Murrah breed of buffalo is an excellent dairy germplasm known for its superior milk quality in terms of milk fat and solids-not-fat (SNF); however, it is often reported that Indian buffaloes had lower lactation and fertility potential compared to the non-native cattle of the country. Recent techniques, particularly the genome-wide association studies (GWAS), to identify genomic variations associated with lactation and fertility traits offer prospects for systematic improvement of buffalo. DNA samples were sequenced using the double-digestion restriction-associated DNA (RAD) tag genotyping-by-sequencing. The bioinformatics pipeline was standardized to call the variants, and single-nucleotide polymorphisms (SNPs) qualifying the stringent quality check measures were retained for GWAS. Over 38,000 SNPs were used to perform GWAS on the first two principal components of test-day records of milk yields, fat percentages, and SNF percentages, separately. GWAS was also performed on 305 days’ milk yield; lactation persistency was estimated through the rate of decline after attaining the peak yield method, along with three other standard methods; and breeding efficiency, post-partum breeding interval, and age at sexual maturity were considered fertility traits. Significant association of SNPs was observed for the first principal component, explaining the maximum proportion of variation in milk yield. Furthermore, some potential genomic regions were identified to have a potential role in regulating milk yield and fertility in Murrah. Identification of such genomic regions shall help in carrying out an early selection of high-yielding persistent Murrah buffaloes and, in the long run, would be helpful in shaping their future genetic improvement programs. Bubalus bubalis) is an imperative livestock species and act as a key component for improving agricultural economy and supplying milk, meat, and draft power. The buffalo population across the world was recently estimated to be 194 million, 97% of which were present in Asia  in buffaloes for lactation traits have been mainly limited to the use of bovine single-nucleotide polymorphism (SNP) chip  and AffyHence, the present study was conducted to identify novel SNPs associated with milk production, composition, lactation persistency, and fertility traits at the genomic level using the genotype-by-sequencing technique in Murrah buffalo, India’s major buffalo breed and milch animal of the nation.A total of 672 test-day records on each trait, i.e., milk yield (TDMY), fat percentage (TDFP), and solids-not-fat (SNF) percentage (TDSNF) were collected from 96 female Murrah buffaloes reared at LRC, NDRI, Karnal, India (29.68°N and 76.99°E). Records of 96 buffaloes on 305 days’ milk yield (305DMY), birth weight (bwt), age at first calving (AFC), calving interval (CI), and age at sexual maturity (ASM) in months were collected. Other traits such as lactation persistency, postpartum breeding interval in days (PPBI), and breeding efficiency (BE) were derived from the primary phenotype records. Breeding efficiency was calculated for female buffaloes as described by Lactation persistency was estimated by following four different methods:(I)(II)(III)(IV)SphI and MluCI restriction enzymes. Adapters (P1 and P2) were prepared as per standard Illumina read multiplexing protocol using an inline barcode along with Illumina index for library preparation. After adapter ligation and size selection, samples were sequenced on a Illumina Hi-Seq 2000 platform.DNA was isolated from 96 Murrah buffaloes selected for the study following the standard phenol-chloroform method . SamplesGCF_003121395.1, was retrieved from the NCBI dataset and used as a reference genome. Index and sequence dictionary files were created using the Burrows–Wheeler algorithm (BWA)   and Picag FastQC , and eacg FastQC . Adapterg FastQC .3 The BWqualimap . Variantqualimap .p < 0.0001 were removed from the dataset. SNPs in LD with r2 > 0.8 were also removed. Only autosomal and X chromosome SNPs were retained for the final analysis. All the quality control operations and data preprocessing were performed using PLINK v1.9  of TDMYs, TDFPs, TDSNFs, and 305DMY; lactation persistency calculated by four different methods; age at sexual maturity in months; postpartum breeding interval (in days); and breeding efficiency. Genome-wide identity-by-state (IBS) for all pairs of individuals was checked. Multidimensional scaling (MDS) based on SNP information was done to check for the presence of any population stratification  at 5 and 10% levels ( ≤ (i/m) × q, where i = rank of the SNPs, m = no. of individual tests performed, and q = either 0.05 or 0.1  instead of direct traits. Principal component analysis (PCA) was performed in the R programming environment (v4.0.3) on the 672 test-day records of each trait separately. As the first two PCs cumulatively account for most of the variation in the dataset, they were selected to be GWAS traits. The traits on which GWAS were performed were PCs . After quality check of raw data, it was observed that the average GC content was 50.54%. The rate of duplication was quite high, i.e., 80.11%, which was later checked during variant calling. The quality score (Q) throughout the dataset varied from 35 to 40.Raw files that were quality checked in FastQC were combined using MultiQC, and the report is provided as 1 and PC2) of TDMY, TDFP, and TDSNF.GWAS were performed with 38,560 SNPs, and the genome-wide significant threshold was set at the 5% FDR level. GWAS were performed on the two foremost variation explaining PCs , 9 (62699319 bp), and 16 (74362861 bp) were significantly associated with PC1 and PC2 of the test day’s milk yield were also in the top SNP list for 305DMY. The Manhattan and Q-Q plots for association results of 305DMY are given in GWAS on 305DMY revealed that there was no significant association between SNPs and the trait. It was observed that five SNPs that appeared in the list with GWAS for PCp-values along with their genomic position and genes within a ± 20-kb region are listed in GWAS on lactation persistency estimated by four different methods revealed no significant association between SNPs and the trait generated. The top 10 SNPs having the lowest p-values and genes within a ± 20-kb region are listed in Upon performing GWAS on age at sexual maturity, postpartum breeding interval, and breeding efficiency, no significant association of any SNPs were observed for the traits; however, the top 10 SNPs with the lowest The test-day model (TDM) as repeated measurements of milk yield traits is the method of choice for predicting 305 days’ milk yield. TDMs account for environmental effects on each test day and is useful to model individual lactation curves . PCA beiGRIA3, was reported by It is important to understand the reasons for considering 96 samples taken in the study as optimum, as in the buffalo farming scenario in Asia, particularly in India, the maximum number of buffaloes maintained at any large organized herd ranges from 250 to 500, with 100–200 breedable buffaloes having complete phenotype information. The National Dairy Research Institute has the second-largest herd in the Indian Council of Agricultural Research (ICAR) with a well-managed herd of 250 breedable buffaloes. Furthermore, there is no buffalo sequencing consortium/project in operation (India). In such a case, one could afford this sample size to genotype with complete pedigree and with sufficient genetic diversity. However, the sample size is less for conducting GWAS and raises the question of whether the results are reliable enough. It was observed that the results obtained under the present study are encouraging and important, as one of the genes identified in the present study, i.e., To identify novel SNPs associated with various economic traits, sequencing through the ddRAD approach was performed. LOC100847171, BCL6, RTP2, SST, PTGS2, LOC100295047, and LOC101908004 are associated with milk production; KCTD8, LOC782855, LOC101904777, ESRRG, TRNAY-AUA, and GPATCH2 are associated with fat percentage; LOC101903483, SART3, ISCU, CMKLR1, WSCD2, MFNG, CARD10, and USP18 are associated with protein percentage. MFSD14A, SLC35A3, PALMD, RGS22, and VPS13B, for milk production in the Italian Mediterranean buffalo through GWAS. In another GWAS in buffalo, D-glucose level in the blood affect milk production in buffalo.An earlier bovine SNP chip was used to study the traits of buffalo. Using a bovine SNP chip (Illumina BovineSNP50 BeadChip) for a GWAS in buffalo population, 1 of TDMY at the 5% FDR level. However, six SNPs present on chromosomes 1, 6, and 9 at 182,059,836, 35,648,660, and 48,559,942 bp, respectively, were above the suggestive threshold of 10% FDR and are presented in 1 of TDMY. However, for the rest of the traits studied, no SNPs were detected to be significantly associated (or as rejections) at 5% or even 10% FDR.In the present study, GWAS performed on the milk yield PCs explaining maximum variation, which revealed that three SNPs were significantly associated with PCGRIA3 gene was found. Glutamate ionotropic receptor AMPA type subunit 3 (GRIA3) is reported to be very significantly associated with daughter pregnancy rate in US Holstein cows  glutamate receptors, thus regulating trafficking of AMPA receptors. AMPA receptors are known to participate in luteinizing hormone (LH) secretion  pathways involved in mammary gland development. In the present study, lactation persistency was calculated using four different methods, and it was observed that the top four SNPs identified by GWAS for persistency as estimated by the DENND3, PSTPIP1, ADAMTS20, PRODH2, NPHS1, RREL2, FAM19A1, CLIC5, GPR12, DEFB125, STPG2, CFAP206, RINT1, EFCAB10, TCERG1, and C18H16orf78. Upon network enrichment, it was observed that TCERG1, RINT1, CLIC5, and PSTPIP1 are co-expressed with several other genes in the breast mammary tissue of human, indicating their possible role in persistency of lactation in dairy animals.In Canadian Holstein cattle, APC, LOC112448352, NDFIP2, LOC107131404, and PDP1. APC gene in the canonical WNT signaling pathway plays a critical role in the regulation of ovarian development. Mis-regulation of this key pathway in the adult ovary is associated with subfertility in mice. The APC gene has also been mapped to the QTL region for conception rate in Holstein cattle . The GRID2 gene is however reported to be associated with growth traits in Simmental cattle and may have role in regulating age at sexual maturity , the results obtained in the present study are bias free, as indicated by the FDR. The findings of the present study in the Murrah population will encourage researchers to come forward for GWAS in buffalo in a holistic manner.Optimum production and reproduction in buffaloes are long-standing questions in the Indian subcontinent. We present the analysis and identification of genomic regions that play role in shaping the selection and breeding decisions of Murrah buffalo for persistency of production and fertility. The dataset information presented in the paper is also submitted so that it can be used to compare and evaluate other breeds of buffalo based on the genomic information generated. The putative identified regions have a potential to improve the existing breeding decisions in this important dairy germplasm.PRJEB47270 (https://wwwdev.ebi.ac.uk/eva/?eva-study=PRJEB47270).The data presented in the study are deposited in the European Variation Archive repository, accession number The animal study was reviewed and approved by the ICAR-National Dairy Research Institute IAEC.VV conceptualized the work and interpreted the data. SC, GG, RA, and AM performed the collection of materials, and performed the data analysis. AV and SD provided the data and resources. VV and SC did writing of the manuscript and its critical evaluation. All authors contributed to the article and approved the submitted version.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.


Electrocatalytic water splitting using renewable energy is widely considered as a clean and sustainable way to produce hydrogen as an ideal energy fuel for the future. Electrocatalysts are indispensable elements for large‐scale water electrolysis, which can efficiently accelerate electrochemical reactions occurring at both ends. Benefitting from high specific surface area, well‐defined void space, and tunable chemical compositions, hollow nanostructures can be applied as promising candidates of direct electrocatalysts or supports for loading internal or external electrocatalysts. Herein, some recent progress in the structural design of micro‐/nanostructured hollow materials as advanced electrocatalysts for water splitting is summarized. First, the design principles and corresponding strategies toward highly effective hollow electrocatalysts for oxygen/hydrogen evolution reactions are highlighted. Afterward, an overview of current reports about hollow electrocatalysts with diverse architectural designs and functionalities is given, including direct hollow electrocatalysts with single‐shelled, multi‐shelled, or open features and heterostructured electrocatalysts based on hollow hosts. Finally, some future research directions of hollow electrocatalysts for water splitting are discussed based on personal perspectives. Hollow nanostructures with diverse architectural features and functionalities have attracted considerable interest as promising electrocatalysts for water splitting. Herein, some recent progress in the design of micro‐/nanostructured hollow structures for electrocatalytic water splitting and beyond is summarized. Special focus is taken on the direct hollow electrocatalysts with single‐shelled, multi‐shelled, or open features and heterostructured electrocatalysts based on hollow hosts.  Water electrolysis is considered as one of the promising and zero‐carbon‐emitting strategies for hydrogen production compared to the traditional steam reforming or coal gasification based on the reactions between fossil fuel and steam. However, it remains challenging to achieve highly efficient production of hydrogen at industrial scale using renewable energy. A crucial step for realizing this goal is the development of cost‐effective electrocatalysts to accelerate the sluggish kinetics of hydrogen evolution reaction (HER) at the cathode and oxygen evolution reaction (OER) at the anode. Currently, noble metals and their compounds are state‐of‐the‐art electrocatalysts for water‐splitting reactions at both ends . Nevertheless, the widespread use of these electrocatalysts still suffers from scarce resource, prohibitive cost of noble metal, and lack of long‐term stability.Along with the depletion of conventional fossil fuels and global warming issue because of the increasing carbon dioxide emissions, major concerns about the energy future have triggered the research community to search a clean and sustainable fuel‐combustion technology from fossil‐free pathways.4, 5 One is to increase the apparent activity of total electrode via structural regulation. The other type is to improve the intrinsic activity of each active site through compositional engineering. Particularly, hollow nanostructures could simultaneously integrate the above approaches to maximize their benefits. To be specific, the high surface area of hollow nanostructures provides plenty of accessible active sites. Compared to other nanostructured electrocatalysts with high surface area, the hollow configurations demonstrate strong confinement effects. To be specific, hollow‐structured electrocatalysts with porous shells show significant improvement in protecting particles from migration and aggregation. More importantly, tunable mass transport during reactions could be achieved by regulating the shell structure. Tunable chemical compositions of hollow materials enable the optimization of binding and desorption of reaction intermediates. Moreover, hollow nanostructures can largely reduce the usage of expensive noble metals. According to the function in water splitting, hollow nanostructures could serve as direct electrocatalysts or hosts for loading active materials. For the former type, hollow materials are the direct active species to catalyze water splitting at their surface. As for the latter one, active components are loaded onto the pores/channels, outer/inner surfaces, interior cavities or encapsulated in the frame of hollow hosts to form heterostructured electrocatalysts. Hollow materials are employed as the conductive network, surface modifier, or structural stabilizer for enhancing the performances of active species.Enormous efforts have been made to rationalize electrocatalyst performances at relatively low cost. Generally speaking, the strategies for improving the activity of electrocatalysts can be classified into two types.21 One Usually, material selections for these two reactions are not the same. The atomic‐level descriptor of two‐electron HER is hydrogen binding energy over the full range of pH conditions. Whereas, the electrocatalytic activities of OER electrocatalysts depend largely on the stabilities of adsorbed intermediates  on the surface of catalysts and evaluated by the descriptor of free energy difference, i.e., ΔGO* – ΔGOH*. In addition, the architecture optimization criterions for hollow structured electrocatalysts and support materials are quite different. As for direct catalysts, maximization of exposed electrocatalytic active sites is the priority. Therefore, structural evolution of building blocks from 0D nanoparticles to 1D or 2D high‐energy facets plays a vital role to increase the number of reactive sites. Construction of complex hollow nanostructures with multiple shells or complex internal voids could be helpful for direct catalysts to enhance the loading of active species per unit area. Beyond that, the employment of hollow structures with open features could effectively facilitate the charge and mass transfer. As for the host structures, modulation approaches focus on the integration of electroactive components into hollow supports. Conductive hollow hosts could not only improve the electronic conductivity of electrocatalysts, but also prevent the particle aggregation during electrocatalytic reactions. Last but not the least, simplification of synthetic route is a crucial issue for the wide application of hollow structured electrocatalysts. Generally speaking, the preparation procedure for the direct electrocatalysts is much more convenient than that for the heterostructured electrocatalysts. For the typical synthesis of hollow structures, hard/soft‐templating methods are usually involved, where the template acts as a physical scaffold for the target material coating and is removed subsequently to form void space. Notably, self‐templated strategies are gaining rapidly increasing attention, which could directly convert reactant into hollow structured product without extra template removal process. Based on the formation mechanism of internal voids, self‐templated strategies can be divided into controlled etching, outward diffusion, and heterogenous contraction. The rapid development of template‐involved strategies from hard/soft templating to self‐templated methods has motivated the evolution and optimization of hollow catalysts.Depending on the reactions in water splitting and functions, design principles of advanced hollow electrocatalysts are quite distinctive. The compositional manipulation for HER and OER catalysts is based on the binding energies of electrocatalyst surface with reactive intermediates.32 Usu In this perspective, we summarize some recent progress in the development of hollow catalysts for electrocatalytic water splitting. Special emphasis will be placed on the structural/compositional evolution of hollow nanostructures as direct electrocatalysts and hosts. Particularly, the architectures for direct catalysts are categorized into hollow materials with single‐shelled, multi‐shelled, and open features. Host materials are categorized into heterostructured electrocatalysts loaded on hollow hosts and integrated hollow nanoreactor. In the final part, we provide a brief discussion about both the challenges and directions toward the future design of hollow electrocatalysts for water electrolysis.Several review and perspective articles have critical discussions of the design strategies and syntheses of hollow structured catalysts for various catalytic applications.47 In 2Figure As illustrated in 2.12.1.1 The employment of hollow structures in electrocatalytic hydrogen production could effectively improve the apparent activity of catalysts and reduce the usage of noble Pt metal. As a typical example, nickel phosphide (Ni2P) hollow spheres synthesized from a thermal decomposition process have been reported by Schaak and co‐workers for HER. One‐pot pyrolysis reaction is applied to transform nickel acetylacetonate into Ni2P without additional templates. Transmission electron microscope (TEM) image clearly demonstrates the well‐defined internal void within these Ni2P quasi‐spherical nanoparticles  of 130 and 180 mV to reach the cathodic current densities (j) of 20 and 100 mA cm–2  image shows that the tubular‐like β‐Mo2C structures are built from ultrathin nanosheets  to reach the j of 20 mA cm–2 in acid  hollow structures with tailorable morphologies using water‐soluble precursors. Through a fast self‐templated precipitation reaction, a single‐shelled hollow structure of M‐MoS4 sample with a smooth surface is obtained  have been obtained via the solvothermal reaction between nickel‐cobalt Prussian‐blue analog (Ni‐Co‐PBA) and (NH4)2MoS4. This template‐engaged reaction changes the solid Ni‐Co‐PBA nanocubes into hierarchical nanoboxes assembled by ultrathin Ni‐Co‐MoS2 nanosheets  based oxides with variable valence state have been widely investigated as candidates for OER. For instance, Qiao and co‐workers have reported the design of hollow Co3O4 microtube array (MTA) with hierarchical macro‐/mesoporosity as OER catalysts. Cobalt hydrogen phosphate (CoHPO4) microwires grown on Ni foam are selected as the starting templates. The obtained Co3O4 sample from the potentiostat treatment inherits 1D morphology of the CoHPO4 template hydroxides/phosphides are also considered as possible OER electrocatalysts. Yan's group has done a pioneering work about the preparation of α‐Ni(OH)2 hollow spheres for efficient OER catalysis from the template‐free strategy. As shown in Figure α‐Ni(OH)2 spheres are composed of interconnected nanosheets. Cyclic voltammetry (CV) curves indicate the activation of α‐Ni(OH)2 into electroactive species γ‐NiOOH from the 1st cycle to 100th cycle 2 hollow spheres over β‐Ni(OH)2 solid nanoplates and nanoparticles  assembled by metal ions/clusters with organic ligands have been regarded as ideal templates for multi‐shelled hollow structures. Lou and co‐workers reported a simultaneous etching and coprecipitation method to obtain Ni‐Fe LDH nanocages with different shells  particles are selected as the sacrificial templates. By regulating the ratio of ethanol to water in the mixed solution, the Ni‐Fe LDH shell number can be delicately controlled. Concretely, more water could lead to the fast etching of MIL‐88A, thus forming single‐shelled nanocages (SSNCs), while more ethanol is responsible for constructing double‐shelled nanocages (DSNCs) due to the slow etching rate. Energy‐dispersive X‐ray (EDX) spectrum of Ni‐Fe LDH DSNCs reveals Ni and O elements concentrate in the inner layer while Fe element is in the outer shell, suggesting different distributions of active components throughout the whole DSNCs. Electrochemical measurements demonstrate that Ni‐Fe LDH DSNCs with effective surface area exposure exhibit superior OER activity and double‐layer capacitance (Cdl) to that of single‐shelled one  to close duplicated shells with a narrow spacing (D‐CoP‐HoMSs) and finally turns into a solid one (CoP‐HoMSs). Electrochemical impedance spectroscopy shows that D‐CoP‐HoMSs possess the smallest semicircle in Nyquist plots, suggesting the faster charge‐transport than B‐CoP‐HoMSs and CoP‐HoMSs  feature. By delicately balancing the formation kinetics of Ir and transition metal (Ni and Cu) precursors, a core–shell‐type alloy@alloy structure could be achieved in the one‐pot synthesis  derived from HEAs manifest great potential as promising electrocatalysts, such as FeNiCoCrMnS2 and CoFeNiMnMoPi. It is believed that the combination of hollow structure with HEMs can shed some light on the design of efficient water‐splitting electrocatalysts.As an emerging type of advanced functional materials, high‐entropy alloys (HEAs) are composed of four or more metal elements.64 In 2.3.2 For example, cage‐like open nanostructures of Ni‐Co mixed oxide are derived from Ni‐Co Prussian‐blue‐analog (PBA) nanocages consisting of pyramidal walls. The structural evolution of the Ni‐Co PBA precursors from nanocubes to nanocages can be described as a controlled etching process along the diagonal with the assistance of internal defects  for OER  strategy is developed for the structural evolution. Figure –2, LSV curves indicate the Co‐Fe oxide NAFS sample has faster reaction kinetic compared with Co‐Fe oxide NFs 6Mo7O24 and [Co‐NxCy] units in Co PBA. After subsequent pyrolysis, the resultant Co3O4‐Mo2N nanoframes inherit the functionalities of individual components and demonstrate outstanding activities toward both HER and OER.The construction of frame‐like structure has also demonstrated the superiority in water splitting for nonalloy catalysts, although further understandings of formation mechanism are still needed.67 Fors Figure. By virtR Figure . Startins Figure . Further3 Other than being as the direct electrocatalysts, highly conductive hollow structures could also serve as hosts in heterostructured electrocatalysts to prevent aggregation of active species or introduce new functions, due to their high surface area and low tap density. The uniform distribution and strong binding with conductive substrates enable the electroactive species better stability and reduced resistive loss. These heterostructured electrocatalysts could be divided into two categories based on distinct or vague spatial position of the active species in hollow structures. It should also be noted that the spatial allocation of catalytic components is vital for the interactions between components. In particular, for the heterostructured electrocatalysts with distinct spatial position, the active components are confined into the interior void space, porous shells, or supported on the surfaces of hollow hosts. Interior cavities significantly decrease the loading of active components per unit area, while porous shells guarantee the tunable mass transport of heterostructured electrocatalysts. In other cases, the hollow materials also act as secondary electrocatalysts or promoters to facilitate tandem reactions together with their entrapped active components.Recently, heterostructured materials have exhibited great potential in electrochemical water splitting, which are generally composed of active electrocatalyst and nonactive substrate.71 Oth As illustrated in Figurex nanoparticles are introduced as electrocatalytically active species onto the surface of MWCNTs. X‐ray absorption near edge structure measurements show strong interaction effects of Ni‐Fe‐LDH nanoplates and MWCNTs, indicating the facilitated charge transfer. As a result, the Ni‐Fe‐LDH/CNT hybrid catalyst exhibits smaller η and faster reaction kinetics for OER compared with control samples  using a step‐by‐step self‐assembly strategy. The extended X‐ray absorption fine structure (EXAFS) fitting analysis suggests the presence of atomically dispersed Ni‐N4 moieties in Ni‐N4/GHSs and Fe‐N4 moieties in GHSs/Fe‐N4, which are responsible for OER and oxygen reduction reaction, respectively  value of about 0.79 V, superior to that of Ni‐N4/GHSs and GHSs/Fe‐N4  have been widely used as hosts for various heterostructured electrocatalysts. As a typical example, multi‐walled CNTs (MWCNTs) have been utilized as substrates for the growth of Ni‐Fe LDH nanoplates through a solvothermal treatment.72 As s Figure . Interess Figure .[73]s Figure . Due to s Figure . Apart fy Figure . These J4 Figure . For instance, Lu et al. reported an effective method to generate Ni‐doped FeP/carbon (NFP/C) hollow nanorods with tailorable length and composition based on self‐engaged etching and coordination reactions. Both the carbon and Ni‐doped FeP species are derived from the thermal treatment of the phytic acid‐treated MIL‐88A x shell, which cannot be clearly distinguished  has been reported as one of the promising candidates to replace OER. In a typical example, Figure2S4 yolk–shell spheres (P‐CoNi2S4 YSSs) for both overall water splitting (OWS) and urea‐assisted water splitting to produce hydrogen.Figure 2S4 YSSs with a thin shell composed of ultrafine nanoparticles, which is beneficial for exposing more active sites. X‐ray photoelectron spectroscopy (XPS) results suggest that more electronic interactions have occurred on the P‐CoNi2S4, generating enriched Ni3+ content, which promotes stronger redox reaction and enhances electrocatalytic performance  heterojunction nanospindle using FeOOH as a self‐sacrificial template for both HER and 5‐hydroxymethylfurfural (HMF) electrooxidation reaction (EOR).As verified by HRTEM, the porous structure enables the direct contact between the inner MoO2‐FeP heterojunction with reactants  production  for the desired products. For example, a catalyst composed of core–shell Cu2O/Cu nanoparticles anchored in N‐doped porous carbon (Cu2O/Cu@NC) was reported for electrochemical CO2‐to‐formate conversion, achieving a maximum FE of 70.5% at −0.68 V versus RHE. The high FE can be ascribed to the advanced hollow structure, high N content, and well‐dispersed Cu2O/Cu species.Considering the cost‐effective storage and transport of Hproducts.86 For5Figure Table Electrochemical water splitting serves as a promising pathway to produce hydrogen as a future fuel. The employment of hollow structured electrocatalysts has aroused tremendous interest as an advanced approach for enhanced hydrogen and oxygen production performances, owing to their advantageous features, such as large surface area and reduced mass/charge transfer path. Besides, the tunable shell architecture and internal construction bring great opportunities in the structural design and composition modification to achieve optimized electrocatalytic activities. Considering different requirements for HER/OER and diverse roles of hollow structures, design principles of advanced hollow electrocatalysts are quite distinctive. In this perspective, we summarize some recent developments in the design of hollow nanostructures as direct electrocatalysts and supporting hosts for electrochemical water splitting and beyond. The schematic summary of the advances of the hollow electrocatalysts toward water splitting and beyond is presented in Despite great advances have been achieved, investigations of hollow structured electrocatalysts for water splitting are still at the proof‐of‐concept level. Based on the research trends of hollow catalysts, herein, we propose several directions to be considered in the near future for new discoveries in this field. 1) In order to meet the actual demand for the industry, economical and scalable synthetic methods should be further developed to prepare hollow electrocatalysts with high activity and durability. 2) From the aspect of structural design, in‐depth understandings of the basic material science and chemical reaction mechanism are still required to identify the complex effects  of hollow structures on the underlying origins for electrochemical water splitting. 3) The role of temporal–spatial ordering in mass transfer, storage, and release in the hollow structured electrocatalysts should be further investigated based on theoretical calculations and experimental data to optimize these unique nanoreactors. 4) From the performance point of view, the in situ/operando high‐resolution characterizations are needed to study the reason of performance degradation for practical utilization of hollow electrocatalysts in electrolyzed water equipment. 5) For innovative strategies for nonconventional water splitting, precise structural and compositional controls are highly demanded to alter the surface chemistry of hollow electrocatalysts for target reactions beyond OER.Given the rapid progress and continuous research activities in this field, we are optimistic that the future design of hollow nanostructures can solve the above challenges to promote their commercial applications.The authors declare no conflict of interest.


Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of  Heavy metals are high-density elements that can cause toxic effects when present in excess quantities. Cadmium (Cd) and mercury (Hg) are two of the most toxic heavy metals to humans as Cd poisoning can cause kidney damage and osteoporosis , while Hvia the roots. High intracellular accumulation of metal ions can lead to the denaturation of proteins, the displacement of essential metals from biomolecules, problems in membrane integrity, and the formation of reactive oxygen species (ROS). Excess metals in leaves can result in chlorosis, disruption of photosynthetic pathways, and breakdown of basic metabolic processes often leading to the death of the plant. Managing heavy metal toxicity by plants requires several genetic loci for integrated transport and tissue detoxification that operate at both the cellular and molecular level. Thus, traits associated with tolerance and accumulation are expected to be polygenic.In plants, heavy metals such as Cd and Hg are non-essential ions that can be taken up from the environment by essential micronutrient transporters  and subsABCC1 and ABCC2 in A. thaliana and poplar, transport chelated Cd and Hg ions into vacuoles (HMA3 (AtCAX2 and AtCAX4 (Oryza sativa), the natural resistance-associated macrophage protein 5  transporter), is the main transporter of Cd into roots through the apoplast  enzymatic pathway . Bindingvacuoles  which rees  that can enrich plants with nitrogen. Symbiosis begins when the roots of the host plant come into contact with soil-borne rhizobia, resulting in nodule formation on the roots  and composite likelihood ratio (CLR) tests. F-statistics are traditionally used to test for natural selection between populations by comparing allele frequencies within and between groups  or genomic divergence associated with trait variation. The variation present in species-wide samples from natural populations (such as the Medicago HapMap panel) may be comprised of adaptive and deleterious alleles that have been tested in highly variable, natural habitats. In addition to identifying genetic loci associated with traits, GWAS can be used to estimate allele frequencies of associated SNPs and their effect size, which may reveal the forces of selection that contributed to the genetic architecture of a trait . In GWASn groups , while Cn groups . These tToxic levels of Cd and Hg in plants can be estimated based on relative root growth (RRG) and leaf accumulation. RRG acts as a measure of tolerance or root growth inhibition , 2021 whM. truncatula genotypes were obtained from the University of Minnesota, Medicago HapMap project1. Two separate heavy metal treatments were applied in parallel to the set of M. truncatula HapMap genotypes at the seedling stage. A modified Hoagland nutrient solution , and 1.35 mL of a solution containing: 11 gL–1 H3BO3, 6.2 gL–1 MnSO4 ⋅H2O, 10 gL–1 KCl, 1 gL–1 ZnSO4 ⋅7H2O, 1 gL–1 (NH4)6 Mo7 O24 ⋅4H2O, 0.5 gL–1 CuSO4⋅5H2O and 0.5 mLL–1 H2SO4. One set of plants was treated with mercury using 4 μM HgCl2 added to the Hoagland solution. A second set of plants was treated with cadmium using 10 sμM CdCl2 added to the Hoagland solution. A third set of plants was given no heavy metal treatment and was used as a control group. Each treatment began with fifteen replicate plant seedlings for each M. truncatula genotype. Four traits were measured on the M. truncatula Hapmap panel following the heavy metal treatments: (1) Relative Root Growth (RRG) in plants treated with Hg and (2) RRG in plants treated with Cd, (3) accumulation of Cd in leaf tissues, (4) and accumulation of Hg in leaf tissues. Seedlings were placed for 24 h in the hydroponic system with 250 mL of nutrient solution for acclimatizing in growth chamber conditions , and then 48 h in the same conditions with or without the heavy metal treatment. RRG is a reliable indicator of metal tolerance in M. truncatula . For leaf accumulation, three samples  were analyzed per genotype. We used R version 3.3.3 to conduct statistical on phenotypic distributions and the lmer4 package to estimate variance components to calculate broad sense heritability (H2). Metal concentrations were determined as previously reported  were imputed using BEAGLE version 4.1 . For each k, average cross validation errors were calculated and the iteration with lowest CV error was plotted in R. The SNP dataset containing 262 HapMap accessions was filtered to contain accessions with phenotype data present in the current study, resulting in a SNP dataset containing 236 genotypes.Genome-wide SNP data (BCF files) called against the Mt4.0 version of the reference genome from 262 sion 4.1  with defsion 4.1 . We usedsion 4.1  to genersion 4.1  using thk = 5), and a second time without the structure covariance matrix. A kinship (K) matrix, to control for genetic relatedness was estimated by the software. The parameters used in the GAPIT model were KI = NULL, PCA.total = 3, SNP.MAF = 0.02, SNP.fraction = 0.6, Major.allele.zero = TRUE and Geno.View.output = FALSE. We also ran the GWAS using GEMMA , were extracted from the gene context files. Additionally, the blastn tool provided by NCBI  and tissue-specific RNAseq expression levels (from six tissue types in the M. truncatula A17 genotype) were further annotated using information from MedicMine  were manually identified using IGV 2.6.3  and effect size of the 100 and 1000 most significant GWAS SNPs was determined using Pearson’s correlation coefficient for both GAPIT and GEMMA output datasets. We permuted genotype-phenotype values to make 100 phenotype datasets, then ran 100 iterations of GWAS iterations to make null distributions of MAF and effect sizes. To run the permutations, we used GEMMA, as it performs far better than GAPIT in terms of computational time. For each iteration, Pearson’s correlation coefficients were calculated for minor allele frequency (MAF) and effect size using the 100 and 1000 most significant SNPs. The resulting distribution of correlation coefficients was compared to the correlation of the unpermuted results for each phenotype.variscan  was equal to 0.61 and 0.72 for the two RRG traits, respectively  (r = 0.39). The heritability estimates and large amount of standing variation suggest that GWAS can identify SNPs and genes underlying these traits due to sufficient genetic differences between genotypes, and that both tolerance and susceptibility alleles are present in the M. truncatula HapMap panel.We measured seedlings from the Medicago HapMap collection for relative root growth (RRG) and metal ion accumulation in leaf tissues following 48 h exposure to Cd and Hg treatments . We founectively . We foun for Hg) . The higM. truncatula using a dataset of ∼12 million SNPs after imputing missing data. Among these, we sampled 843 k SNPs to generate a covariance matrix (Q) to control for confounding effects of population structure in our GWAS. Based on our analysis of population structure, we determined that k = 5 was the most strongly supported clustering (Q. Including only kinship (K) co-variance in the GWAS had similar empirical and expected distributions of p-values  in the quantile-quantile plots as did K and Q together  for each of the four traits  was among the top 100 SNPs and an ATP-binding cassette protein, MtABCC3 [Medtr5g094830, orthologous to AtABCC3 (MtABCC2 (Medtr2g019020) and MtABCC8 (Medtr8g015980), and Gamma-Glutamyl Transpeptidase 3, MtGGT3 (Medtr6g090280), which is localized on tonoplasts in A. thaliana (AtVPS45 (Medtr3g090170), and another Fe (II)-dependent oxygenase superfamily protein. We also found associations in the heavy metal transport/detoxification superfamily protein MtHIPP3 (Medtr2g095480). Heavy-metal-associated Isoprenylated Plant Proteins (HIPP’s) are a family of metallochaperones found only in plants , we found a large region (∼1.3 Mb) of interest associated with Cd RRG on chromosome 2. A highly significant non-synonymous SNP was found at position 15,656,689  in an ankyrin repeat protein (Medtr2g438720), and the surrounding region contained multiple ankyrin repeat genes within 1 kb of many top SNPs , a Fe (II)-dependent oxygenase superfamily protein, ATPase transporter MtHMA7 (Medtr2g035840), and a potassium transporter , and three SNPs in the Pleiotropic Drug Resistance 3 gene, MtPDR3 (Medtr5g070320). In the Mt4.0 reference genome, MtCAX3 and MtPDR3 are adjacent , and an undecaprenyl pyrophosphate synthase MtCPT7 (Medtr5g070270), which is orthologous to AtCPT7 . We found several intergenic SNPs with low p-values in high LD in the region containing a cluster of duplicated undecaprenyl pyrophosphate synthase genes . In the M. truncatula A17 genotype, MtNRAMP6 is expressed most highly in the roots (MtHMA2 (Medtr2g036380) and MtABCC3 (Medtr5g033320). We found a large region (∼2.3 Mb) on chromosome 8 (MtCAX9 (Medtr8g085260) and several surrounding genes with ATP binding/ATPases, and transmembrane GO-terms (r2 = 0.62), suggesting a large locus with several linked genes that may be involved in Hg accumulation. This region overlaps with high Fst windows spanning the same chromosomal region, which indicates higher Hg accumulating genotypes are genetically differentiated in this large genomic region. Among the genes in the highest Fst peak included a duplicated cation amino acid transporter  a magnesium-translocating P-type ATPase (Medtr8g089870), an ATPase amine-terminal autoinhibitory domain protein (Medtr8g090120) and an adjacent membrane calcium-translocating P-type ATPase (Medtr8g090125) , and among the top 20 SNPs was the Natural Resistance Associated Macrophage Protein 6 gene (he roots . Genes fmosome 8  that conGO-terms . The SNPg090125) . The pop+ ATPase 2 (Medtr1g084780), non-intrinsic ABC protein 12 (Medtr1g063920), and an ABC-2 type transporter family protein (Medtr4g076900) . This gene was shown to be most highly expressed in roots in the A17 genotype . Among tg076900) , were alp-values are shifted significantly toward larger effect sizes . Nevertheless, the correlations between MAF and effect size in all four traits were consistently negative. We used the Tajima’s D statistic to test for significant differences in the top SNPs detected in GWAS compared with the rest of the genome . We found significantly lower mean Tajima’s D for the top SNPs from the GWAS compared with background SNPs for three of the four traits  which did not include sequence data (r < 0.5), but existing genetic diversity for root length may also be a contributing factor.Heritability was sufficiently high for GWAS and our heritability estimates showed similar levels of phenotypic/genetic variability for the two RRG traits (about 10% difference) as well as for the leaf accumulation traits. Overall, heritability was higher for RRG than for leaf accumulation. Variability in Cd accumulation found in thaliana , barley thaliana , Brassicca napus , and ricca napus . Interesin maize . Our stuaves see . It is taves see . The strnce data . The posp-values for each trait association because we expected RRG and leaf accumulation of Cd and Hg to be highly polygenic. This allowed us to look at gene annotations and biologically relevant GO-terms among a list of several hundred genes for each trait. Our findings for Cd accumulation are considerably different than a similar GWAS in A. thaliana, where a single genomic peak containing the HMA3 locus was identified, which drowned out the signal at any other genomic region  proteins associated with each of the traits. Many of these are orthologous to ABC-transporters in A. thaliana  and rice that are known to interact with metal-binding phytochelatins that chelate Cd and Hg ions allowing for transportation into vacuoles by membrane bound ABC-proteins  which are well-known for their roles in root to shoot transport  associated with Hg RRG. Interestingly, no HMA’s were associated with either of the Cd traits. Clearly, ABC- and ATPase transporters have widespread roles that range from general to specific metal responses in plants, which include transport of essential and non-essential metal ions , the heavy metal transport/detoxification superfamily protein AtHMP10, and a Fe-oxidoreductase gene. Interestingly, this region has very high LD and contains several gene families with known metal tolerance roles and gene ontologies that support metal transport mechanisms or metal-induced stress responses. Expression enhancement through the triplication of genes is a common mechanism in metal ion transport in hyperaccumulating plants , which was shown to inhibit the expression of IRT1 and PDR8 . AtCPT7 is involved in dolichol synthesis and was shown to be upregulated after exposure to Cd in A. thaliana  suggests this genomic region possesses biologically relevant candidate genes involved in transport of Hg across membranes, including into vacuoles. On this chromosome, the highest Fst peak that is upstream from the GWAS peak includes multiple calcium and magnesium related P-type ATPases. This finding fits the recurring theme that P-type ATPases are involved in multiple metal ion transport mechanisms, including for highly toxic ions, such as Hg. Chromosome 8 contains some highly promising candidate genes to pursue using functional genetics approaches such as knock-out mutations using Hg accumulation to quantify the effects of mutants.To date, few QTL associated with Hg accumulation have been identified in crops or crop relatives . Using GM. truncatula, Cd tolerance has been associated with the efficiency of the plant’s antioxidant defenses  is a zinc-finger transcription factor shown to be triggered by salt stress and in maize (Zea mays), ZmOXS2 homologs are able to enhance Cd tolerance (AtUGGT in A. thaliana were shown to have delayed root growth induced by ion stress, which involved disruption of endoplasmic reticulum transmembrane integrity (M. truncatula, UDP-glycosyltransferases are involved in the biosynthesis of plant secondary metabolites, including flavonoids and isoflavonoids (Together, cellular responses that increase tolerance or accumulation to heavy metals are complex and involve not only ion transporters, but also mechanisms that deal with toxicity such as oxidative stress and ROS production. In excess amounts, all heavy metals trigger oxidative stress responses, resulting in increased levels of ROS in cells and tissues. As storage capacity is exceeded and toxic ions reach the leaves, plants alter leaf structure and chemistry leading to an accumulation of hydroxyl radicals. These hydroxyl radicals will indiscriminately oxidize biomolecules, including vital proteins and plant enzymes. In defenses . The stry metals . In A. tolerance . The mosntegrity . In M. tavonoids . Flavonoavonoids .The top SNPs detected in our GWAS showed a strong negative relationship between MAF and effect size, consistent with expectations of mutation-selection balance  and stabst and XP-CLR was similar to By combining GWAS analysis with population genomics, we were able to find signals of trait associations that overlapped with genomic regions detected by GWAS. Our population genomics approach used the tails of quantitative trait distributions to provide a contrast for genome-wide scans of genomic divergence, similar to the QTL approach described by The original contributions presented in the study are included in the article/TP and JP conceived of the study. JP supervised laboratory and phenotypic experiments. MS and ML conducted phenotypic measurements and data analysis. TP and BH conducted bioinformatics, GWAS, population genomics, and statistical analyses and drafted the manuscript, with editorial contributions from all authors. MC conducted bioinformatics and statistical analyses.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.


In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos.We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively.The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.The online version contains supplementary material available at 10.1186/s12864-022-08294-1. Mosaicism refers to the phenomenon wherein two or more cell lines with different chromosomes exist in an embryo simultaneously, usually as a result of abnormal chromosomal segregation during mitosis . The incPreimplantation genetic testing for aneuploidies (PGT-A) is an important method for avoiding the transfer of aneuploid embryos and thus reducing the miscarriage rate and shortens the time to achieve live birth. As the third type of embryo, other than euploid and aneuploid embryos, the transfer of mosaic embryos into the uterus as well as the risks associated with transfer have increasingly attracted the attention of clinicians and patients undergoing PGT-A cycles. At present, there is increasing evidence suggesting that mosaic embryos have definite reproductive potential and can result in healthy live births after transfer –7. HowevArray-based comparative genomic hybridization (aCGH), single-nucleotide polymorphism (SNP) arrays, and next-generation sequencing (NGS) are the platforms currently used for comprehensive chromosome screening (CCS). In comparison with PGT-A 1.0, which relies on fluorescence in situ hybridization (FISH) and cleavage biopsy, the utilization of PGT-A 2.0, which relies on CCS and trophectoderm biopsy, has been demonstrated to yield significantly better clinical outcomes . Among CA total of 88 couples were included in this study. The mean age of the female participants was 31.2 ± 4.5 years, and the mean age of the male participants was 33.1 ± 4.9 years  embryos showed whole-chromosome aneuploidy mosaicism, 55 (52.38%) showed segmental chromosome aneuploidy mosaicism, and one (0.95%) embryo showed both segmental chromosome aneuploidy mosaicism and whole-chromosome aneuploidy mosaicism.Since the SNP array cannot report the mosaic level of the embryos, a separate aliquot of the multiple displacement amplification (MDA) products of the alleged mosaic embryos was sampled and tested using the NGS platform. Clear NGS diagnoses were obtained for 100% (105/105) of the embryos, and NGS evaluation diagnosed mosaicism in 80 of these embryos  and segmental chromosome aneuploid mosaic embryos (n = 55) determined using SNP  (Table n = 82) and ≥ 35 years (n = 23), the complete concordance rate of the two methods was still similar  (Table n = 80) and low-quality (n = 25) blastocysts, the complete concordance rate of the two methods were 47.50 and 48.00%, respectively, and the difference was still not statistically significant (P = 0.965)  had a mosaicism level ≤ 50, and 28.75% 23/80) had a mosaicism level > 50%. Thus, the proportion of transferable embryos (euploid embryos or embryos with ≤50% chromosome mosaicism) was 64.76% (68/105), and the proportion of untransferable embryos (aneuploid embryos or embryos with > 50% chromosome mosaicism) was 35.24% (37/105)  were transferred back to the uterus in subsequent frozen-thawed embryo transfer (FET) cycles Table . Of thesFor patients who undergo the PGT cycle for assisted reproduction, every embryo is precious. Thus, it is very important to ensure the accuracy of PGT diagnosis and avoid wastage of embryos with reproductive potential. The clinical significance of embryos diagnosed as mosaic using PGT-A is currently unclear. In 2015, Greco et al. reported for the first time that transferring mosaic embryos could result in live births . Recent Zore et al. observed a significant decrease in live birth rates for mosaic embryo transfer (30.0% vs. 53.8%) . AnotherNGS, also known as high-throughput sequencing, has the characteristics of high output, high sensitivity, and high automation. Current research shows that NGS, aCGH, and SNP arrays have a high concordance rate (> 99%) in screening aneuploid embryos , 22. HowIn addition, NGS offers the advantage of detecting the level of mosaicism. Previous studies have verified that NGS can be used to diagnose mosaicism reliably up to 20–80% through a series of cell mixing experiments , 18. A sIn this study, 105 embryo biopsy samples that were identified as showing mosaicism on the SNP array platform were tested using NGS, and the two platforms showed a large discordance rate (34.29%), with some of these embryos identified as aneuploid (13.33%) and euploid (10.48%) using NGS. Our study suggests an inconsistency between NGS and SNP array in the detection of embryo mosaicism. It is necessary to answer the possible causes of this inconsistency and how to solve this problem. In 2018, Li et al. compared the SNP array and NGS data of 254 human DNA samples, from the Hapmap Project and 1000 Genomes Project, respectively . They foOn the other hand, there is room for improvement in both platforms to help narrow the differences further. For SNP array, due to the low quality of SNP data and insufficient training of segmentation algorithm, fragments or breakpoints detected using CNV detection software of SNP array often need manual review to avoid false positives. However, the graphical representation of the results of the SNP array are not as good as those of the NGS platform, which causes some problems for users. To this end, optimizing the visualization of SNP array data to more intuitive visual examination of CNV has been an important trend in recent years . For NGSCurrently, our clinical decisions are based on the assumption that NGS can accurately diagnose mosaic embryos. However, if the SNP array is more accurate in the diagnosis of mosaic embryos, NGS may cause some mosaic embryos to be misdiagnosed as euploid and mistakenly transferred or as aneuploid and discarded, which may damage the clinical outcomes of patients, especially for those with a poor ovarian response. Our study supports the need to further explore its accuracy in mosaicism detection.Importantly, the diagnosis of mosaicism (using either an SNP array or NGS) is not directly obtained by witnessing the chromosome karyotype of each cell in the biopsy sample, but is indirectly inferred from the occurrence of intermediate copy number signal on the platform profile  . HoweverNotably, 71.6% of the patients in this study received PGT-SR because of structural rearrangements . These structural rearrangements typically appear in PGT-A results as partial aneuploidies (deletions/duplications) , so it iThe major strength of this study was that in both tests, MDA was used for whole-genome amplification, and the biopsy and detection were carried out by the same laboratory and the same group of professionals at our center, avoiding the deviations caused by different amplification methods, differences in personnel experience levels, and changes in laboratory conditions. Notably, due to the low initial DNA amount, two biopsies were often required for two PGTs in the past. However, in 2017, Gleicher et al. established a mathematical modeling method to show that single-point biopsy of six cells in a 300-cell trophectoderm cannot accurately reflect the multiplication of embryo mathematically, and at least 27 cells are required to achieve the minimum diagnostic predictability . In 2020The major limitation of this study is that the sample size was small. Moreover, only a small number of FET cycles were performed, and the results of prenatal diagnosis and chromosomal detection of abortion tissues were lacking. Besides, we only evaluated the consistency of the two platforms based on the consistency of the chromosome arms in which the mosaicism is located, regardless of whether the results of segmental chromosome abnormalities were consistent. However, even in this rough assessment, our study showed a very low consistency between the two detection platforms, indicating the significance of our study. Additionally, due to the retrospective nature of this study, a third technique was not introduced to confirm which platform’s test results are correct. In the future, a third aneuploid screening platform could be introduced to detect embryos with conflicting SNP array and NGS results. For example, qPCR can be considered, as described by Tan et al.  In additWith the improvement in the sensitivity and resolution of PGT-A platforms, the detection rate of mosaic embryos is increasing. However, these improvements have also complicated clinical decision-making: discarding mosaic embryos may result in the wastage of many embryos with the potential to achieve a live birth. However, in comparison with euploid embryo transfer, the pregnancy outcomes of mosaic embryo transfer are not ideal. Therefore, accurate evaluation of mosaic embryos is very important for improving the clinical outcomes of patients undergoing PGT-A, especially those with a poor prognosis. This study is the first to compare the detection ability of the two most commonly used PGT-A platforms, NGS and SNP array, for mosaic embryos. The results of this study preliminarily show that the consistency between the two detection platforms for the diagnosis of mosaic embryos is low, and some of the embryos diagnosed as mosaic using SNP array are detected as aneuploid or aneuploid using NGS. Our results raise relevant questions about the accuracy of the two platforms in mosaicism detection, especially for NGS, which is becoming the mainstream testing platform in the field of PGT-A. Thus, larger and well-designed studies should be performed in the near future to verify the accuracy of the SNP array and NGS in the diagnosis of mosaic embryos, so as to facilitate more accurate clinical decision-making.This retrospective study was performed using data from PGT-A or PGT-SR cycles conducted at the reproductive medicine center of the First Affiliated Hospital of Sun Yat-sen University from January 2018 to October 2020. We obtained the data for embryos detected as showing mosaicism with an SNP array, verified them via NGS detection, and then compared the two detection results.We used conventional protocols for controlled ovulation hyperstimulation, intracytoplasmic sperm injection, and blastocyst culture , 43. ForFor each embryo, 4 μL of the corresponding MDA products were hybridized with the Human CytoSNP-12 chip (Illumina), which contains nearly 300,000 genetic markers. The hybridized chip was scanned using the iScan system (Illumina), and the data were analyzed using GenomeStudio software 2.0 (Illumina). We used median LogR deviation and median call rate to evaluate cytosnp-12 chip data. Median LogR deviation < 0.2 and median call rate > 0.98 were defined as the criteria for good data. The SNP array uses the log R ratio and the B allele frequency (BAF) of SNPs to determine the ploidy of chromosomes. When the BAF scatter of heterozygous SNP loci is not concentrated at 0.5, Log R > 0.2 or < − 0.2 indicates the occurrence of mosaicism .For embryos diagnosed as mosaic using the SNP array, another 4 μL of the corresponding MDA products were sampled and tested with NGS. The Veriseq PGS-MiSeq kit (Illumina) was used to construct the library of MDA products, and the samples were sequenced on the MiSeq Sequencer (Illumina) using the MiSeq Reagent Kit V3 (Illumina). Secondary analysis was performed using MiSeq Reporter software (Illumina). For each sample tested with NGS platform, no less than 0.7 million single-end (SE) 36 bp reads are generated, and Map_Ratio, Duplicate, GC_Content, and SD was statistically calculated for data quality control for data quality control. Map_Ratio < 80% indicates that there may be laboratory pollution of non-human molecules, or the sample to be tested has not been successfully obtained, or the sequencing instrument fails. Duplicate reflects the quality of amplified products and libraries and is closely related to library diversity. GC < 39% or GC > 45% represents the possible genome-wide amplification imbalance. Segmental duplication, deletion above 4 MB and whole chromosomal aneuploidy can be detected only when SD < 3.5. NGS evaluates chromosome ploidy by detecting copy number variation (CNV) values. That is, when the CNV value is between 1.20–1.80 and 2.20–2.80, the embryo is diagnosed as mosaic, when the CNV value is < 1.20 or > 2.80, the embryo is marked as pure aneuploidy, and when the CNV value ranges from 1.80–2.20, the embryo is diagnosed as euploidy .Due to the difference in the ability of the two methods to detect segmental chromosomal aneuploidy , the consistency of the two platforms was evaluated by judging the consistency of the chromosome arms in which the mosaicism is located, regardless of whether the results for segmental chromosome abnormalities are consistent. Complete concordance was concluded when the results obtained with NGS and the SNP array showed complete agreement for the chromosome arms involved in mosaicism. Partial concordance was concluded when the results obtained with the two platforms showed partial agreement for the chromosome arms involved in mosaicism. Partial concordance was concluded when the chromosome arms involved in mosaicism detected using the two platforms were partially but not completely consistent. If NGS detected additional chromosome arms involved in mosaicism while detecting all of those present in the SNP array, it was also concluded as partial concordance. Discordance was concluded when the results obtained with the two platforms identified completely different chromosome arms involved in mosaicism or when an embryo diagnosed as showing mosaicism using the SNP array showed a euploid status or aneuploidy on NGS.It should be noted that even mosaic deletion and mosaic duplication which occurring on the same chromosomal arm belonged to different types of mosaicism, which was also applicable to mosaic monosomy and mosaic trisomy of the same chromosome. Even on the same chromosome, mosaicism occurring on the whole chromosome and mosaicism occurring only on the chromosome arm belonged to different types of mosaicism.Blastocysts diagnosed as euploid or showing low-level (≤50%) mosaicism using NGS were defined as transferable embryos, and those with high embryo quality (on the basis of the Blastocyst score proposed by Gardner and Schoolcraft in 2000) were given priority for FET . BlastocOn the 14th day after embryo transfer, blood levels of human chorionic gonadotropin were examined to determine whether pregnancy had occurred. After 4–6 weeks of embryo transfer, if the intrauterine gestational sac and fetal heartbeat were observed, clinical pregnancy was confirmed. Live birth was defined as 28 weeks of gestation with at least one surviving newborn. Miscarriage that occurred before 12 weeks of pregnancy was defined as early miscarriage, and miscarriage that occurred after 12 weeks to less than 28 weeks of pregnancy was defined as late miscarriage.P < 0.05 was considered to indicate statistical significance.Continuous variables were expressed as mean ± SD. The Student’s t test was used to compare continuous variables with a normal distribution, and Mann–Whitney U test was used to compare continuous variables without a normal distribution. The count data were expressed as n (%) and analyzed using Pearson’s chi-square test. When any expected value was less than 5, a Fisher’s exact test was used. This study used IBM SPSS 23.0 software  for data analysis. Additional file 1: Table S1. The detailed SNP array and NGS results of the 105 alleged mosaic blastocysts diagnosed by SNP array.


Vigna unguiculata [L.] Walp., diploid, 2n = 22) is a major crop used as a protein source for human consumption as well as a quality feed for livestock. It is drought and heat tolerant and has been bred to develop varieties that are resilient to changing climates. Plant adaptation to new climates and their yield are strongly affected by flowering time. Therefore, understanding the genetic basis of flowering time is critical to advance cowpea breeding. The aim of this study was to perform genome-wide association studies (GWAS) to identify marker trait associations for flowering time in cowpea using single nucleotide polymorphism (SNP) markers. A total of 368 accessions from a cowpea mini-core collection were evaluated in Ft. Collins, CO in 2019 and 2020, and 292 accessions were evaluated in Citra, FL in 2018. These accessions were genotyped using the Cowpea iSelect Consortium Array that contained 51,128 SNPs. GWAS revealed seven reliable SNPs for flowering time that explained 8–12% of the phenotypic variance. Candidate genes including FT, GI, CRY2, LSH3, UGT87A2, LIF2, and HTA9 that are associated with flowering time were identified for the significant SNP markers. Further efforts to validate these loci will help to understand their role in flowering time in cowpea, and it could facilitate the transfer of some of this knowledge to other closely related legume species.Cowpea ( Vigna unguiculata [L.] Walp., diploid, 2n = 22) is a major crop grown worldwide for food and nutritional security  using SNP and simple sequence repeat (SSR) markers in recombinant inbred lines (RILs). Five QTLs related to time of flower opening and three QTLs related to days to flower were identified in a RIL population of 524B × 219-01 using SSR markers . SNP andExisting genetic diversity of cowpea needs to be assessed in order to strengthen breeding programs for developing high yielding dual-purpose cultivars with good grain and fodder yields. In this study, we phenotyped the UCR Minicore in Ft. Collins, CO and Citra, FL and performed GWAS for days to flowering; and identified candidate genes related to flowering time in cowpea.A total of 368 accessions from the cowpea UCR Minicore  were pla2O5 content, and low K2O, S, and Mg content. Seeds from each accession were planted in single row of 10 plants per plot, and the experiment was set up as a row/column design with two replications and augmented representation of 10 control lines. Each experimental unit (3 m × 0.6 m) consisted of 10 plants manually seeded and spaced at 0.3 m within row and 0.6 m between row spacing , Citra, FL  on September 7, 2018 . The soi spacing . List ofH2), genotypes were treated as random to estimate the genotypic variance (VG), and the residual variance (VE), and applying the formula H2 = VG/(VG + VE)  were conducted in the R statistical package . VariancVG + VE) .r2) were plotted against genetic distance using R statistical package (r2 between pairs of SNPs intercepted the threshold of 0.2.Single nucleotide polymorphism (SNP) genotyping is previously described . Briefly package  and a LOqqman (Marker trait association (MTA) using all SNP markers were evaluated based on the BLUE values for days to flower. MAF threshold of 5% was used to remove rare variants and avoid false–positive associations. Multiple algorithms were applied for GWAS. For all SNP loci and phenotypic data, we applied the generalized linear model (GLM) and MLM implemented in GAPIT . Furtherqqman  in R staqqman .Arabidopsis were identified and functionally characterized using TAIR database1 and their molecular functions were elucidated. Gene models whose gene ontology (GO) function was related to flowering were selected as candidate genes and their function was searched in the literature.For candidate gene identification, the reference genome of cowpea IT97K-499-35 v1.0  and the p < 0.05) showing that early flowering lines in Florida also flowered early in Colorado in both years.There was a significant variation in days to flower in all the datasets evaluated . The aveDaily maximum and minimum temperatures were lower in Colorado than in Florida –3. MinimAll SNP markers after filtering for MAF were used for GWAS. We identified 30 MTAs corresponding to 20 unique SNPs for days to flowering that explained 8–12% of phenotypic variance in the GWAS conducted using four software in the three datasets . These sArabidopsis gene network identified a total of 12 genes that were related to flowering (FT), GIGANTEA (GI), Cryptochrome-2 (CRY2), LIGHT-DEPENDENT SHORT HYPOCOTYLS 3 (LSH3), REBELOTE (RBL) that are known to control flowering time in Arabidopsis and other species   harboredlowering . These g species . These cH2 estimates (0.72–0.95) for flowering time in cowpea, which is similar to the estimates reported in other species like soybean (0.77–0.95) (H2 of flowering time shows the inherent genetic control of flowering as seen in other species. A H2 of 84.5% was reported for days to flower in cowpeas (h2) of 86% was reported in a cross between photoperiod-sensitive and photoperiod-insensitive varieties with at least seven major gene pairs estimated to control time of flowering in this population  , and alf77–0.95) . High H2 cowpeas  and a napulation . Since fArabidopsis, induced mutations revealed the existence of up to 80 loci that affected flowering time (7 lines obtained by crossing a short duration variety (524B) to a long duration variety (219-01) (8 RILs derived from a cross between cultivated (IT99K-573-1-1) and wild (TVNu-1158) cowpea accession (Flowering time is a complex trait  and is ging time . In cowp(219-01) . The lin(219-01) , therefo(219-01) . One majccession . Studiesccession . Droughtccession . Researcccession  and seveccession  models tccession . Few GWAccession , root arccession , black sccession , and seeccession . The avaccession . The Cowccession .For GWAS, we used four algorithms implemented in GAPIT , namely,RBL gene was 197 kb upstream of the significant SNP (2_46442) and this gene redundantly influences floral meristem termination (FT was located 124 kb downstream of the most significant SNP (2_55402). FT, together with LEAFY (LFY), integrates environmental signaling for induction of flowering  that delays transition to flowering and has been identified in legumes like pea, Medicago, and lotus (FT is expressed in leaves and is induced by long-day treatment in Arabidopsis (GI was located 70 kb downstream of the most significant SNP (2_27454). GI-mediated integration of photoperiodic and temperature information shapes thermo-morphogenic adaptation responses in plants that optimizes plant growth and fitness in warm climates  in our study could not be directly compared to previous QTL studies  because Plant breeders develop new varieties with increased yield by improving the crop’s adaptability and stress tolerance . FloweriEarly flowering can be transferred to cultivated cowpea through hybridization with early flowering accessions. Selection of early flowering cowpea that performs well in subtropical regions will undoubtedly help to increase the global production of cowpea as well as help to develop climate resilient cowpea accessions. On the other hand, extended vegetative period in late maturing varieties can provide higher biomass production which would be ideal for forage and cover crop cultivation, where the crops can be terminated before they flower and seed, thus avoiding potential invasiveness. Vegetative growth and rate of plant production have been shown to have additive and epistatic relationships with flowering time QTLs in common beans using comparative QTL mapping, suggesting pleiotropic effects between these traits . Furtherhttps://doi.org/10.1002/leg3.95).Publicly available datasets were analyzed in this study. This data can be found here: ER conceived the project. ER and RD collected the phenotypic data in Florida. MM-A and JR collected the phenotypic data in Colorado and provided the genotypic data. DP analyzed the data and wrote the manuscript. All authors reviewed 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.


Vrindavani) cows using 50K SNP Chip. After quality control, a total of 41,427 SNPs were retained and were further analyzed using a single-SNP additive linear model. Lactation stage, parity, test day milk yield and proportion of exotic inheritance were included as fixed effects in GWAS model. Across all traits, 13 genome-wide significant (p < 1.20 x 10−06) and 49 suggestive significant (p < 2.41 x 10−05) SNPs were identified which were located on 18 different autosomes. The strongest association for protein percentage, calcium (Ca), phosphorus (P), copper (Cu), zinc (Zn), and iron (Fe) were found on BTA 18, 7, 2, 3, 14, and 2, respectively. No significant SNP was detected for manganese (Mn). Several significant SNPs identified were within or close proximity to CDH13, BHLHE40, EDIL3, HAPLN1, INHBB, USP24, ZFAT, and IKZF2 gene, respectively. Enrichment analysis of the identified candidate genes elucidated biological processes, cellular components, and molecular functions involved in metal ion binding, ion transportation, transmembrane protein, and signaling pathways. This study provided a groundwork to characterize the molecular mechanism for the phenotypic variation in milk protein percentage and minerals in crossbred cattle. Further work is required on a larger sample size with fine mapping of identified QTL to validate potential candidate regions.In this study, genome-wide association study (GWAS) was conducted for identifying significantly associated genomic regions/SNPs with milk protein and minerals in the 96 taurine-indicine crossbred ( Milk and dairy products are considered a nutrient-rich diet, consisting of fat, protein, lactose along with several essential micronutrients including vitamins and minerals. Minerals representing a small fraction (~9 g/L) of bovine milk, are important for nutritional and technological properties of milk . The minMinerals play an important role in several physiological and biological activities. For instance, calcium, phosphorus, and magnesium are involved in the development of connective tissues such as bones, muscles, cartilages, and teeth in humans . CalciumThe concentration of milk minerals influences the stabilization of casein micelle in milk. Calcium, phosphorus, and magnesium are an important components of casein micelle and positively regulates the coagulation properties of the milk . The milThe genome-wide association studies may improve our understanding of the genetic architecture and underlying molecular mechanism for variation in protein and minerals content in bovine milk. Studies have revealed about several potential candidate genes associated with milk protein percentage in different cattle breeds , 12. Howad libitum with mixed ration, and milked in a free-stall barn using automated milking systems. The cows were in milk (24-403 days) and from parity 1-8.Morning milk samples (50 ml) were collected from 96 crossbred Vrindavani cattle of the ICAR-Indian Veterinary Research Institute, Bareilly. Vrindavani is a composite breed developed at Indian Veterinary Research Institute by crossing indicine (Hariana) with three taurine breeds . Details of population structure and synthetic breed information of the Vrindavani population were described in a prior study by . The cowThe protein percentage in milk was determined using a LactoScan milk analyzer . The milBlood samples were collected from individual cows, with the approval from the Institutional Animal Ethics Commit- tee (IAEC) on ICAR-Indian Veterinary Research Institute, Bareilly. Genomic DNA was isolated using Qiagen DNeasy Blood Mini Kit  according to the manufacturer's instructions. The quality and quantity of DNA were evaluated using NanoDrop spectrophotometer, agarose gel electrophoresis and Qubit fluorometer. The extracted genomic DNA were genotyped using Illumina BovineSNP50 Bead Chip platform  consisting of 53,218 SNPs across the genome. The quality control of the SNP genotypes was performed using PLINK v 1.9 . SNPs wihttp://widde.toulouse.inra.fr/widde/widde/main.do?module=cattle) and KRISHI (https://krishi.icar.gov.in/jspui/handle/123456789/31167) web portals. The fixed effect of lactation stage, parity, test day milk yield, and the proportion of exotic inheritance  were tested with lm function in R. A single-SNP linear regression of phenotype on genotype was fitted for GWAS analysis using snpStats package in R following Gondro  of the fitted SNP; α is the vector of effects of the regression coefficient for SNPs, and e is the vector of residual effects with a normal distribution N ~ . Since Bonferroni correction was stringent, a suggestive significance threshold of P = 2.41 x 10−05  was calculated  was used to search for genes flanking a region of 500 kb from the genome-wide significant and suggestive SNPs using BEDtools  in the milk of Vrindavani cattle are presented in p < 1.20 x 10−06) and 7 suggestive significant SNPs (p < 2.41 x 10−05) were detected on BTA 3, BTA 6, BTA18, and BTA22 (BCO1), C-Maf inducing protein (CMIP), N-terminal EF-hand calcium binding protein 2 (NECAB2), Cadherin 13 (CDH13), oxidative stress induced growth inhibitor 1 (OSGIN1) and Solute carrier family 38 member 8 (SLC38A8) genes.For protein percentage, two genome-wide significant SNPs , Versican (VCAN), EGF like repeats, and discoidin domains 3 (EDIL3). A total of three genome-wide and 14 suggestive significant SNPs were found to be associated with P. The most significantly (p = 6.89 x 10−09) associated SNP (Hapmap45428-BTA-47987) was located 0.10 Mb downstream from Inhibin subunit beta B (INHBB) gene and 0.81 Mb downstream from Cilia and flagella associated protein 221 (CFAP221) gene on BTA2. Total 1 genome-wide and 9 suggestive significant SNPs were observed to be associated with Cu. The most significantly (p = 1.68 x 10−07) associated SNP (ARS-BFGL-NGS-1062) with Cu was identified at 0.63 Mb downstream from the 24-dehydrocholesterol reductase (DHCR 24) gene on BTA3. The SNP (BTA-59703-no-rs) significantly associated with Cu (p = 4.73 x10−06) on BTA25 was located within Calcium voltage-gated channel auxiliary subunit gamma 3 (CACNG3) gene. A total of 2 genome-wide and 5 suggestive significant SNP were associated with Zn. Out of these 7 SNPs, 5 significant SNPs were located within 5.29 Mb (4.17-9.47 Mb) region of BTA14. The most significant (p = 3.50 x 10−07) SNP (ARS-BFGL-NGS-76248) associated with Zn was located 0.09 Mb upstream from the ST3 beta-galactoside alpha-1 (ST3GAL1) gene. A total of 2 genome-wide and 15 suggestive significant SNPs were significantly associated with Fe. Total six significant SNPs were identified within 4.10 Mb region (65.60-72.17 Mb) on BTA2. The most significant (p = 3.26 x 10−07) SNP (ARS- BFGL-NGS-96204) associated with Fe was located 0.45 Mb away from NCKAP5 gene on BTA2. The most significantly (p = 1.02 x 10−04) associated SNP (Hapmap58269-rs29018185) with Mn was located on BTA 19, although it was below the cut-off threshold level of significance.A total three genome-wide significant and 1 suggestive significant SNPs on BTA7 (86.04-89.65Mb) were found to be associated with Ca. The region contains several genes including Hyaluronan and proteoglycan link protein 1 , metal ion binding (GO:0005509), integral and transmembrane protein (GO:0086010), and signaling pathway .In the present study, several significant SNPs found to be associated with protein percentage and individual mineral concentration in the milk of Vrindavani cattle. To our knowledge, this is the first study to identify genomic regions associated with milk mineral content in composite Vrindavani cattle. Detection of several previously reported genes and genomic regions associated with different milk composition traits indicates their potential role in regulating the concentration of minerals and protein percentage in bovine milk.CDH 13 (Cadherin-13) gene which is expressed in mammary tissues and associated with the protein content of the milk  on BTA 18 was partly overlapping with previously reported QTLs associated with milk coagulation . The milthe milk .INHBB) gene on BTA2. In humans, INHBB gene plays a major role in regulating calcium and phosphorus during bone formation  is involved in metal ion binding  gene. The USP24 gene belongs to the cysteine proteases family and plays role in protein deubiquitination of the proteins which influences the stability of the casein micelle in the milk  and are involved in tissue calcification and bone mineralization in vertebrates (EDIL3) gene bind with the calcium ion to guide vesicular transportation of minerals during eggshell calcification  associated with Zn, is located at 0.12 Mb away from the Zinc finger and AT-hook domain containing (ZFAT) on BTA14, which is involved in the transcription of immune-related genes.The SNP named Hapmap3 1987-BTC-062044 was significantly  can be found at: Figshare, The animal study was reviewed and approved by Institute Animal Ethics Committee of Indian Veterinary Research Institute, Bareilly.AK conceived and designed the experiments. AS and AK performed the experiments, analyzed the data, and wrote the paper. CG, AP, and TD contributed reagents/materials/analysis tools. All authors contributed to the article and approved the submitted version.This project work was supported by the CAAST-ACLH project of NAHEP. AS was recipient of fellowship from ICMR fellowship during her PhD programme.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.