each direction. This means that in the whole sample we observed more cases both reliably declining and reliably increasing than what would be expected by measurement error and chance. These proportions were markedly different in the subsample with elevated levels of CU traits at baseline, where 43.3% had a reliable decline, 53.3% had no reliable change and 3.3% had a reliable increase. This represents a much larger proportion of cases reliably declining and a similar proportion of cases reliably increasing, compared to what would be expected by chance. This therefore provides some support for the therapeutic malleability of CU traits in adolescence.The increased proportion of reliable reductions in CU traits observed for the high CU group could entail that baseline CU traits act as a moderator for the malleability of CU traits in the context of FFT. For those with higher levels of CU traits at the start of treatment, we are more likely to observe a reliable decline during treatment. This is clinically relevant, as one might argue that only those with higher scores are in need of reducing their CU traits. This potential moderating effect of baseline CU might explain the somewhat mixed results from previous research . In addiChanges in CU traits were positively correlated with changes in externalizing problems and family functioning as reported by parents. This indicates that, from a parental perspective, changes in CU traits coincide closely with changes in externalizing behavior and to some extent with changes in family functioning. By youth-report, however, there was only a weak positive correlation between changes in CU trait and the change in family functioning. This could be due to the lower level of cross-rater correlations between CU traits and externalizing behavior as demonstrated in the baseline correlations and other studies . The facThis study has its strengths in investigating the association between CU traits and treatment outcomes in a large clinical sample of adolescents with behavior problems that received a well-established evidence-based treatment. There are, however, some limitations of the study pertinent to the interpretation of its findings. In the first place, this study used a single group pre-post-treatment design, which limits causal inferences. Secondly, a good proportion of the sample did not complete the study questionnaires at post-treatment, which lowers the study’s statistical power and generalizability. Thirdly, this study assessed the affective dimension of psychopathy, but not the other dimensions of psychopathy which could have provided improved predictions . FourthlThe results of this study indicate that CU traits do not seem to impede treatment completion or improvements in externalizing behavior and family functioning observed in FFT treatment. However, a relationship between CU traits and lower improvements in parental supervision and a lower number of generalization sessions was observed. This suggests that for adolescents with CU traits these treatment areas might need additional interventions and tailoring, than what is already embedded within the FFT program as implemented at these Danish sites. With respect to the malleability of CU, this study demonstrated that a good portion of adolescents with high levels of CU traits at baseline gained reliable reductions in CU traits. Changes in CU traits were furthermore related to changes in externalizing behavior and family functioning.These findings raise several questions and ideas for future research. First of all, further research is needed to determine whether CU traits predict diminished improvements in parental supervision in other contexts, and identify interventions that might be more suited to increase parental supervision for adolescents with CU traits. Second, the potential long-term predictive effect of CU traits on treatment improvements should be studied, to see if CU traits can indicate reduced longevity of treatment results or increased long-term risk of relapse. Third, more research is needed to understand the therapeutic processes and interventions that can result in attenuation of CU traits. Although plausible explanations could be given for FFT , as has been given for a mental models approach (positive emotions and prosocial strategies; css@vive.dk. Requests to access the quality assurance dataset can be directed to DT, d.m.thogersen@nubu.no.The datasets generated for this study will not be made publicly available because: the data contains sensitive data related to personal mental health information that is considered pseudonymized data by the legal advisory board at the University of Southern Denmark. Requests to access the main dataset can be directed to CS, Ethical review and approval was not required for this study in accordance with the local legislation and institutional requirements. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Participants received information about the survey in an invitation letter and voluntary completion and return of the survey questionnaires constituted an implied consent.CS, DT, ME, and GB contributed to the conception and design of the study. CS and DT organized the database. DT performed the statistical analysis with guidance from ME and GB. DT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.DT is occasionally hired to provide training, supervision, and consultation in the Functional Family Therapy model in Denmark, which could be construed as a potential conflict of interest. The remaining 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.


Abnormally excessive growth results from perturbation of a complex interplay of genetic, epigenetic, and hormonal factors that orchestrate human growth. Overgrowth syndromes generally present with inherent health concerns and, in some instances, an increased risk of tumor predisposition that necessitate prompt diagnosis and appropriate referral. In this review, we introduce some of the more common overgrowth syndromes, along with their molecular mechanisms, diagnostics, and medical complications for improved recognition and management of patients affected with these disorders. Characteristic facial appearance includes hypertelorism (widely spaced eyes), large ears, and a “stuck on” protruding chin. Newborns and infants often have truncal hypotonia (~50%) and extremity hypertonia with limited elbow extension. Hypotonia is associated with poor feeding and may also be associated with a characteristic weak, low pitched cry affecting about 50% of patients. Developmental delays are common, both in gross motor  and fine motor skills (poor coordination). Ventriculomegaly is frequently seen on brain imaging. Advanced bone age is uniformly reported, exceeding the chronological age by a factor of 1.5–2 . It is inherited in an autosomal dominant manner. Interestingly, pathogenic variants in EZH2 have been found in individuals with overgrowth without other clinical features of Weaver syndrome, indicating a wide spectrum of the disorder  (617561)  and microretrognathia seen in WS. Intellectual disability has been reported in one out of three published cases  , 75 and (617561) , respected cases . In our 615879). It is caused by pathogenic variants in DNA methyltransferase 3A. Despite sharing a similar mechanism with Weaver syndrome, that is, a heterozygote mutation (pathogenic variant in one copy) in DNA methyltransferase, this syndrome bears more similarities to Sotos syndrome. Both include features of macrocephaly noticed at birth, joint hyperlaxity, scoliosis, hypotonia, and seizures. However, the facial dysmorphism is different and includes round facies, straight and thick (bushy) eyebrows, and prominent maxillary incisors. These features evolve over time and may not be present in early childhood. Moreover, unlike Sotos and Weaver syndromes, patients' length at birth is usually normal and increases to >2 SD only later in life. About 16% of individuals are reported to have congenital heart defects; therefore, an echocardiography should be considered upon diagnosis  syndrome, similar to Sotos and Weaver syndromes, is a DNMT3A-related overgrowth syndrome, also known as Tatton–Brown–Rahman syndrome OMIM 6158. It is cCompared with the Sotos and Weaver syndromes, the “average patient” is more intellectually impaired. A cohort of 55 patients from 41 families  found thDNMT3A can be found in patients with acute myeloid leukemia (AML), myelodysplastic syndrome, and myeloproliferative syndromes. Interestingly, 26 out of the 40 variants described (63%) in DNMT3A, (DNMT3A) were also documented in affected patients with the overgrowth syndrome. Two out of the reported 77 patients with this OGID syndrome developed AML at the ages of 12 and 15  is a syndrome characterized by macrosomia, macrocephaly, round facies, hypotonia, and visceromegaly. Visceromegaly most commonly involves the kidneys  or liver  but can also include the heart, spleen, and pancreatic islet cells . Nephromegaly, seen in 80–100% of affected individuals, is frequently accompanied by nephroblastomatosis (diffuse persistence of metanephric blastema), a characteristic finding in Perlman syndrome, that predisposes to Wilms tumor seen in about a third of the patients. Other common findings are cryptorchidism and inguinal hernias. Newborns commonly present with abdominal distention as a result of nephromegaly, hepatomegaly, ascites, and/or abdominal wall muscular hypoplasia. The abdominal distension induces, in turn, hypoplastic lungs. About 87% of the affected infants develop respiratory distress and/or renal failure and die within the first hours or days of life. In surviving patients, growth parameters typically decline rapidly to reach the lower end of normal.Perlman syndrome (OMIM DIS3L2 exoribonuclease (RNAase); DIS3L2 has been shown to lead to IGF2 overexpression, as seen in the Beckwith–Wiedemann syndrome and is strongly associated with tissue overgrowth and Wilms tumor development  is a complex syndrome with variable presentation consisting of progressive segmental overgrowth of the feet and/or hands, cutaneous connective tissue nevi, and cranial hyperostosis. Affected individuals have minimal manifestation at birth and are born with normal growth parameters. Starting around 6–18 months of age, they can develop excessive asymmetric growth that can reach twice its normal size by age 6 years. The growth is of the bones and the soft tissue, and although the hands and feet are most commonly involved, any bone can be affected. Leg length discrepancy of up to 20 cm and scoliosis of more than 90° have been reported. Final height is normal as skeletal growth plateaus at adolescence. In childhood, patients can develop cutaneous connective tissue nevi most commonly involving the feet, hands, abdomen, or nose. These nevi are highly collagenized connective tissues that are firm with their surface resembling the gyri and sulci of the brain, thus named cerebriform. These nevi are pathognomonic for Proteus syndrome however they should be differentiated from the neurofibromas seen in neurofibromatosis syndrome; neurofibromas are smooth, soft, movable, and rarely involve the plantar aspect of the foot. Another cutaneous finding seen in Proteus syndrome is epidermal nevi. These lesions can present as early as the first few months of life and tend to follow the lines of Blaschko. Adipose tissue overgrowth is common in this syndrome; focal growth tends to occur from early infancy to early adulthood. They are not encapsulated lipomas but their histology is almost always benign. Along with growth, there are areas of adipose atrophy giving a combined presentation of adipose dysregulation. Skeletal abnormalities can occur anywhere in the body and add further to the striking asymmetry. Restrictive pulmonary disease is therefore common. Other distinctive features of Proteus syndrome include cranial hyperostosis, condylar (mandibular) hyperplasia, and rarely craniosynostosis  complicates these vascular anomalies and can lead to early death.The majority of individuals with Proteus syndrome have normal intelligence; however, a subgroup (30%) of patients with dysmorphic facial features has seizures and intellectual disability. Dysmorphism includes dolichocephaly , long face, downslanting of the palpebral fissures , and open mouth at rest. Other organs that are commonly affected include the eye , lungs (13% with cystic changes), and kidneys  . It has AKT1 gene, which is part of the PI3KA/AKT1/mTor proliferation pathway  occurrence, and progressive course. Lesions can be either the pathognomonic cutaneous (cerebriform) connective tissue nevi, or other findings such as asymmetric growth, dysregulated adipose tissue growth, lung cysts, and/or the specific tumors . These criteria were constructed to avoid over-diagnosis of this syndrome. A review of 205 published cases of Proteus syndrome found that only 95 of them fulfilled these criteria, and 80 cases (39%) “clearly did not” . It is rPTEN (Phosphatase and tensin homolog)—characterized by unregulated cellular proliferation leading to the formation of hamartomas. PTEN is a phosphatase that removes a phosphate from the second messenger phosphatidylinositol triphosphate and, by doing so, inhibits the Akt (Protein kinase B) pathway, a cardinal pathway of cell proliferation and angiogenesis. Not surprisingly, somatic inactivating mutations in PTEN are found in breast, prostate, lung, endometrial carcinomas and glioblastoma. In cancer, PTEN mutations are acquired in adulthood in a single cell which proliferates to create a tumor (“somatic” mutations). Alternatively, PTEN mutations that are either inherited from affected parent or formed in the parental gamete prior to conception affect all cell populations (“germline” mutations). In such cases, cells with an additional PTEN mutation (a “second hit”) results in hamartomata and cancer predisposition. The two-hit theory, also referred to as Knudson hypothesis, also explain the nature behind the observed segmental overgrowth: affected individuals are susceptible to Akt overactivation with only one active allele of PTEN; a second spontaneous deactivating mutation in PTEN in a post-zygotic developing tissue may occur, leading to discretely affected areas. The segmental overgrowth is manifested differently in each phenotype of PHTS: newborns with Bannayan–Riley–Ruvalcaba have striking macrocephaly (≥4.5 SD), out of proportion to their birth weight and length; those with Proteus-like presentation exhibit mosaic pattern of rapidly progressive overgrowth of different tissue types; Cowden syndrome, typically manifests in the second decade of life, and is associated with hamartomata and macrocephaly. Cowden syndrome bears an increased lifetime risk of benign and malignant tumors in the breast , papillary or follicular thyroid carcinoma (35%), renal cell carcinoma (35%), endometrial (28%), colorectal (9%), melanoma (5%), and rarely Lhermitte–Duclos disease (cerebellar dysplastic gangliocytoma).Phosphatase and tensin homolog (PTEN) Hamartoma tumor syndrome (PHTS) is a disorder encompassing three seemingly distinct clinical syndromes—Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome, and Proteus-like syndrome—all of which are characterized by mutations in the tumor suppressor gene Bannayan–Riley–Ruvalcaba is further characterized by hypotonia, intellectual disability (50–70%), proximal myopathy (60%), scoliosis (50%), hamartomatous polys in colon (45%), which may cause intussusception or rectal bleeding, seizures (25%), and joint hypermobility. A distinctive clinical finding in most affected individuals is pigmented macules on the penile shaft and if seen on physical examination should raise immediate concern for PHTS. The risk for tumors and malignancy is currently perceived to be similar to Cowden syndrome, especially breast and thyroid cancers, and merits similar surveillance. Proteus-like syndrome is clinically similar to Proteus syndrome and exhibit vascular malformations, lipomas, connective tissue nevi, epidermal nevi, and cranial hyperostosis. Cowden syndrome, named after the first reported patient, is rarely expressed in children and has distinctive trichilemmomas (benign neoplasm derived from the outer root sheath epithelium of the hair follicle), papillomatous papules (benign neoplasm of epithelium), and acral and plantar keratosis seen in 99% of patients by the third decade of life.PTEN (10q23), can give rise to either one of the above phenotypes—making these three conditions allelic disorders. Furthermore, it has been shown that the very same PTEN variant in an affected family can be expressed as either Cowden or Bannayan–Riley–Ruvalcaba syndrome in different family members. Another allelic disorder is macrocephaly with autism. In fact, 10–20% of autistic children with macrocephaly harbor germline mutation in PTEN. Mutations in PTEN includes gene sequencing, analysis of deletions and duplications and the promoter region. PHTS is an autosomal dominant disorder, meaning that only one pathogenic variant in PTEN gene is enough to express the disorder. Affected individuals have a 50% chance of transmitting the pathogenic variant to each one of their children. We recommend referring to genetics post-pubertal individuals for discussion about family planning. In vitro fertilization (IVF) techniques with preimplantation genetic testing for monogenic disorder (PGT-M) for fetuses can be offered to significantly reduce the transmission risk.Genetic testing for Management of PHTS include symptomatic management for the mucocutaneous manifestations of CS  and close follow up on development with neuropsychological evaluation (including IQ test) if suspicion for intellectual disability arises. Genetic testing for other family members is also recommended , 89–91. BRCA1 or BRACA2 carriers: clinical breast examination starting at 25 years of age or 5–10 years before the earliest known breast cancer in the family (whichever comes first). Starting at age 30–35 or 5–10 years prior to first known breast cancer in the family, annual mammography, tomosynthesis (“3D mammography”), or MRI with contrast should be performed until the age of 75. A discussion about risk reduction with double mastectomy should be conducted (sparing oophorectomy). Endometrial cancer screening is symptom-based  only. For both men and women, colonoscopy should be performed at least every 5 years starting at 35 years of age or 5–10 years prior to first known familial case (whichever comes first). Neck ultrasound for thyroid cancer should be obtained at the age of 7, then if negative every 2 years. Renal US is recommended at 40, then every 1–2 years subsequently  of PTEN, while testing cells from affected tissue will show variants in the two alleles. On the contrary, proteins that participate in the growth-promoting pathway that PTEN inhibits, may harbor only one variant which allows their escape from inactivation, resulting in overgrowth. PTEN is the opposed enzyme of phosphatidylinositol 3-kinase: the former removes and the latter adds phosphate to the aliphatic second messenger phosphatidylinositol di/tri-phosphate. The catalytic subunit of phosphatidylinositol 3-kinase is p110α, encoded by the gene PIK3CA. Variants causing overactivation of p110α shifts the balance between p110α and PTEN toward creation of phosphatidylinositol -trisphosphate, which activates AKT  and the second is megalencephaly-capillary malformation (MCAP) syndrome. CLOVES is associated with complex lipomatous overgrowth of the thoracic and abdominal wall, macrodactyly (enlargement of a digit), and plantar or palmar overgrowth which results in wrinkling of the overlying palmar or plantar skin. The lipomatous masses can be found on the skin or extending into the paraspinal and intraspinal spaces which may cause compression of the cord or nerve roots. The lesions are commonly covered by capillary, venous, lymphatic, or arteriovenous malformations. Skeletal malformations can be severely deforming; it includes scoliosis and asymmetric bony overgrowth. Structural anomalies are found in the CNS  and kidneys. A very similar presentation of asymmetric overgrowth of bony, adipose, fibrous, and vascular overgrowth due to PIK3CA over-activation is called Fibroadipose hyperplasia. Milder presentation may include only asymmetric overgrowth of limbs with overlying vascular malformations and can be expressed merely as isolated macrodactyly  seen perinatally by elevated occipitofrontal circumference. It is accompanied by secondary overgrowth of specific brain structures, and ventriculomegaly with polymicrogyria  are common. Patients with MCAP are hypotonic and prone to seizures (30%). Similarly to CLOVES, vascular malformations are common  and asymmetric overgrowth of limbs may be seen. Not surprisingly, mutations causing CLOVES and MCAP syndromes are seen in the COSMIC database, as shed yet . Managemshed yet . Experimshed yet , 96. Sirshed yet . In two shed yet , 98. Sevshed yet . A dual in vitro .PIK3CA is expressed in gonads; therefore, transmission to next generation is expected to be unlikely. Similarly, because this is a post-zygotic mutation, the risk for recurrence in a family is not elevated.Genetic testing should be obtained from the affected tissue and not blood. Prognosis depends on the degree of the severity of the phenotype ranging from guarded to dismal . Rarely Somatic overgrowth with overlying vascular malformation is shared by many other syndromes making this clinical finding an important sign for an underlying disorder. Combined capillary, venous and lymphatic malformation  along with limb enlargement is seen in Klippel–Trenaunay syndrome (KTS); arteriovenous (fast-flowing) fistulae without lymphatic malformation along an enlarged limb are seen in Parkes–Weber syndrome . In KTS, the affected limb is the lower extremity in 95% of cases and upper extremity in close to 5%. It is uncommon to have hypertrophy of the trunk. Capillary malformations appear bluish-purplish in color and may extend to the trunk but rarely to the face. They are commonly accompanied by lymphatic malformation causing lymphatic leak and lymphedema of the involved extremity. In 80% of patients, significant varicosities extending from the dorsum of foot to the popliteal or even the gluteal venous system are noticeable beginning in infancy or early childhood. It is often complicated by thrombophlebitis (up to 50%) and pulmonary embolism (10%). Surgical correction may be complicated by incompetency of the deep vein system seen in some patients. The affected limb may exhibit bone and/or soft tissues hypertrophy and thus the limb can be asymmetric in both length and girth . Toes maybe significantly more affected than the rest of the limb. The sole may have wrinkled appearance to it but lacks the firm consistency of cerebriform connective tissue nevi seen in Proteus syndrome , 102. ClOvergrowth syndromes can present with excessive post-natal growth, tall stature that is out of proportion to the individual's genetic potential in a child, isolated increased head circumference, or somatic asymmetry. Clues for overgrowth syndrome can come from the following: (i) physical examination such as omphalocele, abnormal ear creasing, and macroglossia for BWSp, dolichocephaly and frontal bossing in Sotos syndrome or lipomas, cerebriform cutaneous nevi or vascular anomalies in segmented overgrowth syndrome; (ii) imaging findings such as accelerated bone age and visceromegaly in the prenatally presenting overgrowth syndromes, or hemi-megalencephaly and paraspinous lipomas seen in PIK3CA-related overgrowth; and (iii) neurobehavioral assessment showing learning difficulties, developmental delays, or autism as seen commonly in Sotos, Weaver, and PTEN-hamartoma syndromes. Overgrowth syndromes can predispose affected individuals to hypoglycemia, embryonal tumors, seizures, developmental delay, intellectual disability, and musculoskeletal complications; therefore, timely diagnosis is important. Careful history taking, physical examination, anthropometric measurements, and developmental follow-ups, all of which are tools used on routine office visits, can provide significant evidence for suspected overgrowth syndrome. Maintaining a high index of suspicion for these disorders can assist in timely referrals to genetics, which can assist in the evaluation and testing of these patients. We encourage the primary clinician to be alert of the “large” end of the weight, length and FOC measures, and become familiar with BWSp, which is the most common overgrowth syndrome requiring vigilant tumor surveillance see .Based on expert opinion, stature larger than 2 standard deviations from the mean should be considered tall and raise suspicion for an overgrowth syndrome.Based on expert opinion, workup for tall stature that exceeds the individual's mid-parental height should include assessment of growth velocity and should consider full blood counts, complete biochemical analysis, IGF-I, IGFBP-3, free T4, and TSH, as well as a karyotype and bone age.Based on expert opinion, newborns with either macroglossia, exophthalmos, lateralized overgrowth, persistent hyperinsulinism, multifocal or bilateral Wilms tumor, or adrenal cortex cytomegaly should be tested molecularly for Beckwith–Wiedemann syndrome.Based on published guidelines, individuals with BWSp or SGB syndrome should undergo routine cancer surveillance every 3 months for hepatoblastoma (until the age of 4) and Wilms tumor (until the age of 7).Based on expert opinion, individuals with FOC > 4 SD and autism should be tested molecularly for possible PHTS.Based on published guidelines individuals with PTEN-hamartoma Tumor syndrome should undergo breast cancer surveillance similar to BRCA 1/2, colonoscopy surveillance every 5 years, biennial thyroid US, renal US every 1–2 years.Based on expert opinion, individuals with intellectual disability and tall stature should be evaluated for suspected overgrowth syndrome.JM conceptualized, prepared, and wrote the manuscript and made the tables and figures. SL reviewed, edited, and revised the manuscript and provided the patients' figures.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.


Through experimental and theoretical results, it is discovered that the outstanding performance of V8O20·nH2O is ascribed to the Mn2+/Mn3+‐induced high electrical conductivity and Na+‐induced fast migration of Zn2+. Other cathode materials derived from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are explored to confirm the unique advantages of transition metal ions. With an increase in Mn content in NaV8O20, V8O20 ·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles, which may open new opportunities for the development of high‐performance aqueous ZIBs.Rechargeable aqueous zinc‐ion batteries (ZIBs) show promise for use in energy storage. However, the development of ZIBs has been plagued by the limited cathode candidates, which usually show low capacity or poor cycling performance. Here, a reversible Zn//V 8O20 is synthesized via a one‐step hydrothermal reaction. The Mott–Schottky plots, Tafel curves and calculation results are given to explain the improved cycling performance of Mn‐doped NaV8O20. Other cathode materials derived from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are explored to confirm the unique advantages of transition metal ions in NaV8O20.Mn‐doped NaV However, the toxicity and flammability of organic electrolytes cause environmental problems and safety issues. Reliable strategies such as the utilization of solid‐state electrolytes or aqueous electrolytes seem to overcome these dilemma. Aqueous rechargeable batteries are competitive alternatives because aqueous electrolytes are characterized by lower cost, higher safety, greater environmental friendliness, and higher ionic conductivity (up to 1 S cm−1) than organic electrolytes or solid‐state electrolytes. Aqueous Zn‐ion batteries (ZIBs) using metallic Zn as the anode have attracted surging interest due to their high specific capacity of 819 mA h g−1 at a low redox potential (−0.762 V versus the standard hydrogen electrode). Unfortunately, exploiting suitable cathode materials with high specific capacity and stable cycling performance is still an urgent task, and it limits the practical and large‐scale applications of ZIBs.Given the looming energy crisis and environmental pollution, renewable green energy resources such as wind and solar have attracted widespread attention in recent years.3, 4 such as Mn‐based oxides, Prussian blue analogs, transition metal sulfides, and vanadium‐based compounds for aqueous ZIBs. MnO2 displays a high operating voltage around 1.3 V but suffers from the irreversible phase transition and Mn cations dissolution during the cycling. Prussian blue analogs deliver a low capacity of ≈100 mA h g−1 and are hindered by their sluggish kinetics. Layered vanadium‐based oxides have high capacity exceeding 300 mA h g−1 but suffer from structural degradation. Incorporating metal  cations and/or molecular water (H2O) into V2O5 has been proven to be an effective method for solving the poor long‐term cycling performance of vanadium‐based oxides. Structural H2O in bilayer V2O5 reduces the electrostatic interactions within the V2O5 framework, thus effectively promoting Zn2+ diffusion. Metal ion‐preintercalated V2O5 usually expands the interlayer spacing more than V2O5. The expanded interlayer spacing could provide fast channel for Zn2+ diffusion and improve the reaction kinetics, and the chemically connected layers become more robust to alleviate the structural degradation, resulting for a long‐term cycling stability as well as excellent rate capability. Apart from V2O5 derivatives, other layered vanadium‐based oxides, such as NaV3O8 composed of V3O8 layers and inserted sodium ions, delivers a capacity of 380 mA h g−1 at a current density of 0.05 A g−1. However, NaV3O8 gradually dissolved into the ZnSO4 electrolyte and caused a rapid degradation in capacity during cycling. The addition of high concentration Na2SO4 into the electrolyte can suppress the dissolution equilibrium of Na+ from NaV3O8 because of common‐ion effect. Apart from adding electrolyte additives, exploring other strategies for improving the structural stability of these cathodes during cycling is of essential importance in ZIBs. Until now, many metal ions pre‐intercalated strategies have focused on V2O5‐based cathodes. However, the different role of transition metal cations and alkali metal cations in improving the electrochemical performance of vanadium‐based materials is unclear. New compounds intended as cathode materials for ZIBs could be explored by intercalating transition metal ions such as Mn into NaV8O20, forming V8O20·nH2O.Considerable research efforts have been devoted to exploring cathode materials,33 suc8O20 nanobelts as cathode materials for ZIBs, and we show strong experimental and theoretical evidences that the outstanding performance of V8O20·nH2O is ascribed to the Mn2+/Mn3+‐induced high electrical conductivity and Na+‐induced fast migration of Zn2+. The Mn ions stabilize the NaV8O20·nH2O (NVO) structure during the charge/discharge process. As the positive electrode for aqueous ZIBs, NVO delivers a capacity of 140 mA h g−1 and exhibits a capacity retention of 69% after 1000 cycles at 4 A g−1, while V8O20·nH2O delivers a reversible capacity of 146 mA h g−1 and a capacity retention of 88% after 1000 cycles. With an increase in Mn content in NVO, V8O20·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles. Other cathode materials that were derived from V8O20·nH2O by substituting Na with K, Li, and Zn are explored and deliver long‐term cycling stability. Furthermore, derivative cathode materials from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are also explored to confirm the unique advantages of transition metal ions.Here we report a hydrated, monovalent and divalent/trivalent cations co‐pre‐inserted V28O20·nH2O with different amounts of Mn content was obtained by setting the molar ratio of MnSO4 and V2O5 to 1:13, 13:13, and 20:13 during the hydrothermal process. The as‐prepared Mn‐doped NaV8O20·nH2O was denoted as Mn1‐NVO, Mn2‐NVO, and Mn3‐NVO, respectively. Figure 8O20·nH2O with C2/m space group (JCPDS No. 45‐1363). With the intercalation of a small amount of Mn ion in the NVO, the resulting XRD peaks of Mn1‐NVO were in accord with the structure of NVO, suggesting that the intercalated Mn ions would not produce a new crystal structure. It can be speculated that Mn1‐NVO has two possible structures, which are V8O20·nH2O and Na8O20·nH2O. The average analytical formula can be written as V8O20·nH2O or Na0.97O20·nH2O for Mn1‐NVO, as the molar ratio of Na:Mn:V was calculated to be 0.97:0.02:8 based on the electron microprobe analysis (EPMA). The molar ratios of Na:Mn:V for Mn‐doped NVO are shown in Table S1, Supporting Information.The NVO and Mn‐doped NVO were synthesized via a one‐step hydrothermal method. Va = 11.699(6) Å, b = 3.647(8) Å, c = 11.119(2) Å, α = γ = 90°, and β = 104.05°. The refinement data in detail are shown in Tables S2 and S3, Supporting Information. After the Mn ion was doped, Na(1) moved away from their average positions and Mn(1) occupied the Na(1) positions, indicating that Mn1‐NVO had the crystal structure of V8O20·nH2O instead of Na8O20·nH2O , showing a mixture of 83% Mn3+ and 17% Mn2+ for Mn2‐NVO and Mn3‐NVO . Noted that the average valence of V in the synthesized NVO was 4.876. While for the Mn‐doped NVO, the average valence of V in V8O20·nH2O was 4.871, revealing an increased V4+:V5+ ratio in this structure. On the contrary, the average valence of V in Na0.97O20·nH2O was 4.883, suggesting an decreased V4+:V5+ ratio. From the XPS results , the V4+:V5+ ratio of NVO was 0.44:1, and the V4+:V5+ ratios of Mn1‐NVO, Mn2‐NVO, Mn3‐NVO were 0.47:1, 0.5:1, and 0.53:1, respectively. The increased V4+:V5+ ratio confirmed that the Mn ion doped NVO had the formula V8O20·nH2O. With a thermogravimetric‐differential thermal analysis study, the structural water content in Mn1‐NVO was obtained , and the stoichiometric formula for the prepared Mn1‐NVO was then confirmed as V8O20·0.32H2O.To confirm the position of the Mn ions in NVO, Rietveld refinement of Mn1‐NVO was conducted. The chemical composition of the compound was analyzed by inductively coupled plasma optical emission spectroscopy (ICP‐OES) before progress the Rietveld refinement. The molar ratio of Na:Mn:V was 1.1:0.03:8. Based on the Rietveld refinement results in Figure O Figure .34]34a = 2+ insertion/extraction in aqueous ZIBs.The scanning electron microscope (SEM) image in Figure ous ZIBs.2m aqueous Zn(CF3SO3)2 electrolyte, the electrochemical performance of the Mn1‐NVO nanobelts as the positive electrode were investigated. The Mn1‐NVO delivers a high capacity of 363 mA h g−1 at a current density of 0.1 A g−1  curves of Mn1‐NVO captured at a scan rate of 0.1 mV s−1 are shown in Figure 2+ insertion/extraction could be inferred, which is quite different from the reported V2O5 based cathodes that have two pairs of redox peaks. Some peaks disappear at a faster scan rate of 0.5 mV s−1, leading to one obvious cathode peak at 0.5 V and two anodic peaks at around 0.65 and 1.05 V. Figure b in the range of 0.5–1 represents the slope of the log(i) versus log(v) curve. Ionic diffusion controls the reaction process when the value of b is 0.5, and pseudocapacitance dominates the electrochemical process completely when the value of b is 1. The calculated b values of peaks 1, 2, and 3 are 0.69, 0.54, and 0.76, respectively . In addition, electrochemical impedance spectrum analysis of Mn1‐NVO and NVO batteries after 20 cycles was carried out . It shows lower charge‐transfer resistance of the Mn1‐NVO than the NVO (Rct 4.8 ohms for Mn1‐NVO versus 14 ohms for NVO) after 20 cycles, which suggests a higher surface electron mobility and improved electrochemical activity for Mn1‐NVO.Using a Zn negative electrode and Ti foil (20 µm) as the positive current collector in 3 1 Figure 2a. With 1 Figure . The speox peaks.23 Somy Figure , meaning2+ diffusion at the Mn1‐NVO and NVO electrodes were studied via the Galvanostatic Intermittent Titration Technique (GITT) measurement, and the corresponding Zn2+ diffusion coefficients for the Mn1‐NVO and NVO electrodes in the third cycle are illustrated in Figure 2+ diffusion coefficients in Mn1‐NVO ranged from 1.9 × 10−9 to 2.5 × 10−10 cm2 s−1, while the DZn2+ for NVO ranged from 1.2 × 10−10 to 7.0 × 10−10 cm2 s−1 upon discharge. Interestingly, the Zn2+‐diffusion coefficient in Mn2‐NVO and Mn3‐NVO  were only slightly reduced with an increase in Mn content . The high Zn2+ diffusion coefficient may have been responsible for the improved battery performance of Mn1‐NVO. Figure S9, Supporting Information shows Ragone plot of Zn//Mn1‐NVO and Zn//NVO battery. Mn1‐NVO and NVO electrodes achieve an energy density of 249 and 239 Wh kg−1 at 100 mA g−1, respectively. Even though the Zn//Mn1‐NVO battery cycled at 1 A g−1, the energy density still reaches up to 157 Wh kg−1 at an outstanding power density of 628 W kg−1, which is higher than that of Zn//NVO battery (144 Wh kg−1at the power density of 598 W kg−1).To determine the roles of introducing Mn, the kinetics of the ZnFigure −1, the diffraction peaks for Mn1‐NVO located at 8.3°, 25.6°, 33.5°, and 43.2° shift to lower angle at 8.2°, 25.3°, 33.8°, and 42.4°, respectively. Particularly, the calculated interlayer spacing of the (001) plane shifts from 10.62 Å to 10.70 Å during the initial Zn2+ insertion process, which is ascribed to the insertion of a certain amount of Zn2+ into the interlayer of Mn1‐NVO, expanding the interlayer spacing. Notably, the interlayer spacing of the (001) plane shifts to 10.46 Å and 10.31 Å upon the following discharge to 0.6 and 0.3 V, indicating that lattice contraction occurs. At the subsequent charging from 0.3 to 0.5 V, the diffraction peaks located at 8.6°, 25.8°, 34.6°, and 43.8° shift to a lower angle at 8.5°, 25.9°, 34.5°, and 43.6°, respectively, suggesting an increase in interlayer space corresponding to the deintercalation of Zn2+. When first charged to 1.25 V, the interlayer spacing of the (001) plane increased from 10.31 Å to 10.54 Å. The fully charged electrode at 1.25 V after 400 cycles showed the same XRD pattern as the electrode cycled at the tenth cycle , suggesting that a reversible phase transition occurred during long‐term cycling.To investigate the structural evolution and reaction mechanisms of the Mn1‐NVO, ex situ XRD was performed during the first discharge/charge process. As shown in −1 is ascribed to V‐O‐Zn vibrations in Mn1‐NVO upon discharge, and the Raman shifts at 253, 310 and 371 cm−1 are assigned to the Zn‐OH vibrations, implying the presence of Zn(OH)2 or Znx(OTf)y(OH)2x−y·nH2O upon fully discharge. The OH− in the compound is generated from the decomposition of water, thus accompany with the formation of H+, and H+ may intercalate in the Mn1‐NVO electrode to maintain a neutral charge state. Therefore, the reaction mechanism can be inferred that the H+ is co‐intercalated with Zn2+ into Mn1‐NVO upon discharge from the presence of the above precipitates. However, the low crystallinity and small quantity of the precipitates may be the reason why XRD cannot detect the phase. As shown in the enlarged XRD patterns in Figure S12, Supporting Information, there are no extra signals besides peaks of Mn1‐NVO, suggesting that the main structure is maintained during the charging/discharging process. The lowering of the relative intensity and the broadening of the XRD peaks during the discharge was mainly due to the formation of Zn(OH)2 and Znx(OTf)y(OH)2x−y·nH2O precipitates and the insertion of Zn2+ into the Mn1‐NVO. In addition, the peak associated with V—O—Zn bond and the characteristic Raman peaks of precipitates disappear at the fully charged state, and the spectra is similar with the pristine electrode, suggesting there is no Zn complex formed upon charge. Upon fully charged state, the recovery of the XRD peak was due to the extraction of Zn2+ and H+ from the Mn1‐NVO cathode, which is in accord with the previous studies. Noted that the energy storage mechanism of Mn1‐NVO involves mainly Zn2+ (de)intercalation with minor H+ (de)intercalation, which is in accord with the previous report. For Zn anodes, the Raman shifts at the fully charged/discharged state are similar with the pristine Zn anodes, and the peak associated with O‐Zn bond is stable during the cycling, suggesting there is no Zn complex formed upon charge.To confirm whether there is new phases formation upon discharge/charge, Raman spectra were provided to further show structural information on the Mn1‐NVO electrode at the fully discharged/charged states. As shown in Figure S11, Supporting Information, the strong Raman shift at around 846 cm2+ in the Mn1‐NVO electrode was further demonstrated by ex situ high‐resolution XPS analysis. Characteristic peaks at 517.5 and 516.2 eV in Figure S3b, Supporting Information showed the V5+ and V4+ for Mn1‐NVO. As shown in Figure 5+ become weak, while the peaks for V4+ and V3+ increase during the intercalation of Zn2+, indicating the electrochemical reduction of V cations in the V8O20 framework during the first discharge. Upon charging in Figure 5+ increases as V cations are oxidized to +5, and the molar ratio of V5+:V4+ recovers to that of the pristine V spectrum . Meanwhile, the Zn 2p peaks emerge at the discharge state of 0.3 V 2 or Znx(OTf)y(OH)2x−y·nH2O) formed on the cathode upon the fully discharge state. The Zn 2p signals located at 1022 eV at fully discharged state was caused by the intercalated Zn and the zinc hydroxide triflate precipitation. Upon charging, the intensity of the Zn peaks remarkably decreases  and absorbed Zn features remain . As a result, the NVO crystal structure consists of a small amount of lattice‐trapped Zn after the first cycle, which might be the reason for the rapidly declining capacity in the second cycle. Mn ions were detected by XPS in the fully discharged and charged states both in the first cycle and the 400th cycle , meaning Mn ions keep stable in the crystal structure during the cycling. The Mn ions existing in the NVO crystal structure can act as pillar and stabilize the structure during the long‐term cycling. Furthermore, the Mn ions can change the electronic structure by strengthening the chemical bonds, thus causing more stable cycling performance than that in NVO. To confirm the electronic structure change of Mn1‐NVO, conductivity of the samples was carried out. The Mn1‐NVO exhibits higher electrical conductivity than NVO . To show the role of Mn ion and Na ion in Mn1‐NVO more clearly, the theoretical calculations of density of states and Zn2+ diffusion pathways for the NaV8O20, V8O20, MnV8O20 (MnVO) are provided in Figure 2+ (de)intercalation, thus contributes to the excellent performance of Mn‐doped NVO. Possible migration pathways for Zn2+ in Mn1‐NVO is shown in Figure 2+ mainly diffuses along b axis. Energy barriers along Zn2+‐migration pathways for NVO and Mn1‐NVO is lower than that of MnVO  images in Figure  Upon charging, the lattice spacing increased to 0.21 nm with Zn2+ extraction, which was in accord with the ex situ XRD results.To gain better insight into the electrochemical process during charging/discharging, the morphologies after the first discharge and charge were observed by TEM. The nanobelts was maintained during the cycling, and the element distributions of V, O, Na, Mn, and Zn was collected by EDS mapping with TEM Figure 4a,b. Theanalysis.45 The4, CoSO4, NiSO4, CaSO4 and K2SO4, were used as raw materials to replace MnSO4, producing V8O20, V8O20, V8O20, V8O20, and V8O20 , respectively. The XRD results in Figure −1 was investigated. An initial decrease in capacity was observed for these cathodes, which might be attributable to the slight surface dissolution of materials and the irreversible reactions during the first few cycles. Taking Fe1‐NVO as an example . To further understand the enhanced cycle stability of NVO via transition metal insertion, Mott–Schottky plots and Tafel curves are shown in Figure  Furthermore, there is a decreasing trend in the absolute value of the slope for transition metal‐doped NVO compared with pristine NVO, Ca1‐NVO, and K1‐NVO V8O20·nH2O by substituting Na with Li, K, and Zn  were also explored to confirm the catalytic effect of Mn ion. As shown in Figure S19a,b, Supporting Information, the crystal structures of these derivates were similar to that of Mn1‐NVO, and these cathodes exhibited more outstanding cycling performances compared with that of NVO.To explore the reasons why the Mn ions stabilized the NVO structure, other metal sulfates, such as FeSOe Figure , a dischn Figure 40 FurFigure 0.33V2O5 appeared in the Mn3‐NVO. Interestingly, Mn2‐NVO as well as the mixture of V8O20 and Na0.33V2O5 as cathodes exhibited more outstanding cycling performances than Mn1‐NVO  lattice spacing, that is, the lattice spacing is 10.46 Å in Mn2‐NVO and 10.62 Å in Mn3‐NVO, respectively. Thus, the combined effects of proper lattice spacing, as well as the stable crystal structure are key factors that determine electrochemical performance. In addition, the capacitive contribution of Mn2‐NVO was calculated to be 37.2%, 45.5%, 50.6%, 56.9%, and 62.6% at the scan rate of 0.1, 0.2, 0.3, 0.5, and 0.8 mV s−1, respectively, which is higher than that of Mn1‐NVO or NVO . The capacitive contribution increased with the increase in Mn content in NVO, and this was also responsible for the excellent cycling performance of the Mn2‐NVO and Mn3‐NVO batteries.To further improve the cycling performance of the Zn//Mn1‐NVO battery, the content of Mn in NVO was carefully investigated. With an increase in Mn content in NVO , we found that the (003) plane disappear Figure 6a. AnothO Figure . ObviousO Figure . From th3O8 in the ZnSO4 electrolyte and the formation of Zn dendrites caused rapid capacity fading, we carefully studied the dissolution of NVO and Mn‐doped NVO electrodes. To confirm the dissolution of NVO and Mn‐doped NVO, the NVO and Mn1‐NVO electrodes were dipped in the 1 m ZnSO4 electrolyte for 12 h. ICP‐OES results confirmed the dissolution of NVO and Mn1‐NVO . Furthermore, the dissolution of Mn1‐NVO could be suppressed via the addition of 2 m Na2SO4 to the 1 m ZnSO4, and therefore the capacity retention of Zn//Mn1‐NVO is superior to that of the case without Na2SO4 , which was in accord with the previous report. Noted that a high level of Na2SO4 additive in the electrolyte decreases the energy density of the batteries when they are used in practical applications. As shown in Figure S21, Supporting Information, increasing the concentration of Na2SO4 can suppress the dissolution of Mn1‐NVO and show high capacity retention, but the capacity of Mn1‐NVO was decreased. The dissolution of Mn1‐NVO in 1, 2, and 3 m ZnSO4 electrolytes was also carried out. As shown in Figure S22a, Supporting Information, a high concentration of electrolytes can delay the dissolution process. More importantly, it was noted that the dissolution of Mn‐NVO in ZnSO4 electrolytes was suppressed with the increase in Mn content in NVO, which is likely attributed to the increased structural stability. The increasing Mn in NVO causes the electrolyte colors to become faint, as shown in Figure S22b, Supporting Information. Interestingly, the dissolution of Mn1‐LVO, Mn1‐KVO, and Mn1‐ZVO in 1 m aqueous ZnSO4 was slower than that of Mn1‐NVO , suggesting that Na ions play a significant role in the dissolution process. Furthermore, different kinds of doping transition metals also affect the dissolution process of NVO electrodes , as Fe1‐NVO and Ni1‐NVO have a slower dissolution rate than Mn1‐NVO or Co1‐NVO. The dissolution of Mn1‐NVO in 3 m aqueous Zn(CF3SO3)2 was also carried out to show the advantage of Zn(CF3SO3)2 electrolyte for Mn1‐NVO . As shown in Figure S23, Supporting Information, a large number of vertical and harsh Zn dendrites formed on the surface of Zn negative electrode in the ZnSO4 electrolyte after 500 cycles at 4 A g−1, while no obvious Zn dendrites in 3M Zn(CF3SO3)2 is observed. Therefore, compared with ZnSO4 electrolyte, 3M Zn(CF3SO3)2 can not only suppress dissolution of Mn1‐NVO, but also suppress the formation of vertical and harsh Zn dendrites during the cycling.Since the dissolution of NaVy fading,27 we 38O20 framework. The Mn1‐NVO used as a cathode delivered a high capacity retention of 88% after 1000 cycles at a high current density of 4 A g−1, which was much better than NVO, Ca1‐NVO, or K1‐NVO. With an increase in Mn content in NVO, V8O20 ·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles. Importantly, the dissolution of Mn‐doped NVO was suppressed with an increase in the Mn ion content, providing another way to stabilize the cathodes in the electrolyte. When combined with improved ion diffusion kinetics, charge carrier concentration, and catalysis of transition metal ions, the electrochemical performance of NVO‐based cathodes significantly improved. This strategy may pave the way for the potential application of doping in cathode materials and expand the choice of cathodes for energy storage devices.In summary, we reported a series of transition metal‐doped NVO, in which transition metal ions were intercalated in the interlayers of V44.14.1.14.1.1.12O5, 1.99 g of Na2SO4, and 0.676 g of MnSO4·H2O were dispersed in 50 mL of DI H2O, then CH3OOH was used to adjust pH to 3. After that, the solution was transferred into a 100 mL Teflon‐lined stainless‐steel autoclave. The sealed autoclave was heated to 180 °C for 72 h. Finally, the Mn1‐NVO sample was collected by centrifugation and washed with water and ethanol for three times. The sample was dried at 60 °C in an electric oven for 6 h, then further dried at 60 °C in a vacuum oven for 12 h. NVO was synthesized with the same procedure and processing conditions without MnSO4 source.The Mn1‐NVO nanobelts were prepared by one step hydrothermal method. Briefly, 4.73 g of V4.1.1.2α radiation to determine the phase of samples. The morphologies were characterized by SEM (S‐4800) and TEM (JEM‐2100F). XPS (ESCALAB 250) with Al Kα X‐ray radiation was carried out to measure the oxidation states of the elements. The structural water was detected by the thermogravimetric analysis (LABSYS EVO) under nitrogen atmosphere with a rate of 10 °C min−1 from room temperature to 800 °C and holding at 800 °C for 30 min and then cooling naturally to room temperature. ICP (iCAP Q) performed to analyze the elementals ratio.XRD was performed using a SmartLab‐3KW with Cu K4.1.1.3−2. Zinc foil as the anode and glass fiber membrane was used as the separator. LAND battery testing system (CT2001A) was used to evaluate the electrochemical performance of the assembled cells with a voltage range from 0.3 to 1.25 V versus Zn/Zn2+. CV, Tafel plot, and Mott–Schottky plots were collected by using a CHI 600E electrochemical workstation.The electrodes were assembled into coin cells (CR2032) to investigate the electrochemical performance. Typically, the active material, acetylene black and polyvinylidene fluoride with a mass ratio of 7:1.5:1.5 were fully mixed in N‐methyl‐2‐pyrrolidone solvent for the electrode preparation. Then the slurry was pasted on a Ti foil and dried in a vacuum oven at 60 °C for 12 h. The active material mass loading was around 2 mg cm6The authors declare no conflict of interest.Supporting InformationClick here for additional data file.