Umbilical hernia is one of the most prevalent congenital defect in pigs, causing economic losses and substantial animal welfare problems. Identification and implementation of genomic regions controlling umbilical hernia in breeding is of great interest to reduce incidences of hernia in commercial pig production. The aim of this study was to identify such regions and possibly identify causative variation affecting umbilical hernia in pigs. A case/control material consisting of 739 Norwegian Landrace pigs was collected and applied in a GWAS study with a genome-wide distributed panel of 60 K SNPs. Additionally candidate genes were sequenced to detect additional polymorphisms that were used for single SNP and haplotype association analyses in 453 of the pigs.P < 0.0001) explaining up to 8.6% of the phenotypic variance of the trait. The region is rather broad and includes 62 significant SNPs in high linkage disequilibrium with each other. Targeted sequencing of candidate genes within the region revealed polymorphisms within the Leukemia inhibitory factor (LIF) and Oncostatin M (OSM) that were significantly associated with umbilical hernia (P < 0.001).The GWAS in this report detected a highly significant region affecting umbilical hernia around 50 Mb on SSC14 (LIF and OSM highly associated with the trait. However, because of extended LD within the region, studies in other populations and functional studies are needed to determine whether these variants are causal or not. Still without this knowledge, SNPs within the region can be used as genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs.A highly significant QTL for umbilical hernia in Norwegian Landrace pigs was detected around 50 Mb on SSC14. Resequencing of candidate genes within the region revealed SNPs within  Hernias are of the most common congenital defects in pigs which often leads to poor animal welfare and severe economic losses for pig producers. The most common types of hernias in pigs are umbilical and inguinal/scrotal hernia. Umbilical hernia is diagnosed by the protrusion of abdominal contents beneath the skin at the navel (umbilicus). It is generally accepted that weakened supportive muscles around the umbilical stump or navel area of the animal causes the umbilical opening not to close properly and intestines protrude through the intestinal wall to form the “ball-like” structure. The threat of a hernia is the potential entrapment of intestines through this opening. Physical injury, nutrition, excessive pressure, muscular weakness and heredity have been offered as causes of hernia . Umbilic2 estimates of umbilical hernia are reported to be very low in pigs (0.06–0.08) [2 = 0.4) are reported to be much higher [KIP2, a regulator of cell proliferation [It is generally accepted that genetics influence congenital umbilical hernias, but mode of inheritance and genetic architecture are more or less undiscovered, and h06–0.08) , 8. The h higher , 10 thanh higher  observedh higher  detectedh higher  publisheh higher –15. Addiferation , 17.Studies on genetic control of umbilical hernia so far is not clear, although indicating that the disorder is complex and affected by multiple causative genes and variants. Therefore, the aim of this study was to collect a proper case/control material and identify genomic regions affecting the frequency of umbilical hernia in pigs, using GWAS on a high resolution SNP panel followed up by candidate gene approaches.2) was also estimated to be very low (0.065). Due to the low incidence of umbilical hernia in Norwegian Landrace, samples had to be collected over time-periods of altogether 4 years. To generate sufficient number of case/control samples we collected data and samples from all the nucleus herds in Norsvin . Blood samples were collected from a total of 369 purebred Norwegian Landrace piglets displaying umbilical hernia, along with blood from 202 phenotypically unaffected fullsibs within all affected litters. The affected pigs were distributed on 259 litters, with each litter containing 1 to 6 affected piglets from 110 sires. Altogether, 168 parents were genotyped. Samples were obtained from 35 pig breeding farms, where several sires were used on different farms. The farmers reported the cases and thereafter the diagnostic procedures were performed by a Norsvin-breeding technician. The umbilical hernias were subjectively classified into two categories by approximate size “small” or “large” . Based on this, 165 hernias were classified as “small” and 267 were classified as “large”. Additionally, the time of first observation was recorded, together with records on whether the piglet previously had a history of navel inflammation or not. The size-score of the umbilical hernias was considered not to be valuable as a covariate in the analysis. The first observation of hernias ranged between 2 and 24 weeks, with the average of 14 weeks. Only 2 piglets were previously observed to have an inflammation, and this was therefore not accounted for in the statistical analysis. All animals were cared for according to laws and internationally recognized guidelines and regulations controlling experiments with live animals in Norway , and the Animal Protection Ordinance Concerning Experiments with Animals of January 15th, 1996), according to the rules given by Norwegian Animal Research Authority.The overall frequency of umbilical hernia for Landrace in Norwegian nucleus herds was found to be only 0.55%, and the heritability . The concentration and quality of samples were measured on a Nanodrop, ND-1000 spectrophotometer , and on a 1420 Victor plate reader  using PicoGreen fluorescence , and thereafter normalized to 50 ng/ul in 96-well plates.www.cigene.no), Norwegian University of Life Sciences, Norway. Genotyping for the GWAS was performed using the iScan  platform with the PorcineSNP60 array according to manufacturer’s instructions. Image intensity, data processing, clustering and genotype calling were performed using the genotyping module in the Genome Studio software . Altogether, including the hernia pigs, their full-sibs and their parents, 739 Norwegian Landrace were genotyped for 64,232 SNPs. The SNP markers passing the quality control had call rate above 0.9, minor allele frequency (MAF) above 0.01, proportion of genotyped above 0.25, and proportion of genotype errors below 0.025. The average call rate across samples was 0.997, and no samples were excluded from analysis due to unacceptable call rates from the genotyping. Animals with pedigree errors  were removed from the study. After quality control, 554 animals were available for the case/control, in addition to 168 parents. The most frequent failing category is non-informative markers . Beagle v. 3.3.1 was used to impute sporadically missing genotypes in the final genotype file [The genotyping was performed at CIGENE (ype file . In totaLeukemia inhibitory factor (LIF), Oncostatin-M (OSM), Macrophage migration inhibitory factor (MIF), cyclin-dependent kinase inhibitory protein p57KPI2 (CDKN1C), Pyrroline-5-carboxylate reductase 1 (PYCR1), Versican (VCAN), Matrix metallopeptidase-13 (MMP13), and Vimentin (VIM). Names, positions from www.ensembl.org and references are listed in Table Candidate genes were chosen based on GWAS results in this study and putative role in development of umbilical hernia from literature , 19–25. MIF and CDKN1C, but 50 SNPs were obtained in six other genes. Primer assays for use in the Sequenom MassARRAY system were designed using MassARRAY Assay Design Software, with multiplexes between 12 and 19. Genotyping was done according to the manufacturer’s instructions in the IPLEX protocol. The MassARRAY Typer software was used for the automated genotype calling . Altogether, 46 SNPs in six different candidate genes were successfully genotyped for 463 animals; 201 pigs with umbilical hernia, 135 healthy siblings and 127 parents. Due to high SNP density in VCAN (16 SNPs) the six SNPs with highest MAF were selected, and additional five SNPs located in other genes were removed due to low MAF (MAF < 0.01). Therefore, 31 SNPs were used for the final statistical analyses . Gender-factor have not either previously been reported in pigs, but in cattle, however, umbilical hernia have been found to be more frequently occurring in female compared to males [The association analyses were carried out for the 49,049 SNPs mapped on the 18 porcine autosomes using pig genome assembly Build10.2. The minor allele frequencies of the selected SNPs were uniformly distributed between 0.01 and 0.5. Genome-wide association analysis was performed with the GenABEL package (version 1.6–5) in R  using thp-values corrected for genomic control (GC) of a 1-df test were accepted to represent proof of genome-wide association at p < 0.001 (−log10(p) = 3). Collection of samples was done during intensive periods of altogether four years, but it was seven years between the first and final collection of samples. Therefore, a population stratification analysis (“strata”) was performed to find whether the population during some generations of selection was grouped into several genetic populations or not. However, only one population was defined and correction for stratification was not added in the final analysis.The For the single SNP-marker and haplotype association analysis the ASReml v.2.00  was usedp-value of 0.001. The genetic variance explained by a SNP was calculated from the estimated genotype effects and the observed genotype frequencies. It was expressed as a percentage of the total phenotypic variance obtained from the model without the genotype effect.where Y is the binomial trait “case” or “control”. The SNP, coded as 0, 1, or 2 for homozygote allele 1, heterozygote, homozygote allele 2, respectively, was fitted as a fixed effect. In case of the haplotype associations, each haplotype allele combination was assigned its own level and fitted as a fixed effect. Sex was fitted as a fixed effect whereas animal id was treated as a random effect, using a pedigree based relationship matrix to account for population structure. The F-statistics was calculated for each SNP or haplotype and the significance level was set at a corresponding P < 0.001, 129 SNP markers reached the significant level. Almost all of them, 126 SNPs, were located on SSC14 between positions 47.16 Mb and 58.91 Mb. The other three single SNP associations were obtained on SSC1 around 102 Mb at the Porcine Build10.2 genome sequence (http://www.ensembl.org), on SSC8 around 88 Mb, and on SSC17 around 68 Mb, respectively. The Manhattan plot of all chromosomes is shown in Fig. P < 0.0001, 62 SNP markers were significant and all of them were located between 48.04–50.99 Mb at SSC14. The most significant SNPs (p < 7 × 10–5) are listed in Table P < 0.0001).Assuming a corrected significance level of OSM) and Leukemia inhibitory factor (LIF) was the two genes located within the SSC14 QTL. For OSM, all the eight single SNP markers, and the haplotype combining all the eight SNPs, were significantly associated with umbilical hernia (P < 0.001) and found to explain up to 8.6% of the phenotypic variance  on SCC14. This marker was also significantly associated with umbilical hernia (p < 0.001) and found to be in perfect LD with OSM  obtained significant associations with umbilical hernia . OSM is located at SSC14 base pair position 50,281,701–50,286,188, LIF is located at SSC14 base pair position 50,263,722–50,266,807 and MIF at position 53,282,553–53,283,313. The three genes were sequenced at the genomic level in eight hernia pigs and eight healthy pigs, but SNPs were only detected in the OSM and LIF genes. OSM and LIF have similar gene structure and functions, both are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family, and they can interact with each other [LIF is shown to be the inducer of the acute phase protein synthesis affecting the cell recruitment into the area of damage or inflammation . MIF isPYCR1 was added as a biological candidate gene due to its importance in conversion of glutamate to proline and suggested role in cell growth regulation [PYCR1 and hernia [MMP13 on SSC9, is shown to obtain an important role in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis [VCAN gene, located on SSC2, was chosen as a biological candidate gene. Finally, vimentin (VIM), which is located on SSC10, was added as a biological candidate gene due to its function of being a marker of immature smooth muscle cells and being involved in collagen structure [gulation . Prolinegulation . Diaphragulation , 39. Mord hernia . Severalteolysis . MMP13 wteolysis . The VCAteolysis . In thistructure , as welltructure .Since there are variability in level of expression incidences of umbilical hernia, we are aware that defining the trait as a case or control trait is one of the limitations in this study. However, no other way of recording the trait have successfully been applied, in either this study or others. Statistical packages suitable for analyzing binary traits were therefore performed for GWAS study as well as for the single SNP/haplotype association study. Limited number of genomic studies have investigated associations between genes/genetic markers and umbilical hernia. A genome wide scan in White Duroc and Erhualian, using 194 microsatellites and two different statistical methods, obtained significant QTL regions for umbilical hernia in altogether 11 chromosomes . The mosP < 0.00015) explaining up to 8.6% of the phenotypic variance for umbilical hernia. Resequencing of candidate genes provided SNPs and haplotypes for association analyses and SNPs within positional candidate genes LIF and OSM in the SSC14. Due to the large degree of LD in the region, future functional studies are needed to assign the causal variant(s) and further studies should be conducted to investigate the effect of this QTL region in other pig breeds. SNPs within the QTL region can be used as genetic markers to reduce incidences of umbilical hernia in Norwegian Landrace pigs.A highly significant QTL for umbilical hernia in Norwegian Landrace pigs was detected between 48 and 51 Mb on SSC14 (


QTL‐seq approach, therefore, was used to identify candidate genomic regions for 100‐seed weight (100SDW) and total dry root weight to total plant dry weight ratio (RTR) under rainfed conditions. Genomewide SNP profiling of extreme phenotypic bulks from the ICC 4958 × ICC 1882 population identified two significant genomic regions, one on CaLG01 (1.08 Mb) and another on CaLG04 (2.7 Mb) linkage groups for 100SDW. Similarly, one significant genomic region on CaLG04 (1.10 Mb) was identified for RTR. Comprehensive analysis revealed four and five putative candidate genes associated with 100SDW and RTR, respectively. Subsequently, two genes (Ca_04364 and Ca_04607) for 100SDW and one gene (Ca_04586) for RTR were validated using CAPS/dCAPS markers. Identified candidate genomic regions and genes may be useful for molecular breeding for chickpea improvement.Terminal drought is a major constraint to chickpea productivity. Two component traits responsible for reduction in yield under drought stress include reduction in seeds size and root length/root density.  Annual global production of chickpea is around 13.1 million tons from 13.5 Mha FAOSTAT, . India iFusarium wilt  of 58.20% for 100SDW, an important trait that has relevance to yield and 16.67% PVE in case of root dry weight/total plant dry weight ratio (RTR) that has relevance to drought tolerance  and candidate genes prioritized for analysis  have seen the rapid decrease in cost of sequencing, enabling the cost‐effective application of genomics in crop improvement possible  were constructed and subjected to whole‐genome resequencing using Illumina MiSeq. In total, 38.46 million paired‐end (PE) reads  for 100SDW and 35.52 million PE reads  for RTR were generated. A total of 29.29 million PE reads were generated for ICC 4958  responsible for differences in 100SDW and RTR, between the low and high RIL bulks, we compared the ‘SNP‐index’ between them. Focusing on a particular SNP position of the genome, the SNP‐index is calculated as a ratio of PE short reads aligned to that position with a nucleotide different from that of the reference sequence. The SNP‐index represents frequencies of parental alleles in the population of bulked individuals. In this case, the ICC 4958 genome was used as the reference, where a SNP‐index of 1 indicates that reads in the population are derived only from the ICC 1882 genome, whereas SNP‐index = 0 indicates the reads are derived only from ICC 4958. A SNP‐index of 0.5 indicates an equal genome contribution from both parents. A significant deviation from SNP‐index of 0.5 could indicate the contribution of that SNP to the phenotypic difference observed in the bulks  deviation from equal inheritance of the two parental genomes had five SNPs with ∆SNP‐index = −1 (Table S3). However, none of the SNPs was from predicted genic regions. The lower number of SNPs may be due to the narrow candidate region and/or low coverage. Further, the second 2.70 Mb region (11.12–13.82 Mb) on CaLG04 harboured 21 SNPs with ∆SNP‐index = −1. Of these 21 SNPs, five SNPs were present in four putative genes  to isoleucine (ICC 1882).Sequence analysis for 100SDW revealed two genomic regions on two linkage groups (CaLG01 and CaLG04) . Interestingly, two SNPs (at 13666705 bp for synonymous change and 13666728 bp for T to I amino acid change) were identified in the exon of a putative gene, Ca_04586 predicted to be coding for cytochrome P450 monooxygenase  on linkage group 4 (CaLG04) revealed 26 SNPs with ∆SNP‐index = −1, suggesting a biased inheritance of parental genomes in the two populations . Of 11, eight primers amplified prominent fragments of expected size in two different parental combinations . Of these eight primer pairs, six (three for each 100SDW and RTR trait) were found polymorphic using their respective restriction enzymes. Of six polymorphic markers, four Ca_04364_11311944 and Ca_04607_13822453 for 100SDW and Ca_04586_13666705 and Ca_04586_13666728 for RTR were validated in the high and low DNA pools. The fragment size of high DNA pools of 100SDW and RTR followed the similar pattern of the tolerant line ICC 4958 and the low DNA pool of 100SDW and RTR bulks followed the similar pattern of the sensitive line ICC 1882. The details on amplified fragment size and digested product size of each primer pairs are presented in Figures P < 0.01%) for all four markers. The candidate markers for 100SDW, Ca_04364_11311944 and Ca_04607_13822453, explained high phenotypic variation of 28.61% with LOD score of 64.58 and 19.25% with LOD score of 45.61, respectively  were used to develop genetic linkage map. As a result, a map length of 25.18 cM was obtained for linkage group 4. Single‐marker QTL analysis showed a high significance  8′ hydrolase which involved in ABA catabolism  identified a comparatively large number of SNPs , which were used for calculating genomewide SNP‐index. Analysis of SNPs located in this region revealed a candidate gene, et al., et al., et al., Ca_04607 on CaLG04, coding for a ‘transmembrane protein’. Role of transmembrane proteins in controlling grain weight, grain length, grain width and thickness has been earlier reported in rice . This might be due to low‐coverage sequencing of RIL population (average 0.72 X per RILs) or because of errors in genotyping of RILs or resequencing of parental genotypes.Similarly for 100SDW, two genomic regions on CaLG01 (1.08 Mb) and CaLG04 (2.70 Mb) were identified. It is interesting to note that several studies reported the presence of QTL for 100SDW on CaLG04 in chickpea  explaining 16.67% of phenotypic variance was reported between TAA170 and NCPGR21 on CaLG04  population (ICCRIL03) comprising 262 lines developed from a drought tolerant genotype, ICC 4958 (with high 100SDW and high RTR) and a drought‐sensitive genotype, ICC 1882 (low 100SDW and low RTR) was used in the present study. Extensive phenotyping data under rainfed conditions for several drought component traits (20 traits) in 1–7 seasons and 1–5 locations along with marker data enabled by Varshney et al.  to proviExtreme bulks were prepared for 100SDW and RTR traits based on precise phenotyping data obtained for 5 years  and 2 years (2005 and 2007), respectively, under rainfed conditions. For developing the extreme bulks for each trait, 15 RILs with high mean phenotypic values and 15 RILs with low mean phenotypic values were selected. The equimolar concentration of DNA from 15 RILs with high phenotypic values was pooled together as one bulk, and similarly DNA from low mean phenotypic values was pooled together as another bulk. Thus, four extreme bulks two each for both the traits were prepared for sequencing.® NGS , end repaired and adapter ligated. Size selection of libraries was performed using 2% agarose gel to get a target insert size of 500–600 bp and purified for further analysis. Further, the libraries were enriched using adaptor compatible PCR primers. The size distribution of amplified DNA libraries was checked on an Agilent Technologies 2100 Bioanalyzer using a High Sensitivity chip. The DNA libraries were sequenced on Illumina MiSeq platform with MiSeq Reagent Kit v2 (500‐cycles)  to generate 250‐base pair‐end reads.A total of five Illumina libraries (four from extreme bulks mentioned above and one from ICC 4958 the drought tolerant parent) were prepared using TruSeq DNA Sample Prep kit LT, (set A) FC‐121‐2001. Two microgram DNA from each sample was sheared using diagenode Bioruptoret al., http://genome-e.ibrc.or.jp/home/bioinformatics-team/mutmap, developed by Iwate Biotechnology Research Center, Japan) was used for calculating SNP‐index. Briefly, the cleaned reads of donor parent (ICC 4958) were first aligned to the reference genome  = count of alternate base/count of reads aligned.et al., The positions with read depth <7 in both the bulks and SNP‐index <0.3 in either of the bulks were filtered out for ∆SNP‐index calculation. ∆SNP‐index can be calculated by subtracting SNP‐index of low bulk from SNP‐index of high bulk. Only SNP positions with ∆SNP‐index = −1  were considered as the causal SNPs responsible for the trait of interest. The possible effects of the identified SNPs were inferred using SnpEff v3.0 open source program  and four bulks (High and low bulks of RTR and 100SDW). For this purpose, the SNPs were converted into CAPS/dCAPS markers using dCAPS Finder 2.0  was genotyped with all the four candidate CAPS/dCAPS markers. The genetic map was constructed using JoinMap V4.0 with the default parameters. Single‐marker analysis (SMA) was carried out using the map along with the phenotypic data by QTL Cartographer. The mean phenotyping data generated under rainfed conditions used for making the pool as described earlier for 100SDW and RTR were used for this analysis. Based on the p‐value, the marker was determined to control the trait of interest.V.K.S., A.W.K. and D.J. performed most of the experiments; V.K. and A.C. generated sequence data; A.W.K., H.T. and R.T. performed QTL‐seq analysis; P.M.G. contributed genetic material; V.K.S., A.W.K., D.J., M.T., M.R., V.G., T.S., R.T. and R.K.V. analysed and interpreted the QTL‐seq data; D.J., V.K.S. and R.K.V performed validation of candidate SNPs; V.K.S., D.J., M.T., M.R., T.S., R.T. and R.K.V. wrote the manuscript; R.K.V. conceived, designed and supervised the study and finalized the manuscript. All authors read and approved the manuscript.The authors declare that they have no competing interests.Figure S1 QTL‐seq approach used in chickpea.Figure S2 Frequency distribution of 100SDW of the 262 RILs derived from a cross between ICC 4958 and ICC 1882 for different years  in field conditions.Figure S3 Frequency distribution of RTR levels of the 262 RILs derived from a cross between ICC 4958 and ICC 1882 over two different years (2005 and 2007) with mean values in controlled conditions.Figure S4 (a) Based on the 2 year phenotyping of 262 RILs for RTR, a total of 15 RILs with high RTR and 15 with low RTR were used to develop high RTR and low RTR bulks, respectively. (b) Similarly, based on 5 years data of 100SDW a total of 15 RILs with high 100SDW and 15 with low 100SDW were used to develop high 100SDW and low 100SDW bulks.Figure S5 SNP‐index plots for eight chromosomes of high 100SDW bulked DNA.Figure S6 SNP‐index plots for eight chromosomes of low 100SDW bulked DNA.Figure S7 The Δ(SNP‐index) plot obtained by subtraction of high 100SDW SNP‐index from low 100SDW SNP‐index for RILs obtained from a cross between ICC 4958 and ICC 1882.Figure S8 SNP‐index plots for eight chromosomes of high RTR bulked DNA.Figure S9 SNP‐index plots for eight chromosomes of low RTR bulked DNA.Figure S10 The Δ(SNP‐index) plot obtained by subtraction of high RTR SNP‐index from low RTR SNP‐index for RILs obtained from a cross between ICC 4958 and ICC 1882.Table S1 Chromosome wise SNPs distribution between low and high 100SDW bulks.Table S2 Chromosome wise SNPs distribution between low and high RTR bulks.Table S3 List of putative SNPs identified for 100SDW based on SNP‐index values.Table S4 List of putative SNPs identified for RTR based on SNP‐index values.Table S5 List of primers used for validation of candidate genes for RTR and 100SDW.Table S6 List of primers successfully validated for 100SDW and RTR.Table S7 Comparison of the identified QTLs from QTL‐seq with earlier studies.Click here for additional data file. Click here for additional data file.


Ketone body therapy and supplementation are of high interest for several medical and nutritional fields. The intake of ketone bodies is often discussed in relation to rare metabolic diseases, such as multiple acyl-CoA dehydrogenase deficiency (MADD), that have no alternatives for treatment. Case reports showed positive results of therapy using ketone bodies. The number of ketone body salts offered on the wellness market is increasing steadily. More information on the kinetics of intake, safety, and tolerance of these products is needed. β-hydroxybutyrate (βHB) as well as food additives. The blood samples drawn in the study were tested for concentrations of D-βHB, glucose, and electrolytes, and blood gas analyses were done. Data on sensory evaluation and observed side effects of the supplement were collected. The product also went through chemical food analysis.  In a one-dose kinetic study, six healthy subjects received an intervention (0.5 g/kg bw) using a commercially available ketone body supplement. The supplement contained a mixture of sodium and calcium D-/L-βHB concentration in blood 2.5 and 3 h after oral intake . The first significant effect was measured after 2 h with a mean value of 0.598 ± 0.300 mmol/L at the peak, which was recorded at 2.5 h. Changes in serum electrolytes and BGA were largely unremarkable. Taking the supplement was not without side effects. One subject dropped out due to gastrointestinal symptoms and two others reported similar but milder problems.  The supplement led to a significant increase of D-βHB salt shows a slow resorption with a moderate increase of D-βHB in serum levels. An influence of βHB salts on acid-base balance could not be excluded by this one-dose study. Excessive regular consumption without medical observation is not free of adverse effects. The tested product can therefore not be recommended unconditionally. Intake of a combination of calcium and sodium D-/L- The basis of all three “ketone” bodies is acetoacetate. Acetoacetate can then either be reduced to beta-hydroxybutyrate (βHB), or acetone is generated by spontaneous decarboxylation of acetoacetate. Only acetoacetate and βHB are relevant for energy expenditure [βHB serum levels are less than 0.1 mmol/L [Interest in the importance of ketone bodies has risen in the recent past. Ketone bodies are an alternative fuel produced in the liver, in a process referred to as ketogenesis, in the event of reduced availability of glucose . Insulinenditure , 6. The enditure . Normal 1 mmol/L . They in1 mmol/L . The ter1 mmol/L , 11.βHB serum concentration of 0.4 mmol/L, ketone bodies and specific diet forms like the ketogenic diet are of high scientific interest [The human organism has two nutrition-related ways of reaching ketosis. The first is starvation and the second is a high fat and at the same time low carbohydrate (HFLC) diet which is also known as a ketogenic diet , 12. Ketinterest , 15.There are different types of clinically relevant ketogenic diets. The milder forms are the low glycaemic index diet (LGID) and the modified Atkins diet (MAD) where carbohydrate intake is limited to 15 grams per day. More stringent are the medium chain triglycerides (MCT) diet as well as the 3 : 1 and the 4 : 1 classical ketogenic diets. The ratio depicts the fat contents in relation to the sum of carbohydrates and proteins based on their weight . A well-The ketogenic diet has proven to be an effective therapy for epilepsy, although not only the anticonvulsive effect is used in medicine. In addition, some inherited metabolic diseases of glucose uptake or metabolism, for example, the GLUT1 or pyruvate dehydrogenase deficiency, are treated using ketogenic diets to ensure sufficient energy supply .β-oxidation, like multiple acyl-CoA dehydrogenase deficiency (MADD), a direct intake of ketone bodies for energy supply is necessary. Inducing ketogenesis using normal food products is not possible due to the impairment of fatty acid oxidation. The genetic defect in the electron transport flavoprotein or the electron transport flavoprotein oxyreductase causes a dysfunction of all acyl-CoA dehydrogenases in β-oxidation by the impaired oxidation of FADH2 to FAD [βHB in serum was detectable within one hour of intake and led to dramatic clinical improvements. After 2 and 9 months, MRI investigations showed a progressive decrease of leukodystrophy, a typical problem in MADD patients [βHB as a supplement has been used without any adverse side effects and increased the serum concentration of D-βHB [In special severe metabolic diseases of 2 to FAD . For thipatients . In caseof D-βHB .βHB [βHB and an alcohol, for example 1,3-butanediol [Good efficacy of ketogenic diets is clouded by poor practicability and the necessity of maintaining a constant ketosis during the day. Therapeutic levels can be achieved more easily through oral intake of βHB . The dirβHB . Newer pβHB , 28. Howtanediol .Supplements containing ketone bodies have potential applications in cases of severe metabolic diseases, cancer, neurodegenerative disease, and many more. Apart from the medical use, such supplements are also of interest for lifestyle applications such as weight loss .βHB as the main ingredient are available since 2015. The first available product was a simple mixture of calcium and sodium βHB salt with an added flavor. There is currently no scientific opinion or direct testing published about products with a calcium and sodium βHB salt as the main ingredient. The aim of this study is to provide additional scientific information for an easily available ketone body salt mixture and discuss the potential benefit of such products in clinical application.In an Internet search of the worldwide supplement market, a lot of products with 2. The criteria for inclusion were absence of metabolic diseases (like diabetes), not pregnant, medically healthy with a normal medical history, and no intake of drugs  or nutritional supplements in the previous 30 days prior to the start of the study. Subjects were excluded if they suddenly got ill or consumed a restrictive diet, low carb, or ketogenic diet, in the 60 days before the day of testing. Recruitment was done by putting up a notice on a board at the University Hospital of Muenster. After filling in a precheck questionnaire, a short clinical examination was performed by a medical doctor. On the day of testing, those that had qualified for inclusion had to appear with an empty stomach. The intake of food and caloric beverages was not allowed for 12 hours, 24 hours for alcoholic beverages, before testing began. Black unsweetened coffee and water were allowed on the testing day. All subjects were informed in detail about the study and signed the consent before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Medical Association of Westfalen-Lippe and the University of Münster (project identification code: 2017-402-f-S).A total of six healthy adult subjects  aged between 18 and 57 years (40 ± 15.9 years) were selected for this study. The participants, 4–normal, 1–overweight, and 1–obese, had an average BMI of 25.44 ± 5.99 kg/mt0), the subjects ingested the prepared test solution. The solution was prepared for each subject by using the formula 0.5 grams of βHB-salt supplement per kilogram body weight dissolved in 250 milliliters (8.45 fl oz) of water. This translated to 30–57.5 g of the supplement per subject depending on their bodyweight. The taste of the beverage was recorded in a sensory interview after intake. Blood samples were drawn every 30 minutes over a period of 5.5 hours. Throughout testing, subjects were free to drink mineral water without gas and black unsweetened coffee or tea as well as move within the building. Each subject recorded observed side effects in a predetermined protocol. The concentrations of βHB and glucose were determined; blood gas analyses (BGA) in a two-sample series was performed every 2.5 hours, and the content of the minerals Na+, K+, and Ca2+ in serum was quantified. A medical doctor was available all the time and monitored the course of the study.A single-center one-dose kinetic study was conducted at the Muenster University Hospital, Germany, in accordance with the guidelines of good clinical practice. The present design of a one-dose kinetic study does not require randomisation, placebo-control, and blinding of the medical staff or subjects. The subjects fasted from 6 pm on the day before the test until the test began at 8 am. After weighing, placing a venous access and drawing the first blood sample . Additional ingredients are citric acid, natural flavor, and stevia as sweetener. The supplement facts are shown in The supplement, with the trade name KetoCaNa Orange, is produced by a manufacturer in the USA (Ketosports) and was purchased from an online shop in the Netherlands. The main ingredient is a combination of sodium and calcium At the start of study, a venous catheter was placed and the first blood sample was drawn. For further sampling, the catheter was flushed with physiological saline. To avoid dilution of the sample, five milliliters of blood were discarded up front each blood sample. In case of a blocked venous access, a new catheter was placed. The samples were immediately cooled at −20°C for analysis on the same day. Otherwise, storage temperature was −80°C.βHB were determined by using an enzymatic assay kit produced by Sigma-Aldrich . The kit is designed to produce a compound whose colorimetric intensity, determined at a wavelength of 450 nm, is proportional to the concentration of D-βHB. Glucose and mineral  content in serum were determined using the analyzer Cobas 8000 manufactured by Roche Diagnostics  and carried out in the clinical laboratory of the university hospital in Muenster.Serum levels of D-Blood samples were transported in a cooling box to the central laboratory immediately after sampling. The fully automated blood gas analyzer ABL800 Flex manufactured by Radiometer  was used to measure pH, electrolytes, and metabolites.+ was determined photometrically at 340 nm. Determination of pH was done using a pH meter and an electrode manufactured by Mettler Toledo , after performing a three-point calibration. The quantification of minerals was carried out by a certified food laboratory. For sodium, potassium, calcium, and magnesium, ICP-MS and ICP-OES according to DIN EN ISO 11885/DIN EN ISO 17294-2 were used.Content of citric acid was determined using an enzymatic test kit for food, manufactured by Boehringer Mannheim . The amount of oxidized NADH is stoichiometric to the citrate content. Difference between NADH and NADThe taste of the beverage was recorded in a face-to-face interview. All subjects had to describe the flavor and mouth-feel of the product in their own words supported. The questions of the interview were “Please describe as accurately as possible the taste of the beverage” followed by “How is the mouth-feel?” and “How would you describe the aftertaste?.” The last question was “Did you notice anything else?.” The test person was not to be interrupted when answering the questions, and the interviewer was prompted to interrupt only when responses were ambiguous or it was unclear what the test person meant. Especially positive or negative statements were examined more closely. The interview was recorded in its entirety.Data preparation was performed by using Microsoft Excel 2016, and for the data analysis, IBM SPSS Statistics 24 was used. Kolmogorov–Smirnov test, Shapiro–Wilk test, and graphical analyses were used to evaluate normal distribution. All data were first analyzed using descriptive statistics such as mean value, median, and standard deviation.t0). To calculate the difference between the results, a t-test for paired groups or Wilcoxon test was used. The level of significance was set at p ≤ 0.05. For relation among different sample series, the area under curve (AUC) was calculated.The basis for comparison was the first blood sample drawn from the subjects  with power of 80% and two-sided alpha of 0.05, a total of 5 participants were required. For security in case of dropout, one more subject (20%) was recruited.In order to detect a clinical relevant change in the main outcome variable n=5) successfully completed testing.One of the subjects  dropped out directly after imbibing the solution due to a severe reaction to the intake. These reactions included severe vomiting, nausea, and upper abdominal pain. All other volunteers (βHB/kg BW (3 mmol D-/L-βHB/kg BW) was calculated using the information provided by the manufacturer. The free D-βHB in serum increased after 2.5 hours from 0.232 ± 0.177 mmol/L at t0 to the maximal mean value of 0.598 ± 0.300 mmol/L at t6. The differences between 0 min, 150 min (maximum), and 180 min after treatment were significant . The first effect of the βHB salt intake was noted 2 hours later in the form of an increased concentration of serum D-βHB. Directly after reaching a maximum, the concentration declined continuously to near the baseline. There were large differences in D-βHB concentrations between the test persons as depicted in the rather large standard deviation in AUC . Serum glucose concentrations in the serum of the test subjects remained constant almost right through the study period with an increase at the maximum of D-βHB concentration and after 4-5 hours. Both differences in serum glucose were not significant  and anion gap (1.450 ± 0.778 to 2.350 ± 1.061 mmol/L). The measured electrolytes sodium and calcium presented no difference during the study period. Potassium increased, and the increase was directly proportional to D-βHB levels in tested subjects. Determination of significance was not possible due to the small number of cases.Blood gas analysis showed no difference in pH, pCOn=5) finished the test successfully. One proband reported feeling of fullness directly after beverage intake, which was caused by the volume of the solution. Two others had nausea and slightly upset stomach in the first 30 minutes of testing. One of the subjects developed stomach cramps, diarrhea, and severe nausea after one hour, and a metoclopramide medication was necessary. In further course, one person felt hyperactive and three reported not feeling hungry over the complete study period.One of the subjects dropped out directly after solution intake due to severe vomiting, nausea, and upper abdominal pain. All other volunteers  with a mean pH of 4.32 when prepared according to the recommendation made by the manufacturer. An intake of 0.5 g/kg of the product corresponds to 0.7 mmol/kg citric acid. The content of minerals was analogous to the specifications on the product package. For magnesium and potassium, possible contaminations causing slight deviation were detectable . The choβHB serum levels with a slow decline in healthy humans. Intake of a high concentration of D-/L-βHB supplement caused an average maximum increase of 0.366 mmol/L. Interpersonal variation of ketone body levels in humans is a well-known fact, a publication from 1958 determined a difference of 30 percent in healthy young men after fasting overnight [βHB concentration was around 50 percent which is similar to our findings [The results show a slow and moderate increase of D-vernight . In a cofindings .βHB salt. Only some case reports on the usage of sodium D-/L-βHB in severe metabolic diseases in children and studies testing different sodium and potassium D-/L-βHB salts are available. Van Hove et al. described a peak between 0.19 mmol/L and 0.36 mmol/L after 30 minutes to 1 hour caused by intake of 0.150 g/kg BW D-/L-βHB [βHB concentration increase of 0.055 mmol/L within 1-2 h (150 mg/kg BW) and a measured maximum of 0.343 mmol/L after 2 h (200 mg/kg BW) [βHB concentration after the intake of 0.9 g/kg BW, and only after increasing the intake to 2.6 g/kg BW was there a measurable change in D-βHB levels [βHB within 0.5–3 h measured in two children with hyperinsulinism [βHB salt when compared to our data [βHB [A direct comparison with other data is not possible due to absence of publications on the mixture of sodium and calcium D-/L-D-/L-βHB . Gautschg/kg BW) . Anothersulinism . Especiaour data . In all ata [βHB , 33, 34.βHB (3-hydroxybutyl-3-hydroxybutyrate) exhibited a concentration peak between 1,5 and 2.5 h into testing. The recorded cmax D-βHB was 1.00 mmol/L achieved using 357 mg/kg BW of ester; this value corresponds to 2.80 mmol/L achieved using 1 g/kg BW of ester. In the single intake study, no adverse effects using a maximum dose of 714 mg/kg BW were reported. Only in a repeated dose study with a concentration of 2142 mg/kg BW per day did gastrointestinal side effects like vomiting, nausea, diarrhea, or abdominal pain occur [βHB concentration in 10 minutes after drinking a solution made using 573 mg/kg BW of the 3-hydroxybutyl l-3-hydroxybutyrate ketone ester. In different experimental setups with young athletes, no adverse effects of βHB were reported [βHB concentration after 1.5 h to 1.00 ± 0.1 mmol/L after the intake of 282 mg/kg BW of a sodium and potassium D-/L-βHB salt. At the same time, tested βHB-ester leads to higher values at the same concentration (2.8 ± 0.2 mmol/L) [βHB salts to athletes during exercise. The levels of D-βHB concentration were between 0.60 and 1.00 mmol/L after the intake of the supplements [βHB [βHB was not high enough for specific side effects caused by ketone bodies. A possible reason is the high content of cations and citric acid in this product, causing the intense sour taste. As a whole, use of combination of calcium and sodium βHB salt is not free from adverse effects and exhibits a slow resorption kinetic. Just like in other supplements, such as the ester of 1,3-butandiol and βHB, the combined sodium and calcium salt exhibits a fast decrease in serum concentration. A direct comparison between the salts and the ester is not possible due to the structural differences and the metabolization of 1,3-butanediol being unclear. There are indications that 1,3-butandiol is metabolized to ketone bodies. This and the hydrolysis to D-βHB, not D-/L-βHB as in the salts, are possible explanations for the reported high βHB concentration in subjects after intake of this ester [βHB concentration in serum and a higher maximum using nearly the same dose when compared to our testing [βHB concentration [βHB. The results of athletes during exercise are not comparable to our population [Testing of a ketone body ester consisting of D-1,3-butanediol and D-reported . In heal mmol/L) . Two furplements , 28. In plements , 28, 37.nts [βHB . The tesis ester , 39. Thentration . It seempulation , 28.βHB. D-βHB acts as the main energy substrate in fasting humans and is therefore of high therapeutic relevance [βHB, acetoacetate, and acetone were not analyzed in this study. Accordingly, an underestimation of total ketone bodies is possible. The effect of active substrates is considered to be low because there was an intake of βHB without longer fasting time period and a resulting short-time increase of βHB in serum. L-βHB is the nonphysiological enantiomer of D-βHB. There is an indication that L-βHB can be converted to physiological active ketone bodies (acetoacetate and D-βHB) and lipids in animal models but only in a limited amount [βHB showed a much lower metabolic rate and conspicuously higher elimination in urine than the D-βHB after intake of a D-/L-βHB salt [βHB in humans.A limitation of the measurements is the usage of an enzymatic assay specific for clinically relevant D-elevance . L-βHB, d amount , 42. In βHB salt . More reA one dose intake did not result in an increase of calcium and sodium. The relatively high amounts of both salts had no direct effect on their serum levels. In an example, a person weighing 70 kg would take around 2.100 g (91 mmol) sodium and 2.170 g (54 mmol) calcium with one dose (0.5 g/kg) of the supplement. The three-times daily dosage of 19 g product recommended by the manufacturer would lead to an intake of 3.420 g 149 mmol) sodium and 3.534 g (88 mmol) calcium. The calculated loads of both elements are above the tolerable upper intake level (UL) for adults recommended by the Institute of Medicine (IOM) (sodium 2.3 g/d (100 mmol/d); calcium 2.5 g/d (62 mmol/d)) , 44. In  mmol sodβHB and potassium levels needs further research. In our testing, we can exclude the influence of the test product (16 mg K+/100 g) or a pseudo hyperkalaemia by venous catheter.The finding of a similar increase between D-+ and Ca2+) is a potential risk factor for alkalosis. On the other hand, a low SID and at the same time high strong ion gap (SIG) triggered by ketoacids can cause a metabolic acidosis [βHB mixture, but no similar effect in a corresponding test using βHB-ester. Both products had a mild influence to the acid-base balance [2, and product specific cations, are necessary to avoid adverse metabolic effects.The excessive intake of ketone body salt or acid could have consequences for the acid-base balance. According to a theory by Stewart, a shift of the strong ion difference (SID) can cause alkalosis or acidosis. On one hand, a high SID generated by intensive resorption of cations . No interpersonal factors have been included in this simple calculation of our results. In combination with a higher intake, the cation load and proportion of additives increases, therefore increasing the potential risk of side effects. For treatment with high doses of external ketone bodies, further clinical trials on healthy adults are needed to gather more information on metabolic utilization.


Here we compare for thefirst time the utility of 33 Single Nucleotide Polymorphisms (SNPs) and ninemicrosatellites (MS) in isolation and in combination for assigning parentage incaptive White Rhinoceros. We found that a combined dataset of SNPs andmicrosatellites was most informative with the highest confidence level. This studythus provided us with a useful set of SNP and MS markers for parentage andrelatedness testing. Further assessment of the utility of these markers over multiple(> three) generations and the incorporation of a larger variety of relationshipsamong individuals  is strongly suggested.The white rhino is one of the great success stories of modern wildlife conservation,growing from as few as 50-100 animals in the 1880s, to approximately 20,000 whiterhinoceros remaining today. However, illegal trade in conservational rhinoceros hornsis adding constant pressure on remaining populations. Captive management of Ceratotherium simum simum) have increased from a population of lessthan 100 at the end of the 19th century, to an estimated population of over 20,000 represent the most abundant type of DNA variationin the vertebrate genome and are distributed across the entire genome providing broadergenome coverage as compared to mitochondrial DNA or microsatellites (MS) . Clones containing fragments ranging from 200-700bp were selected and sequenced utilising a Big Dye V3.1 Terminator Kit and an ABI 3500XLgenetic analyser. The potential SNP loci were amplified in the 5 isolates used for theinitial DNA pool. Amplification reactions were done in a final volume of 25 μLcontaining 30 ng DNA, 25 pM of each primer and 2X DreamTaq® Green Master Mix(Thermo Scientific). Thermal cycling consisted of initial denaturation at 95 °C for 5min, 45 cycles of denaturation at 95°C for 30 s, annealing at 55-59 °C for 30 s,extension at 72 °C for 90 s, followed by final extension at 72 °C for 10 min. Resultingamplicons were inspected on 1% agarose gels followed by purification and sequencing asdescribed above. Sequences were inspected and aligned in CLC Bio Genomics work bench8.0.1 . Twelve resulting SNP markers were further typed in theremaining 27 isolates. GENEPOP version 4.0.10  following the manufacturer'sinstructions. A SNP enriched library was constructed using DNA from 5 individuals anddigestion with Endonuclease V as previously described . Allele sizes were estimated by comparison with aGenescan™500 LIZ™ internal size standard  using the ABI programsGENESCAN (version 1.2.2.1) and GENOTYPER (version 1.1). Sanger sequencing was performedin both directions and SNP calls were only made on bases with quality scores higher thanQ > 20. The SNPs all fall within the central region of the fragments where sequencingquality is the highest. In order to ensure accurate genotyping, the samples wererepeated if they were homozygous, aberrant stutter patterns or spurious peaks wereobserved or if the profiles were below the quality score. Differences in mean observedheterozygosity (Ho), mean expected heterozygosity (He) and mean number of alleles wasdetermined as mentioned above.Nine microsatellite loci: BR6 , RHI7C, Parentage assignment was evaluated using the MS and SNP data sets individually and as acombined dataset. The software program Cervus v3.03  and pairTwelve SNPs (GenBank accession numbers 1416044499-1416044509) were identified in thisstudy across 11 loci (WR1-WR11). The primer sequences and allele frequencies of the 12SNPs developed here together with a further 21 SNPs for the 32 individuals are listed inThe 32 individuals consisted of 11 known mother/offspring groups with two mothers havingtwo offspring as illustrated in Using the SNP data set, six paternal allocations could be made with 95% confidence.Using the MS data set, two paternal allocations can be made with 95% confidence and sixwith 80% confidence. Two of the allocations with positive scores correspond between thetwo data sets. Paternal allocations on the combined dataset were conducted in two ways;where maternal assignments were fixed and without known mothers. Using the combined dataset with unknown mothers, five paternal allocations could be made with 95% confidence. In the Together with the 12 new SNPs identified in this study, 33 SNPs are now available forwhite rhino . The SNPTo our knowledge this is the first study to employ SNP and MS markers for parentageanalysis in white rhino. The current SNP set outperform the MS set during maternalassignment, where all assignments were correct while the MS data set allocated onematernal sample incorrectly. In general, assignments made with the SNP data set hadhigher confidence than those with the MS set. Confidence levels increased when combiningthe two data sets. The increased accuracy of the SNP markers in this study over MSmarkers can be attributed to the greater marker numbers in the SNP data set and lowallele numbers of the MS markers. It is apparent that low levels of genetic diversitycharacterise white rhino populations and the results from our study are consistent with other studies making use of MS markers. Challenges to parentage assessment can arise when family members other than the parentsof the offspring are included in the pool of candidate parents . Inclusi


Oncorhynchus mykiss), SNP discovery has been previously done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL) and RNA sequencing. Recently we have performed high coverage whole genome resequencing with 61 unrelated samples, representing a wide range of rainbow trout and steelhead populations, with 49 new samples added to 12 aquaculture samples from AquaGen (Norway) that we previously used for SNP discovery. Of the 49 new samples, 11 were double-haploid lines from Washington State University (WSU) and 38 represented wild and hatchery populations from a wide range of geographic distribution and with divergent migratory phenotypes. We then mapped the sequences to the new rainbow trout reference genome assembly (GCA_002163495.1) which is based on the Swanson YY doubled haploid line. Variant calling was conducted with FreeBayes and SAMtools mpileup, followed by filtering of SNPs based on quality score, sequence complexity, read depth on the locus, and number of genotyped samples. Results from the two variant calling programs were compared and genotypes of the double haploid samples were used for detecting and filtering putative paralogous sequence variants (PSVs) and multi-sequence variants (MSVs). Overall, 30,302,087 SNPs were identified on the rainbow trout genome 29 chromosomes and 1,139,018 on unplaced scaffolds, with 4,042,723 SNPs having high minor allele frequency (MAF > 0.25). The average SNP density on the chromosomes was one SNP per 64 bp, or 15.6 SNPs per 1 kb. Results from the phylogenetic analysis that we conducted indicate that the SNP markers contain enough population-specific polymorphisms for recovering population relationships despite the small sample size used. Intra-Population polymorphism assessment revealed high level of polymorphism and heterozygosity within each population. We also provide functional annotation based on the genome position of each SNP and evaluate the use of clonal lines for filtering of PSVs and MSVs. These SNPs form a new database, which provides an important resource for a new high density SNP array design and for other SNP genotyping platforms used for genetic and genomics studies of this iconic salmonid fish species.Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout ( Oncorhynchus mykiss) is an iconic salmonid fish species with a remarkably diverse life history, and has a wide interest as a model research organism as well as high economic value for the sport fishing and aquaculture industries. The substantial scientific and economic interests in this species justify the continued development of genomic resources. Much effort has been devoted in recent years for developing genomic resources for research in rainbow trout, including a draft genome assembly .The rainbow trout (GCA_002163495.1), generated a great opportunity for large-scale SNP discovery using whole genome resequencing of target populations. A comprehensive SNP database from genome resequencing data can further improve the design and selection of a new SNP arrays by improving the genome coverage and spacing of the SNPs as well as selecting markers for follow up studies within targeted regions of the genome and for particular populations.Whole genome association analyses (WGA) can be useful for dissecting complex traits in natural populations and in aquaculture breeding programs and a lot of research in rainbow trout has been using WGA for better understanding of the genetics underlying complex traits  round of ancestral genome duplication in their evolutionary history compared to mammals and birds, but the salmonids underwent a further salmonid-specific (Ss4R) whole genome duplication (WGD) event ~95 MYA  and the coastal subspecies (O. mykiss irideus), although most come from hatchery strains at various stages of domestication.In our more recent SNP discovery effort we have demonstrated the effectiveness of using doubled-haploid (DH) homozygous fish in the discovery panel for filtering out likely PSVs  was used as the reference, and BWA-MEM was used to map the sequences to the reference. We employed two pipelines, SAMtools mpileup and FreeBayes, for variant calling, and filtered the SNPs with a series of stringent filters, including using data from the 11 DH lines to filter out PSVs and MSVs. The SNP database generated will be useful for improving the current whole genome SNP genotyping arrays for rainbow trout as well as for targeted SNP selection in smaller studies that are focused on specific genome regions or particular populations.In this study, we used whole genome shotgun resequencing data from DH lines together with aquaculture, hatchery and wild populations to produce a comprehensive SNP dataset for rainbow trout and steelhead. The recently released rainbow trout genome assembly  using BWA MEM  and SAMtools mpileup. In both pipelines, we required a minimum mapping quality score : We only extracted the SNPs that are bi-allelic, not located within 4 bases distance to an indel, and have the phred-scaled variant quality score, QUAL, larger than 30.mdust (https://github.com/lh3/mdust/) as suggested by Li (Low-complexity filter (LC): We removed the SNPs that are in the low-complexity regions in the genome that were identified by the program ed by Li . OverallFreeBayes and SAMtools mpileup report the read depth in slightly different manner due to algorithm differences, we chose the same maximum depth value of 1,500, corresponding to a coverage of 24.6 reads per sample : We removed the sites that were covered by excess number of reads as these sites are very likely to be located within a repeat or a multi-duplicated region. Although mple Li, , for filMinimum sample filter (NS): Only SNP sites with sequence reads coverage from at least 58 of the 61 samples passed this filter, to ensure that the majority of the samples were represented in SNP calling for all the sites. To call genotype for an individual sample, we required at least two reads to support the alternate allele and one read to support the reference allele.Double haploid filter (DH): To filter out putative PSVs and MSVs, we employed the same strategy previously used for RAD sequence data  was used in building the SnpEff database. The SNPs VCF file was used as the input file, and the results of SnpEff contain the classifications of the effects of the SNPs based on the annotated protein-coding genes and their genomic locations.The putative SNPs were annotated with SnpEff (version 4.3p), an automatic pipeline for rapidly categorizing the effects of SNPs based on the annotation data of a reference genome . This is a fairly stringent criteria compared to other published SNP discovery projects in salmonids , the potential impact of the single base substitution and the number of SNPs found in each effect type category. Most of the SNP effects (98%) were classified as unknown with the “MODIFIER” putative impact. However, there were 15,198 SNP effects that were considered as HIGH putative impact and 1,579,314 with MODERATE or LOW putative impact. In addition, SnpEff also classified 7,108, 625,238 and 734,274 SNP effects as NONSENSE, as MISSENSE and as SILENT functional classes, respectively.SNPs that can cause gain or loss of function can modify the protein coded by the gene and have major impact on the phenotype of the organism. Therefore, it is important to annotate the SNPs with a potential functional impact in the new SNP database. The functional annotations of the potential SNP effects are listed for each SNP in a supplemental VCF file , among the 50 outbred or non-DH rainbow trout that we sequenced. In comparison, for the ~31.4M SNPs that passed our filtering criteria among the same set of 50 fish, only 12% of the loci were not in Hardy-Weinberg equilibrium (P < 0.05) and the average observed heterozygosity was 0.12, indicating excess heterozygosity among many of the ~1.7M putative PSV/MSV loci that were filtered out of the SNP database. Higher average observed heterozygosity than expected per locus is a strong indicator for potential MSVs or PSVs among the surveyed loci , and genome resequencing of 61 rainbow trout and steelhead samples, including 11 doubled haploid clonal lines, we discovered 31,441,105 high quality SNP loci. The SNP loci are broadly distributed across the 29 chromosomes with a genome-wide average rate of 15.6 SNPs per 1 kb. The new SNP database, together with their functional annotation, provide an important resource for new SNP array designs, whole genome association analyses, and design of genotyping assays in rainbow trout.Using the new rainbow trout reference genome (GenBank assembly Accession SRP107028. (Project ID: PRJNA386519).Raw sequence data that were generated in this study were deposited in the NCBI short reads archive under accession DNA sequences: NCBI SRA accession SRP107028 (BioProject ID: PRJNA386519).The VCF file for the database of all the SNPs identified in this study including the SnpEff annotation information and a file with allele frequency information for each SNP in the database are available for downloading from a public repository .GG: performed bioinformatics and data analysis and wrote the manuscript draft; TN: assisted with the study design and data collection and contributed to the data analysis; DP: co-designed the study and contributed samples; TM: collected samples and contributed DNA sequence data; KN: collected samples and contributed sequence data; GT: co-designed the study, collected samples and contributed sequence data; SL: co-designed the study and co-directed the work; YP: co-designed the study and directed the work and contributed to the data analysis and first draft writing. All authors reviewed the manuscript draft and approved its content.The handling Editor declared a past co-authorship with several of the authors KN, SL, and YP. TM was employed by company AquaGen. The other 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.


Magnetic hollow spheres with a controlled diameter and shell thickness have emerged as an important class of magnetic nanomaterials. The confined hollow geometry and pronouncedly curved surfaces induce unique physical properties different from those of flat thin films and solid counterparts. In this paper, we focus on recent progress on submicron-size spherical hollow magnets , and discuss the effects of the hollow shape and the submicron size on magnetic and optical properties. Nanostructured magnetic materials have attracted tremendous attention because of their potential for clarifying fundmental nanomagnetism and their technological applications in diverse fields ,4,5,6,7,3O4 nanoparticles have been used for targeted delivery and controlled release of cisplatin, which is a powerful therapeutic agent against numerous solid tumors [Among various morphologies of nanomaterials, hollow spheres are of great interest because of their high ratio of surface to volume, large pore volume and low density, which could be exploited for promising applications in controlled encapsulation-release of drugs and medical diagnostics ,10, enerd tumors . In the context of magnetism, magnetic hollow spheres can show unique physical properties compared to those of flat thin films and their solid counterparts of the same sizes, due to their confined hollow geometry and pronoucedly curved surfaces ,24,25,263O4 [2O3 [2O4 [3O4 [Over the last decade, much effort has been devoted to studying submicro-/nano-size magnetic hollow spheres of metals  have been widely prepared in recent years, and have shown promising applications in biomedical fields because of their superparamagnetic behaviors, it remains important to prepare submicron-size magnetic hollow spheres with highly homogeneous features in terms of size and shape, in order to carry out fundamental magnetic studies, such as investigations of magnetic domain structures and magnetization reversal. The comparatively large size also means that the critical temperatures of magnetic ordering or magnetization blocking would be sufficiently high for practical applications. Further, the submicron size and low-density hollow structure may bring about specific optical response in the visible region. On the other hand, magnetic elements with submicron sizes have shown different physical properties from those with nano- or macro-scale sizes. For example, two characteristic domain states, referred to as “vortex” and “onion” states have been observed in submicron-size ferromagnetic rings, and switching between the two states has been experimentally realized 0 nm have, and curi.e., about several hundred nanometers). In this paper, we first present a facile synthetic strategy for preparing hollow spheres of different phases, such as ccp-Co, hcp-Co, Co3O4, α-Fe, Fe3O4, and α-Fe2O3. Then we describe unusual physical properties in the submicron-size spherical hollow magnets of ccp-/hcp-Co, Co3O4 and Fe3O4, and discuss the effects of the hollow structure and the submicron size. Finally, we provide a conclusion and outlook on future developments in spherical hollow magnets.There are many comprehensive reviews of the synthetic strategies and applications of hollow micron-/nanostructures, especially for nonmagnetic materials ,60,61,62et al. have recently reported various submicron-size magnetic hollow spheres prepared by a homogeneous deposition method using a PS-bead template [3)2 (or FeCl3), urea, PS, and PVP resulted in an increase in pH due to thermal decomposition of urea. As soon as the solution became basic, homogeneous precipitation of basic cobalt carbonate (or iron hydroxides) took place on the surfaces of the PS beads. Aging at 85 °C (or 100 °C) for 24 h allowed inorganic nanocrystals to grow on the surfaces; precursors for cobalt-based (or iron-based) hollow magnets were obtained after centrifugation of the above dispersion and washing with pure water. The optimized conditions are 3.78 mM Co(NO3)2•6H2O, 0.30 wt % PVP, 0.04 M urea, and 0.10 g L-1 PS beads at 85 °C for 24 h for cobalt-salt/PS particles; and 1.48 mM FeCl3, 22.0 mM urea, 0.10 g L-1 PS, 0.90 wt % PVP (K-30), and 0.06 g L-1 HCl (36.0 wt %) at 100 °C for 24 h for iron-salt/PS particles, respectively. As described in the Introduction, the hard template method provides an effective approach to prepare submicron-size hollow spheres with a uniform size distribution; monodisperse polystyrene (PS) beads have been commonly used as templates because they are readily available in a wide range of sizes. Ohnish template . As a fi3O4 hollow spheres. Hcp-Co hollow spheres were obtained by a reduction of the Co3O4 particles under 1:1 H2/N2 gas at 400 °C; in contrast, ccp-Co hollow spheres were obtained by the calcination of the parent cobalt-salt/PS particles under H2-N2 at 400 °C. Hollow spheres of Fe3O4 and α-Fe2O3 were selectively prepared by calcination of iron-salt/PS particles at 500 °C in two different atmospheres, namely under N2 and in air, respectively. Furthermore, the α-Fe2O3 particles were reduced to α-Fe under H2-N2 at 350 °C without any changes in morphology. After calcining the precursors under different atmospheres and temperatures to remove the PS-bead cores, various types (phases) of hollow magnets were obtained, as shown in the schemes in 3O4 and Fe3O4, indicating the formation of hollow structures with a uniform diameter and shell thickness. The mean diameter, shell thickness and average grain size of the various magnetic hollow spheres are summarized in Ms between them, yet their coercive fields Hc differ significantly. The Hc value of 1150 Oe for hcp-Co is much larger than that of 450 Oe for ccp-Co; this finding is consistent with the fact that hcp-Co has greater magnetic anisotropy. Further, the hcp-Co hollow spheres exhibit both enhanced coercivity and a higher remanent ratio (Mr/Ms = 0.42) than the chains of hcp-Co hollow spheres with a diameter of 500–800 nm  reported by He et al. [As one of the advantages of the above synthetic strategy, ccp-Co and hcp-Co, which are usually mixed in conventional cobalt samples, were successfully separated in the hollow magnets. The two phases of cobalt hollow magnets were confirmed by the two sets of XRD patterns presented in e et al. . This ree et al. .Although both the ccp- and hcp-Co hollow spheres were polycrystalline particles, they exhibited great differences in magnetic anisotropy based on their hysteresis behaviors. Recent theoretical calculations on nanosized Co hollow spheres showed that magnetic anisotropy played a significant role in determining the three possible magnetization ground states: the single domain, curling vortex, and three-dimensional onion states ,63. Ther3O4 possesses a normal spinel structure with Co(II) and Co(III) ions respectively locating in the tetrahedral and octahedral interstices of the ccp-lattice of oxide anions and exhibits antiferromagnetism below the Néel temperature of TN = 33 K, since the Co(II) ions at the tetrahedral sites form an AFM sublattice with a diamond structure [3O4 materials, such as nanoparticles [Antiferromagnetic (AFM) nanomaterials have recently drawn increasing attention by virtue of their potential for exhibiting quantum tunneling of magnetization, weak ferromagnetism, and exchange anisotropy. Néel suggested that nanoscale AFM nanoparticles can exhibit superparamagnetism and weak ferromagnetism due to the presence of uncompensated magnetization (UCM) on two sublattices . In nanotructure . Recentlarticles ,73 and narticles . These aet al. have reported greatly enhanced field-cooled (FC) magnetizations in submicron-size Co3O4 hollow spheres [3O4 hollow spheres with diameters of 360 and 680 nm. Below TN = 33 K, they exhibit enhanced magnetizations arising from the UCM. The UCM of the hollow spheres is several times larger than those of bulk samples and nanoparticles of Co3O4, with characteristic size-dependence: the UCM of the smaller hollow spheres is twice as large as that of the larger ones. This is probably caused by the fact that the increases in the surface/volume ratio and in the number of lattice defects are greater in the smaller hollow spheres. Because the UCMs behaved superparamagnetically, the total magnetic spins became easy to order, and hence the magnetization was increased. The field-induced magnetization below NT is believed to be stored at the domain boundaries, where the magnetic moments are readily aligned to the field direction, taking advantage of the fact that the long-range AFM correlation is interrupted there. The cooling field energetically favors the domains with a parallel magnetization alignment and induces a remnant magnetization below NT. Since the Co3O4 hollow spheres probably involve a number of lattice defects that cause such domains, they exhibit a drastic cooling-field dependence; the stronger the cooling field, the larger FC magnetization below TN [Yoshikawa  spheres . Figure below TN . Figure t in a logarithmic scale. The time-dependent magnetizations were recorded at each temperature under a zero field, after the samples were cooled down from 300 to 2 K under 50 kOe and then heated up to the target temperatures with a zero field. At each temperature, UCM showed a gradual decrease that was proportional to the logarithm of t. This dependence was fitted to the following equation [equation ,75(1)M with open pores (~30 nm) in shell layers using dodecylamine micelles as a template [et al. reported the synthesis of single-crystal magnetite hollow spheres with a diameter of 200–300 nm by a template-free solvothermal route [3O4 hollow spheres with a diameter of 100–200 nm have recently been reported by Huang et al. [et al. have prepared magnetite hollow spheres with rather uniform size of outer diameter ca. 650 nm and shell thickness ca. 40 nm [Submicron-size magnetite hollow spheres have been synthesized by many groups. Yu template . Zhu et al route . Furtherg et al. . Howevera. 40 nm , and sysa. 40 nm  and groua. 40 nm . 2+ and Fe3+ ions on the octahedral sites has been proposed to account for the phase transition, although this mechanism is still under debate [Bulk magnetite undergoes the Verwey transition at around 120 K, and the charge ordering of the Fer debate ,82. The r debate . Both thr debate ,82,85. Tr debate . In addii.e., M–H curves) and the hysteresis behaviors of magnetite hollow spheres were carefully studied in the temperature range of 2–300 K [Mrs, and possessed the largest maximum energy product, (MH)max, which corresponds to the area in the hysteresis loop. This result indicates that one way to achieve magnetically hard materials is by assembling soft nanoparticles into spherical hollow structures.Magnetization curves  of the magnetite hollow spheres, dense particles and ground NPs are significantly lower than that of bulk magnetite (97 emu/g), which is attributable to the surface disorder and/or spin canting at the surface of the particles [M-H curves measured at different temperatures. For the three samples, the Ms obtained at 5 T showed a steady decrease with increasing temperature, and the thermal decreases were fitted by the Bloch law [Ms(0) is the saturation magnetization at zero temperature, B0 is the Bloch constant related to the exchange integral and n is the Bloch exponent. The best fitting results for the three samples are listed in n = 2.0), but different from recent reports (n = 1.5) on magnetite nanoparticles [The saturation magnetizations Ms, pseudo-single-domain (PSD), and multi-domain (MD) states are separated from each other. The PSD state implies that the magnetic behavior is intermediate between SD and MD [The unique hysteresis behaviors and possible domain states were further elucidated by using a Day plot ,91,92, i to zero ,94. FiguD and MD , and theHcr/Hc, resulting in the Day plots for the hollow spheres appeared to indicate the SD state.This feature of the hollow sphere was caused by its unique shape. In the case of a spherical hollow magnet with low magnetocrystalline anisotropy, the magnetization was theoretically predicted to predominantly follow the circumference in order to minimize the stray field, and hence the curling-vortex state with the vortex singularities on the poles of the hollow sphere was formed as a ground state ,95,96,97i.e., iron-salt/PS particles) were fabricated by the fluidic-cell method [Magnetite is a low magnetocrystalline-anisotropy material, and its thin films usually exhibit two-dimensional (2D) shape anisotropy; the parallel magnetization is larger than the perpendicular one, reflecting dipole-dipole interactions in the films . Recentll method ,99. And M-H curves of the two films were measured under magnetic fields parallel and perpendicular to the 2D films. The As shown in this section, the magnetite hollow spheres exhibited unique magnetic properties, such as high blocking temperature, enhanced coercivity and curling-vortex magnetization state, as well as magnetic isotropy. These results suggest that the magnetic hollow structures may have advantages for investigating the dynamic behaviors of spin rotation and relaxation by applying magnetic or electrical fields, and therefore have potential applications in spintronics, in which the aim is to achieve a spin-polarized current or current-control magnetization .Hollow structures and submicron sizes induce not only unique magnetic properties but also anomalous optical properties. The green solid curve in R for the highly aggregated particles was theoretically calculated through the Kubelka-Munk approach [Qabs, the scattering efficiency Qsca, and the asymmetric factor r1 and refractive index m1 and a magnetite shell with radius r2 and refractive index m2 [m1 = 1.0 (fixed), m2 = 2.55 + 0.70i, r1 = 285 nm (fixed), and r2 = 325 nm (fixed). The obtained m2 values are consistent with the reported values for bulk magnetite [In general, color results from the interactions between light and matter, namely absorption, scattering and reflection . These aapproach :(3)R=1+g theory ,102. In index m2 . The greagnetite ,104,105.m1 = m2 = m. The black dotted curve in m = 2.55 + 0.70i, which is the value optimized above, and the particle radius r2 = 410 nm. The theoretical simulation well reproduces the weak wavelength dependence of diffusion reflectance of the dense particles. Although the dense particles had a noticeable size distribution, the calculated reflectance spectra did not show large differences by setting the radius r2 from 250 to 450 nm. It is notable that this feature is caused mainly by the light absorption in the visible range, due to the dense structure; the absorption of the hollow spheres is much weaker than that of the dense particle, due to its low-density structure [For the dense particles which are homogeneous media, the simulation of the reflectance spectra was carried out based on Eq. 3 and the Mie scattering theory by setting 3O4 hollow spheres. The Co3O4 hollow spheres with outer diameter of 760 nm and shell thickness of 70 nm displayed a light green color  of hollow spheres were obtained by selectively optimizing the calcination conditions. The synthetic strategy showed advantages for obtaining a uniform outer diameter and shell thickness of hollow spheres, which enabled people to achieve and tune amomalous physical properties. Futher, it is noteworthy that the ccp and hcp phases of cobalt could be separated and a single phase of hcp-Co with high magnetic anisotropy could be selectively synthesized using the synthetic method. The hollow spheres exhibited significantly different physical properties from those of flat thin films and solid counterparters. Weak ferromagnetism arising from uncompensated spins and exchange anisotropy were found in the antiferromagnetic Co3O4 hollow spheres. Magnetite hollow spheres showed significantly enhanced coercivity and an unusual green color. These unique physical properties were interpreted in terms of the effects of the hollow structures, curved surfaces, and submicron sizes, in comparison with submicron-size dense particles and nanoparticles. These results indicate that the spherical hollow magnets may have potential applications in data-storage media, spintronics, and magneto-optical devices. However, some fundamental aspects need to be further studied, such as direct observation on magnetic domain structures, electrical and magnetic transport on individual magnetic hollow spheres, and magneto-optical properties.We have reported on recent progress in the study of submicron-size magnetic hollow spheres. The hollow spheres with uniform size and morphology were fabricated through two steps: first, inorganic precursor materials were homogeneously deposited on the surfaces of PS beads; then various phases  macromolecules via polymerization of 4-Vinylpyridine and N-alkylation reactions [Further, spherical hollow structures are advantageous for various surface modifications. Ohnish eactions . After t2O hollow microspheres show greatly improved sensitivity to ethanol gas [In addition to surface modifications, the interior hollow pore of the hollow spheres may be constructed into multilevel architectures, such as a core-in-hollow-shell (or rattle-type) structure, multishell structure and multichamber structure ,57,58. Sanol gas . However