Within BAP1-positive lesions (n = 20), 8q-gain did not relate to MVD (p = 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (p = 0.041), Von Willebrand Factor (VWF) (p = 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (p = 0.024), and increased numbers of tumour-infiltrating macrophages  and lymphocytes . Concluding, vascular density of UM relates to its genetic profile: Monosomy 3 and BAP1-loss are associated with an increased MVD, while an early event (gain of 8q) is not independently related to MVD, but may initiate a preparation phase towards development of vessels. Interestingly, VEGF-B expression is decreased in UM with a high MVD.Increased angiogenesis is associated with a higher metastasis- and mortality rate in uveal melanoma (UM). Recently, it was demonstrated that genetic events, such as 8q-gain and BAP1-loss, influence the level of immune infiltrate. We aimed to determine whether genetic events, and specific cytokines, relate to angiogenesis in UM. Data from UM patients who underwent enucleation between 1999 and 2008 were analysed. Microvascular density (MVD) and the presence of infiltrating immune cells were determined with immunohistochemistry (IHC) and immunofluorescence in 43 cases. Chromosome status, BAP1 IHC and mRNA expression of angiogenesis-related genes were known in 54 cases. Tumours with monosomy 3/BAP1-loss showed a higher MVD compared to tumours with disomy 3/normal BAP1 expression ( Uveal melanoma (UM) is the most common ocular malignancy in Caucasian adults. The disease is often lethal with up to 50% of patients developing metastases . In receRestricted by the limits of diffusion 1–2 mm), an expanding UM requires new vessels to grow. The concept of the “angiogenic switch” describes the turning point between an initial phase of slow, avascular growth into a phase with more rapid growth and angiogenesis. Angiogenesis can be studied using micro-vascular density (MVD). An increased MVD has been associated with a higher metastasis rate  mm, an e,4. SinceWhile angiogenesis describes the formation of endothelial-lined new vessels, it is important to note that other mechanisms resulting in intra-tumoural vascular channels have been recognized in UM ,10. ThisThe MVD of UM is known to be associated with the tumour’s immune infiltrate. Studies on this topic have focussed mainly on the relation with an increased presence of macrophages, describing their pro-angiogenic properties. An association between a higher MVD and increased numbers of all macrophages (CD68+ cells)  and M2 tWe hypothesize that genetic changes not only influence the immunological microenvironment, but also drive angiogenesis in UM, and that MVD is a consequence of a highly-malignant genetic profile. We set out to test this, and also analysed if several angiogenesis-related cytokines are expressed in relation to the development of tumour vascularity of primary UM.As a high MVD is known to be associated with a bad prognosis in UM, we first determined whether our population confirmed the existing assumptions. The MVD had been determined by counting CD34-expressing vessels in sections of 43 UM, using a well-established technique as presented by Makitie et al. , ,19, 2,19Tumour material was snap frozen using 2-methyl butane and later used for DNA and RNA isolation. Remaining tumour material was fixed in 4% neutral-buffered formalin for 48 h and embedded in paraffin. Haematoxylin/eosin-stained 4 μm sections were reviewed by an ocular pathologist for confirmation of the diagnosis and evaluated for histologic parameters . The eighth edition of the AJCC staging manual was used for tumour classification .2. Numbers of lymphocytes and macrophages were assessed as described previously [2. Macrophages were detected with IF using antibodies against CD68, CD163, and CD68CD163 double-staining. Counts were determined in pixels/mm2. BAP1 status was assessed with IHC as described previously [MVD was assessed in 43 cases with IHC for CD34 as described previously [eviously . T cellseviously . NuclearThe QIAmp DNA Mini Kit was used to isolate DNA for single nucleotide polymorphism (SNP) analysis according to guidelines of the manufacturer . Status of chromosome 3 was determined with SNP analysis performed with the Affymetrix 250K_NSP chip and the Affymetrix Cytoscan HD chip  . The cophttp://cancergenome.nih.gov/ [Findings were corroborated using mRNA data from 80 UM patients from the TCGA project: nih.gov/ . In thisnih.gov/ .U test between 2 groups, and with the Jonckheere test between multiple groups with a trend. Correlations were assessed with the Spearman’s test. Linear regression was performed for univariate and multivariate analyses. Survival data was analysed with the Kaplan–Meier method and log-rank tests; groups of high and low MVD and mRNA gene expression were based on the median. p-values < 0.05 were considered statistically significant.Analyses were performed using SPSS version 23 (I.B.M.). Categorical data was analysed with Chi-square tests. Numerical data was analysed with the Mann–Whitney In conclusion, we demonstrated that the genetic evolution of UM not only involves tumour infiltrate, but also tumour angiogenesis. Late events (such as BAP1 loss) are related to an increase in MVD, while early events (such as 8q gain) are not. Gain of 8q may be related to a preparation phase; however, as several angiogenesis-related genes are already expressed differentially in the absence of monosomy 3/BAP1 loss. We observed new associations with MVD, such as with monosomy 3/BAP1 loss, an increased count of lymphocytes, and a decreased expression of VEGF-B, indicating that more (and other) mechanisms are involved in angiogenesis of UM than previously thought.


Here, we present an approach and its accompanying software for high-resolution reconstruction of founder mosaic genotypes in the intercross offspring from such populations using whole-genome high-coverage sequence data on founder individuals (~ 30×) and very low-coverage sequence data on intercross individuals (< 0.5×). Sets of founder-line informative markers were selected for each full-sib family and used to infer the founder mosaic genotypes of the intercross individuals. The application of this approach and the quality of the estimated genome-wide genotypes are illustrated in a large FTn5-based library preparation protocol and an imputation algorithm that was optimized for this application. In total, 7.6 million markers segregated in this pedigree and, within each full-sib family, between 10.0 and 13.7% of these were fully informative, i.e. fixed for alternative alleles in the founders from the divergent lines, and were used for reconstruction of the offspring mosaic genotypes. The genotypes that were estimated based on the low-coverage sequence data were highly consistent (> 95% agreement) with those obtained using individual single nucleotide polymorphism (SNP) genotyping. The estimated resolution of the inferred recombination breakpoints was relatively high, with 50% of them being defined on regions shorter than 10 kb.We describe how we obtained whole-genome genotype data for hundreds of individuals in a cost- and time-efficient manner by using a https://github.com/CarlborgGenomics/Stripes.A method and software for inferring founder mosaic genotypes in intercross offspring from low-coverage whole-genome sequencing in pedigrees from heterozygous founders are described. They provide high-quality, high-resolution genotypes in a time- and cost-efficient manner. The software is freely available at The online version of this article (10.1186/s12711-019-0487-1) contains supplementary material, which is available to authorized users. Genotyping by next-generation sequencing has emerged as a rapid, high-throughput approach to obtain high-density genotypes in large populations , 2. The Several algorithms have been developed for genotype imputation based on low-coverage sequencing data. Some were developed for general pedigrees and data, while others were targeted for more specific applications, resulting in variation in their performances depending on how well underlying assumptions about the data are met. In populations with unrelated individuals, the individual genomes are often assumed to consist of short mosaics of ancestral haplotypes. Examples of software that have been developed for genotype imputation in such applications include Beagle , MaCH 1, and ShaThere is a dearth of alternatives for analysing data from populations that were bred by mating two or more outbred (heterozygous) founders. For such populations, both genotype imputation and inference of founder mosaics in the offspring are more challenging due to the unobserved phases in the pedigree founders. A software useful for genotype imputation in such data is STITCH , which c2 intercross [Here, we report an approach and its associated software that facilitates cost- and time-efficient inference of founder mosaic genotypes in experimental crosses from outbred (heterozygous) founders using very low coverage (< 0.5×) sequencing data. Although our approach has several similarities to earlier methods that were developed for inbred populations from bi-parental crosses, and uses one of these in part of the implemented pipeline, it provides extensions that are essential for the efficient estimation of founder mosaics in outbred crosses with multiple segregating founders. The properties of our method were illustrated by re-genotyping an Ftercross , 21 betwtercross –24. Our 2 intercross population is founded by intercrossing multiple outbred founders from two parental lines, the population can be divided into nuclear families with one F2 offspring and four F0 parents. The genomes of the offspring in each of these nuclear full-sib families will be mosaics of the eight haplotypes of the four F0 founders, as illustrated in Fig. 0 genomes are, however, unknown, which means that the line origin of the alleles in the F2 offspring cannot be inferred with confidence in regions of the genome where the F0 founders are heterozygous  and intercross individuals to low-coverage (~ 0.5× recommended).Individual-based sequencing libraries were prepared for the F0 individuals, markers fixed for alternative alleles in the divergent founders of each full-sib family (within-family informative markers) were identified across the genome. These markers are highlighted as large capital letters in yellow (HWS informative) and blue (LWS informative) in Fig. Using the high-coverage sequence data on the Fhttps://github.com/CarlborgGenomics/Stripes/blob/master/scripts/fastq2vcf/Snakefile).A computationally-efficient pipeline was implemented for read calling at the within-family informative markers in the offspring from the low-coverage sequence data Fig. . First, 0, two F1 and one F2 individuals, using the polymorphism data obtained by mapping the low-coverage reads from the F2 (Step 3) to the set of founder-line informative SNPs that were identified for the family (Step 2). Because all of these markers were fixed for alternative alleles in the founder lines, the dataset resembles that of an inbred population. Therefore, the founder-line mosaic genotypes of the intercross individuals in the family can be estimated using existing software developed for such populations . In addition, an R package was developed and released that: (i) facilitates reformatting the founder mosaic structure into a recombination mosaic matrix, where every recombination in the offspring is tagged; (ii) performs initial quality control (QC) of the data and removal of double crossover events; and (iii) exports the genotype data across bins of the desired physical length and into R/qtl format for further quality control and downstream linkage and/or QTL mapping analyses (https://github.com/CarlborgGenomics/Stripes_downstream).The complete analysis pipeline outlined in Steps 2 to 4 was implemented in a user-friendly pipeline manager Snakemake . A detai2 intercross between chickens from two divergently selected lines, obtained by bidirectional selection for body weight at 56 days of age (here referred to as the high weight selected “HWS” and low weight selected “LWS” lines) [1 population of the intercross was generated by mating 10 males and 17 females from the HWS to 8 males and 21 females from the LWS line. These F0 founders were from HWS and LWS generation 40. To generate the F2, 8 males and 72 females from the F1 were mated [2 individuals with DNA available for sequence library preparation, and the 56 F0 founders (nHWS = 27 and nLWS = 29) that contributed to these F2 individuals.Data from a reciprocal F” lines) , 24, 26,re mated , 21. Our0 founders contributing to the F2 offspring in the pedigree were prepared using Illumina TrueSeq and sequences obtained by paired-end sequencing (2 × 150 bp) on an Illumina HiSeq X . Mapping, SNP calling, and quality control followed the Broad best practices. Reads were mapped to the chicken reference genome . Base quality score recalibration (GATK 3.7) was carried out before SNP calling with HaplotypeCaller (GATK 3.7). Variants were filtered using the following criteria, a minor allele frequency (MAF) higher than 0.043, and AC and QUAL values greater than 5 and 30, respectively, which resulted in a high-quality set of SNPs for further analyses.Libraries for high-coverage sequencing of the 56 Fgalgal5; ) using tgalgal5; ). AligneTn5-based protocol [2 intercross individuals in the pedigree. Genomic DNA was fragmented using tagmentation by Tn5 transposase from AddGene  [A protocol  for low-ID60240) . Dual inID60240) .2 full-sib families (n = 32) were sequenced to ~ 0.8× coverage to test the quality of the prepared libraries and the implemented pooling strategy . The remaining F2 individuals (n = 805) were then sequenced to ~ 0.4× coverage by pooling ~ 200 multiplexed individuals per lane (Texas A&M Genomics and Bioinformatics Service). Demultiplexing of the dual indexed reads into individual fastq files and trimming of the adapters were done using bcl2fastq v2.17.1.14 .Sequencing of intercross individuals was performed using an Illumina HiSeq 4000. First, the two largest F2 individuals were mapped to the Gallus V5.0 reference genome [https://github.com/CarlborgGenomics/Stripes). This procedure for calling genotypes reduced the time for computation to less than 1/10 of that of the GATK UnifiedGenotyper.The low-coverage sequence data from the Fe genome  using the genome . Only ree genome  was usedFirst, individuals with few called SNPs (genome wide average < 5 SNPs/Mb) were removed from the dataset. This was necessary because accurate genome-wide haplotype mosaic reconstruction requires a reasonably high marker density. Nearby double recombination events are biologically unlikely due to crossover interference. Cytological evidence suggests crossover interference to be absolute in regions shorter than 5 Mb on the largest chicken chromosomes . The rawSample mix-ups, DNA contaminations, and pedigree errors in the data can lead to inaccurate haplotype mosaic reconstruction and increase the number of inferred genome-wide crossover events in affected individuals. To filter out such individuals, we deleted samples for which the genome-wide genotype call rate decreased to less than 90% after removing short (< 3 Mb) double recombination events. An alternative approach would be to remove individuals that were outliers in the distribution of genome-wide recombination events that were inferred using TIGER . This wo2 family, ranged in number from 101 to 140 between families, and were used in this evaluation. For our approach, the genotypes—high weight homozygous (HH), heterozygous (HL), low weight homozygous (LL)—were extracted from the inferred founder mosaics at a 1-Mb resolution. Bins with TIGER imputed recombination events were excluded from the comparisons. The proportions of genotypes in agreement between the two methods per marker across individuals and across all markers per individual were used as measures of genotyping accuracy (https://github.com/CarlborgGenomics/Stripes_downstream).Quality of the reconstructed line origin haplotype mosaics from the low-coverage whole-genome sequence data across chromosomes 1 to 24 was evaluated by comparing them to the individual genotypes of SNPs reported previously . In tota2 population that was bred from the Virginia chicken lines divergently selected for high (HWS) and low (LWS) body weight.Properties of the reported approach to infer founder mosaic genotypes in intercross data from outbred (heterozygous) founders were illustrated by generating and analysing data for an FHWS = 27 and nLWS = 29) that contributed to the F2 individuals. There were 213,946 SNPs that were fixed for alternative alleles in the sequenced individuals from the two founder lines and these were unevenly distributed across the genome  individual sequence data detected 7,608,483 SNPs among the HWS and LWS founders  biologically unlikely numbers of recombination events, and (ii) genotypes resulting from inferred recombination events at locations that were physically too close, the agreement between imputed genotypes using our pipeline and the previously assayed SNP genotypes in Wahlberg et al.  was stro2 individual across chromosome 1. At 95.7% of the marker locations, the estimated founder mosaic genotype agreed well with the estimated genotype probabilities in Wahlberg et al. [Figure g et al. . In addig et al. . This, f2 population that was produced by crossing chicken lines that were divergently selected for body weight from a common founder population.In this paper, we describe an approach and software for estimating founder mosaic genotypes in experimental crosses between outbred (heterozygous) founders from very low-coverage sequencing data. Its properties are illustrated by generating and analysing data from an FWe compared our approach with another genotype imputation software, STITCH . The impAccounting for available pedigree information allows additional quality control to be performed based on the inferred number of recombination events after the founder mosaic genotypes have been estimated. First, the number of estimated crossover events across the genome should agree with its expectation given the length of the chicken linkage map , 34. Sec2 individuals were processed by a low-cost whole-genome sequencing protocol to facilitate low-coverage sequencing of hundreds of intercross individuals . This cost is lower than that of existing strategies for genotyping individuals for a few hundred SNPs, even when accounting for the cost of high-coverage sequencing of the large (n = 56) number of founder birds in our pedigree. In addition to the lower cost, there was a considerable increase in marker density, from a few hundred to several hundred thousands of imputed segregating sites across the genome.Our proposed strategy to reconstruct the founder mosaic haplotype structure using low-coverage sequencing data provides more genotyping information than classical approaches at a considerably lower cost. The founders of the pedigree were sequenced to a high coverage by using standard approaches, while F0 founders from one line but segregating in the two F0 founders from the other line (which here would increase the number of informative markers by ~ 20%), or (ii) deeper sequencing, to obtain reads at a larger proportion of the markers that are informative between the two founder lines  were from a common base population founded by crossing seven partially inbred lines of White Polymouth Rock. It is worthwhile noting that while the pooled founder sequencing approach is more efficient for populations with more divergent founders, even in populations with founders from closely-related lines, it is likely still sufficient for the purpose of building high-quality linkage maps and performing genome-wide QTL analyses.In crosses in which the founder populations are highly divergent, an alternative strategy for genotyping can be considered to reduce sequencing costs. Rather than sequencing individual founders to high coverage, libraries from the founders of each line could be pooled. The two pools, each representing one founder population, can then be sequenced to high coverage (~ 30×) to identify markers that are fixed (or nearly so) for alternate alleles in the two lines. In our F2 cross to illustrate how it provides high-quality, high-resolution genotypes in a time- and cost-efficient manner.In this paper, we propose and evaluate a new method for reconstruction of founder mosaic genotypes from low-coverage genome sequencing in outbred intercrosses. We applied this method to an outbred chicken FAdditional file 1: Figure S1. Visualization of the number of crossovers in all F2 individuals. (a) Histogram of number of imputed crossover events for the 803 genotyped F2 individuals; b) Number of imputed crossover events in each individual, sorted by the 73 full-sib families. Individuals with low call rate  are coloured into red.Additional file 2: Figure S2. Distribution of the between founder line informative SNPs for the evaluated F0-F2 families on chromosome 1. (a) Line graph illustrating the number of SNPs in non-overlapping 1-Mb bins across chromosomes 1 to 24 . The black/tomato lines represent the total number of SNPs segregating in the pedigree/the average number of informative SNPs for the 64 families in the Virgina chicken line F2 pedigree. (b) Histogram illustrating the average number of informative SNPs in the 73 full-sib families in the pedigree .Additional file 3: Figure S3. Comparison of the founder mosaic in one F2 offspring obtained by using individual SNP-genotypes (a), to that obtained from our method (b) and STITCH (c) using the same low-coverage sequence data.Additional file 4: Figure S4. Illustration of relationship between sequencing coverage, SNP density and imputation accuracy. (a/b) Histograms of the sequencing coverage/SNP densities for the 803 genotyped F2 individuals; (c/d) Scatter plots of individual coverage/SNP density vs imputation accuracy measured as proportion of sites that has same genotype with the averaged genotype probabilities estimated by Wahlberg et al. [g et al.  using geg et al. .


Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4  The coatomer protein complex II (COPII) coat assembles on the cytoplasmic surface of endoplasmic reticulum (ER) exit sites to drive the formation of membrane-bound transport vesicles. Efficient recruitment of proteins and lipids into these vesicles occurs via physical interaction with the COPII coat. For carin vivo remains uncertain. Additionally, although SURF4 deletion did not affect the secretion of a control protein, alpha-1 antitrypsin, a broader role for SURF4 in protein secretion remains possible and is supported by the recent identification of other potential cargoes including apolipoprotein B, growth hormone, dentin sialophosphoprotein, and amelogenin and mSurf4-ex2-rev [CAGGTAGCCACAGCTCCAGG]. Sanger sequencing was performed with the same genotyping primers and chromatograms were inspected both manually and by automated deconvolution[nvolution to deterSurf4 allele was quantified by decomposition of Sanger sequencing chromatograms with TIDE indel analysis[Surf4-forward [CTGTTGGCCTCATCCTTCGT], Surf4-reverse [GGCAATTGTCTGCAGTGCG], Actb-forward [CCACTGCCGCATCCTCTTCC], Actb-reverse [CTCGTTGCCAATAGTGATGACCTG], B2m-forward [CATGGCTCGCTCGGTGACC], B2m-reverse [AATGTGAGGCGGGTGGAACTG], Tbp-forward [CCCCACAACTCTTCCATTCT], Tbp-reverse [GCAGGAGTGATAGGGGTCAT], Ppia-forward [CAAATGCTGGACCAAACACAAACG], Ppia-reverse [GTTCATGCCTTCTTTCACCTTCCC].Mice were housed and monitored in accordance with University of Michigan Unit of Laboratory Animal Medicine (ULAM) guidelines. Blood was collected at 6–12 weeks of age by retro-orbital bleeding into heparin-coated collection tubes from mice anesthetized with isoflurane. Plasma was prepared by centrifugation at 2,000 g for 10 min at 4°C. A second blood collection was performed 1 week following the initial collection. Plasma samples were analyzed by total cholesterol colorimetric assay  and ELISAs for PCSK9  and apolipoprotein B . Liver tissue was perfused with PBS and harvested from mice at the time of sacrifice. Liver mRNA was prepared with RNeasy Plus Mini Kit  and oligo(dT)-primed first strand cDNA generated with Superscript III reverse transcriptase  according to manufacturer’s instructions. Quantitative PCR was performed using 20 ng of cDNA per reaction with PowerSYBR Green PCR Master Mix . Normalization of qPCR data was performed using a panel of 4 selected housekeeping genes. The rel analysis. The prig. Protein concentrations of lysates were measured by DC Protein Assay . Equal amounts of lysate were analyzed by immunoblotting with antibodies against Apolipoprotein B , PCSK9 , LDLR , and alpha-tubulin  and densitometry analysis performed with ImageJReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: PLoS One Review of Murine Surf4 is essential for early embryonic development In this manuscript the authors generate a null allele of Surf2, an ER cargo receptor and one they previously identified as interacting with PCSK9 and thus one that may have important roles in cholesterol homeostasis. Overexpression and deletion of Surf2 in cell lines were supportive of this role and they sought to determine if this role was recapitulated in the mouse. They generated 4 lines via a Crispr strategy including 4 putative null lines. Heterozygous animals were generated at expected frequencies but null mice were early embryonic lethal. They find that hets have no changes in plasma levels of cholesterol, PCSK9 or ApoB but do observe significant changes in the abundance of ApoB in the livers of het mice.This paper represents a natural extension of their previous findings and should be of interest to the same audience.Several Major issues include:1) The authors do not present evidence SURF4 is reduced in hets. They do, however repeatedly state that SURF4 (protein) is reduced in heterozygous animals. This information is critical to support the conclusions made herein.2) To make definitive conclusions, the heterozygote data presented with a single line should be performed with more than one of the founder lines. The sex and number of animals used in Figs 2 and 3 should also be provided.3) No methodology is provide for the data presented in Figure 3. The authors state that liver cell lysates were produced in the text but no information on the antibodies or assays used were provided. It would be important to include a blot demonstrating a reduction in SURF4 in Hets in this figure.4) Images of the mutant blastocysts that were created via IVF should be provided in order for experts to decide if there are obvious defects.OtherAbbreviations should be named prior to use: SURF2, ERGIC, LDLR, PCSK9 etc are never defined.Reviewer #2: The manuscript “Murine Surf4 is essential for early embryonic development” by Emmer et al describes the generation of a new mouse knockout model for the anterograde cargo receptor SURF4. The authors used CRISPR/Cas-mediated mutagenesis to target exon2 of the Surf4 gene, generating 4 gene-edited lines . They then analyzed one of those lines in heterozygous adult mice, by assessing plasma samples for total cholesterol, PCSK9, and ApoB . PCSK9 and ApoB are two candidate proteins that are predicted to be trafficked by SURF4 from the ER to Golgi based on published literature, and total cholesterol could have been impacted if PCSK9 and ApoB were not synthesized and secreted properly by SURF4-mutant cells. Seeing no effects on Surf4+/- plasma, they analyzed total liver lysates for ApoB, PCSK9, and LDLR (which is regulated by PCSK9) by western blotting . They found an aberrant accumulation of ApoB in the liver lysates, although this did not affect circulating ApoB levels, as demonstrated by the plasma ELISAs. The authors could not analyze Surf4-/- mice because they were not recovered at weaning from three of their mutant lines (Table 2). They analyzed one of the lines with timed matings to assess when the Surf4-/- embryos died but could not recover live or partially resorbed Surf4-/- embryos at E9.5—the earliest timepoint they analyzed. They also performed in vitro fertilization with Surf4+/- oocytes and sperm and cultured fertilized eggs for 3 days until the blastocyst stage. This approach did generate Surf4-/- blastocysts, so the authors concluded that Surf4-/- embryos died en utero between E3.5 and E9.5 from unknown causes.This manuscript provides the first description of a Surf4-deficient mouse, which is a critical reagent for interpreting the in vitro data that has been generated on SURF4 and its function in different cell types. The authors particularly hoped to validate that the PCSK9 protein—which they had described as being trafficked by SURF4 in vitro —would also be found to be trafficked in vivo, with possible implications for LDLR surface expression and plasma cholesterol levels. However, the Surf4+/- mice they analyzed did not support a critical role for SURF4 in this capacity. They did, however, find accumulation of ApoB in the livers of Surf4+/- mice, which supports published evidence that SURF4 helps to traffic ApoB for proper secretion from hepatocytes . Curiously, this did not alter circulating ApoB plasma levels or cholesterol levels, which the authors could not explain. Altogether, the conclusion from these analyses is that Surf4 haploinsufficiency does not impact total cholesterol levels in circulation.While I appreciate the importance of the new Surf4-mutant lines that this manuscript describes, I would have liked to have seen a better analysis of those lines. Specifically, I wish the authors had made some effort to confirm the reduction of SURF4 in their heterozygous adult mice—either by immunoblotting or qPCR. The Surf4-/- blastocysts they generated also could have been analyzed by qPCR. I believe that more careful validation of the mutant lines and of Surf4 reduction is important because Surf4 is known to be subject to alternative splicing . In addition, I found the developmental analysis of Surf4-/- embryos to be perfunctory. The fact that no partially resorbed Surf4-/- embryos could be recovered at E9.5 suggests that the embryos die much earlier—potentially around implantation. I found the in vitro generation of Surf4-/- blastocysts to be an interesting approach to assessing survivability of Surf4-/- embryos at early stages of development, although the artificiality of this approach could be misleading in interpreting early Surf4-/- embryonic development. Instead, I would have preferred to see the authors flush blastocysts from pregnant mice at E3.5 to assess the feasibility of early Surf4-/- development. Furthermore, analysis of embryos at peri-implantation (E5.5-6.5) would be a significant improvement to this study, since it would clarify whether Surf4-/- embryos can implant. Methods for dissecting peri-implantation mouse embryos are well described . Altogether, better analysis of the timepoint at which Surf4-/- embryos die would assist in future studies that will elucidate SURF4 cargo proteins that are critical for early development.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  14 Nov 2019A full point-by-point response to editor and reviewer comments has been uploaded as a separate file.Attachment2019_emmer_PLOS_ONE_response_to_reviewers.docxSubmitted filename: Click here for additional data file. 19 Dec 2019Murine Surf4 is essential for early embryonic developmentPONE-D-19-17413R1Dear Dr. Emmer,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Given the amount of key information that was missing from the initial submission I suggest that the authors make sure that they are providing all key methodology and reagents for the final paper.Reviewer #2: The revised manuscript now confirms reduction of transcripts in heterozygous mice and embryonic lethality in 3 separate homozygous lines. Although the timing and cause of the embryonic lethality is still unclear, the manuscript represents a sufficient initial description of the importance of SURF4 for embryonic development. Subsequent studies with a floxed Surf4 allele will presumably clarify the cargo and role of SURF4 in development and in specific cell types.**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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Bud sports occur in many plant species, including fruit trees. Although they are correlated with genetic variance in somatic cells, the mechanisms responsible for bud sports are mostly unknown. In this study, a peach bud sport whose fruit shape was transformed to round from flat was identified by next generation sequencing (NGS), and we provide evidence that a long loss of heterozygosity (LOH) event may be responsible for this alteration in fruit shape. Moreover, compared to the reference genome, we identified 237,476 high quality single nucleotide polymorphisms (SNPs) in the wild-type and bud sport genomes. Using this SNP set, a long LOH event was identified at the distal end of scaffold Pp06 of the bud sport genome. Haplotypes from 155 additional peach accessions were phased, suggesting that the homozygous distal end of scaffold Pp06 of the bud sport was likely derived from only one haplotype of the wild-type flat peach. A genome-wide association study (GWAS) of 127 peach accessions was conducted to associate a SNP found at 26,924,482 bp of scaffold Pp06 to differences in fruit shape. All accessions with round-shaped fruit were found to have an A/A genotype, while those with A/T, or T/T genotypes had flat-shaped fruits. Finally, we also found that 236 peach accessions and 141 Prunus species with round-type fruit were found to have an A/A genotype at this SNP, while 22 flat peach accessions had an A/T genotype. Taken together, our results suggest that genes flanking this A/T polymorphism, and haplotyped carrying the T allele may determine flat fruit shape in this population. Furthermore, the LOH event resulting in the loss of the haplotype carrying the T allele may therefore be responsible for fruit shape alteration in wild-type flat peach. Prunus persica L.) is an important model species in the Rosaceae  events are common genetic events found in many types of cancer . Early sa genome . LOH evea genome .A. thaliana  was grown in the orchard of the experimental station of Shandong Agriculture University, Tai’an, China. The base of one branch of this tree produces normal flat-shaped fruit while the top of the same branch bears round-shaped fruit , and two accessions with distinct divergence were all excluded prior to conducting a GWAS. The resulting 217 SRA runs were individually mapped against the peach genome version 2.0  using BWsamtoolsmpileup-B-q1|java-jarvarscanmpileup2snp -min-coverage2 - min-reads21 - min-avg-qual15 - min-var-freq0.25 - min-freq-for-hom0.75 - p-value0.99 - output-vcf 1. The SNP set was then extracted for downstream analysis.2) as calculated by the -r2 command of PLINK v1.9  implemented by EIGENSTRAT v 6.1  on an LDINK v1.9 . An LD h package .P-values  were used for significance testing. The top ten EIGENSTRAT principal components were used as covariates. Manhattan and quantile–quantile plots were generated by the qqman R package  was filtered to retain SNPs with call rates >75% and minor allele frequencies >5%, resulting in 431,028 SNPs. This set was used to conduct the GWAS. Mixed-model association analysis was conducted in EMMAX  using a  package .Prupe.6G292200 of the Lovell reference genome v2.0  databaseA single flat peach with round-type fruits was observed in our fruit orchard . The base of this tree (below Label 1) was bearing just flat-type fruits , Label 1We sequenced the whole genomes of the wild-type and bud sport peaches, generating ∼12 Gb of raw data for each sample. After mapping the reads to the peach reference with mapping quality filter ≥60, we obtained an average coverage depth of ∼28× and ∼26× for the wild-type and bud sport mutant genomes, respectively. More than 85% of the genome sequence was covered by at least 4 reads for each sample. Calling SNPs from short reads remained challenging, and we therefore included reads from the double haploid Lovell reference genotype (SRR502985) to control our SNP calling pipeline. A total of 237,476 high quality SNPs were obtained after quality control procedures were performed. As shown in As shown in Despite the fact that the number of inconsistencies between the bud sport and wild-type genotypes was very low for all scaffolds except Pp06, Pp06 itself attracted our curiosity by showing many more inconsistencies . CompareAs shown in We plotted a genotype distribution pattern along scaffold Pp06 and found that for the sequence running from 0∼22,202,387 bp of scaffold Pp06, there were no significant differences between the wild-type and bud sport mutant genotypes . HoweverFurthermore, a dataset consisting of 10,515 SNPs showed that the genotype at the distal end of scaffold Pp06 was significantly different between the wild-type and the bud sport mutant . In the Because deletions can cause heterozygous to homozygous conversions of runs of DNA, we plotted the genotype depth ratio (wild-type/bud sport) between the homozygous distal end of scaffold Pp06 and whole genome . We founWe further checked the genotype depth at the distal end of scaffold Pp06 between the wild-type and bud sport mutant genotypes . We calcWith the exception of the distal end of scaffold Pp06, all parts of the 8 largest scaffolds of the bud sport mutant genome were derived from the corresponding parts of the wild flat peach genome . With reUsing the LD-reduced set of SNPs from the whole genome, we performed PCA to quantify the population structure of all 127 peach accessions. The principal component score plot showed a continuous distribution without any distinct clusters , indicatPrupe.6G292200. Although 19 SNPs were above the threshold, only the single SNP at 26,924,482 bp of scaffold Pp06 was found in an interval (26.7 M∼27.2 M) reported by a previous linkage analysis study to be within an S-locus. In 11 out of 12 (91.7%) flat accessions in this GWAS population, this SNP is A/T (10/11) or T/T (1/11), while in all round accessions (93 accessions), this SNP was A/A.As shown in As shown in We further genotyped 258 additional peach accessions at this SNP and found that all 236 round-typed peach accessions carried an A/A genotype, while 22 flat-typed peach accessions carried an A/T genotype . BecauseSport mutations are common in many plant species including fruit trees, and are important for plant breeders because they provide novel variants for selection. Although previous works proposed that bud sports might occur at both the chromosome and gene levels , the prePrunus species showed different results, all of them further validated our haplotype phasing result at 26,924,482 bp of scaffold Pp06. The haplotype difference at this SNP may be due to different uses of the reference genome sequence – i.e., the previous study , LOH events in different cell types may result from distinct mechanisms. According to histogram cell theory , the peaIn conclusion, a long LOH event in a flat fruit haplotype in peach was identified in this study; this LOH event may therefore be responsible for the transition from the flat fruit wild-type shape to a round shape.DG conceived and designed the experiments, revised the intellectual content of the manuscript, and supervised the project by correspondence. QT, XL, and HG performed the experiments and analyzed the data. WX, XC, XF, LL, DL, and DG provided technical and theoretical support to the manuscript. QT wrote 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.


Acipenser sturio), however, makes its future genetic monitoring challenging, and the current resolution needs to be increased. Here, we describe the discovery of a highly informative set of 79 SNPs using double‐digest restriction‐associated DNA (ddRAD) sequencing and its validation by genotyping using the MassARRAY system. Comparing with STRs, the SNP panel proved to be highly efficient and reproducible, allowing for more accurate parentage and kinship assignments' on 192 juveniles of known pedigree and 40 wild‐born adults. We explore the effectiveness of both markers to estimated relatedness and inbreeding, using simulated and empirical datasets. Interestingly, we found significant correlations between STRs and SNPs at individual heterozygosity and inbreeding that give support to a reasonable representation of whole genome diversity for both markers. These results are useful for the conservation program of A. sturio in building a comprehensive studbook, which will optimize conservation strategies. This approach also proves suitable for other case studies in which highly discriminatory genetic markers are needed to assess parentage and kinship.The use of genetic information is crucial in conservation programs for the establishment of breeding plans and for the evaluation of restocking success. Short tandem repeats (STRs) have been the most widely used molecular markers in such programs, but next‐generation sequencing approaches have prompted the transition to genome‐wide markers such as single nucleotide polymorphisms (SNPs). Until now, most sturgeon species have been monitored using STRs. The low diversity found in the critically endangered European sturgeon ( This population has been supported since 1995 by a breeding and restocking program  have recently increased time and cost efficiency  detect novel SNP markers for the species using double‐digest sequencing protocol for Ion Torrent sequencing  and select an optimal set for parentage assignment through validation by genotyping using the MassARRAY system, (b) apply the novel set of markers for parentage assignment of 192 juveniles of the French captive stock, for which putative parents are recorded in the breeding database, (c) compare the resolution of SNPs and STRs for parentage testing and for their effectiveness in measuring genetic diversity, relatedness, and inbreeding, for empirical and simulated datasets, and (d) define relevant and precise genetic indicators for the long‐term monitoring of the breeding and restocking program of A. sturio (Studbook implementation) that can be transposed to other sturgeon species.In this study, we characterized novel SNP markers for the critically endangered 22.1A. sturio. Samples included 33 wild‐born breeders, 8 captive‐born adults , 40 juveniles captured by trawling in the Gironde estuary  during 2009–2014 population monitoring campaigns . Two technical replicates were included to estimate repeatability and error rates resulting from library preparation, sequencing, and bioinformatic analyses. Replicates are issued from the same DNA source but were processed independently. DNA quantification was made using the Quant‐iT dsDNA BR Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. ddRADseq library preparation protocol followed the methods described by Pukk, Kisand, Ahmad, Gross, and Vasemagi , with soAll the raw sequences were quality‐trimmed using the default settings of the Ion Torrent BaseCaller (>Q16 with a windows size of 30 bases) and demultiplexed based on their barcodes. Then, stacks  and with a minor allele frequency (MAF) ≥ 0.175  the selection of SNPs present in more than 50% of individuals , N = F1 captive‐born juveniles, and N = 40 recaptured individuals in the Gironde estuary. We included two exogenous positive controls and two negative (water) controls to check reliability. Raw data analyses were performed using the software MassARRAY TYPER 4.0. We filtered out monomorphic SNPs , loci with weak or ambiguous signal (loci displaying more than three clusters or unclear cluster delineation), and those with Mendelian discordance (checked in 11 captive‐born families).Finally, 186 candidate SNPs were submitted for assay design using the MassARRAY2.4N = 37 breeders. To test for the resolution of the panel of SNPs in our context, calculations were also done based on the SNP allelic frequencies of the future breeders . The combined PID over loci was calculated using the analysis module in GenAlEx 6.5 for unrelated individuals (PID‐unrel) or relatives   was assessed and compared to that of STRs, based on the same N = 67), female identity is recorded but two or three males were possible . In this case, male ID deduced by parentage testing will be recorded in the captive breeding database. Only those parents showing 95% Trio confidence and 0/1 mismatch with their putative offspring were validated as “true” parents. To compare parentage efficiency between SNPs and STRs, results were compared for 40 individuals analyzed by both markers (n = 32 captured and N = 8 captive).For parentage assignment, the program CERVUS  and F1 juveniles . Because the F1 generations produced in captivity are issued from only a subset of breeders, some of them being related, we expect inbreeding to be higher in F1 than in wild breeders because of selection and genetic drift. We used Wilcoxon rank sum tests to test for significance. Recent works have also proposed identity disequilibrium (ID) as a measure that may capture variance in the level of inbreeding within a population , the heterozygosity of noninbred individuals , and the distribution of F among the simulated individuals to measure g2. The F values of the simulated individuals are sampled randomly from a beta distribution with mean (meanF) and variance (varF) specified by the user . Also, to infer how well genetic marker heterozygosity reflects the inbreeding level F and whether this correlation could be improved by using an increasing number of markers, we also use and compare the “simulate_r2_hf function” among both markers  and F1 juveniles (F1‐JUV). A value of g2 significantly greater than 0 is interpreted as evidence that those markers contain information about variation in inbreeding.Because inbreeding will be considered for the breeder's selection in captivity, we also evaluated reliability and precision of SNPs and STRs in estimating inbreeding. First, we calculated R) of the individual inbreeding coefficient F and the individual heterozygosity by loci  available in GenAlex  were retained for the species (see Appendix N = 20). When comparing the two technical replicates, we found that 29% of loci was only present in one or the other replicate (mean locus error rate), while mean allele error rate  was of 9.6% .In total, 92,063,509 reads  with a median read length of 206 bp were generated through the sequencing of the 40 individuals. After the STACKS filtering steps, a large number of SNPs . Minor allelic frequency (MAF) varied from 0.214 to 0.489 depending on SNPs , SNPs whose profiles showed two groups of heterozygotes  and SNPs for which amplification intensity was low in most samples (2.6%). Finally, three additional SNPs were further removed for inconsistency in Mendelian inheritance, checked in family groups. The final set after filtering includes 79 SNPs3.3−18 and PID = 1.5 × 10−34 for full siblings and nonrelatives, respectively) than the panel of 18 STRs , based on the allelic frequency of the broodstock. The best PID value for STRs is reached with only 28 SNPs. The PIDs obtained considering the allelic frequency of the future breeders (F1 juv) are high and very similar to that based on BREEDERS , the assignment of males was highly concordant among the multiple choices for all individuals except for one (ID#297).Results of parentage assignment in the captive stock of F1 juveniles indicate a high level of assignment for individuals of known parent pairs. All but four  have been assigned to the parent pairs recorded in the captive stock database of origin with 95% confidence. For three  of these four individuals, the identification has been likely mistaken during sampling and handling in captivity: Individual 149 was genetically assigned to the parents' pairs of individual 150, and individual 209 was assigned to a parent pairs that was also crossed the same year (both assignations with 95% confidence). This may lead us think to family mixing during handling in the tanks at young stages or during tagging procedures. If we accept this “misidentification” hypothesis, genetic assignment then reached 99% success. Among the sturgeons for which only female identification was available . For both SNPs and STRs, the best correlation was found for maximum‐likelihood (ML) estimators. TrioML and DyadML were considered as the most accurate estimators for subsequent analyses for SNPs and STRs, respectively.Best correlation coefficients  among the seven relatedness estimators and the true simulated values were obtained for SNPs. The correlation coefficients were significantly lower for STR (varied between 0.476 and 0.677) than for SNPs  were not significantly different from expectations . Also, as expected, there were no significant differences  between the relatedness coefficients of PO and FS categories for SNPs; while using STRs, we observed significant differences between FS OBS and either FS SIM, PO OBS, and PO SIM  were significantly lower for SNPs than for STRs . Furthermore, unlike for STR, IQRs of SNPs did not overlap among the different relationship categories .A gradient of relatedness coefficient relating to kinship was found for both SNPs and STRs Figure . It is wH . For both markers, mean inbreeding in F1 juveniles  was significantly higher than that in BREEDERS .We analyzed the relationship between individual heterozygosity H Figure a and F eg Figure b at SNPsg2 estimates is higher for STRs than that for SNPs, and decreases when a higher number of markers are used  are both concordant in higher inbreeding level in F1 juveniles than wild‐born breeders for both marker types: At SNPs, g2 values were slightly positive and significant for F1‐JUV and BREEDERS  while at STR values  were only statistically significant for F1‐JUV .The analysis from INBREEDR package showed that the variation around d Figure a. The pry Figure b is almoy Figure b. The es4The use of high‐throughput sequencing techniques in nonmodel organisms has opened the way for obtaining numerous SNP markers that may compensate the limited power of other markers, especially in scenarios of small and/or decreasing populations composed of related individuals. Our study showed that double‐digest RAD sequencing  to ensure the maximum reliability of genotypes although this has consequently reduced the number of loci retained . Our proportions of ddRADseq error rates were high between replicates  and higher than other similar libraries using Ion Torrent platform  had sufficient power to discriminate even between closely related individuals , the fact that the sensitivity of the methodology may be at play could not be excluded. Here, we showed that limited resolution of STRs was probably the key impediment in discriminating among related breeders. Indeed, SNP markers were successful in assigning known parent pairs to 17 of these 20 individuals which reduce the number of fish that might be originated from natural reproduction. For 11 of these individuals, the males identified by STRs are the offspring of the male (Justin) identified using SNPs. In this case, it proved that when candidate parents are highly related (parent–offspring or full sibs), SNPs are better at assigning fathers than STRs.The main aim of this work was to develop a set of SNPs with higher resolution than STRs and primarily with enough efficiency to resolve situations in which individuals might be closely related. This study is probably one of the most extensive comparisons of STRs and SNPs that estimates reliability and precision on parentage, relatedness, and inbreeding. This work clearly showed that the full panel of 79 SNPs (four times the number of STR loci) was more powerful and reliable than the previous 18 STRs to determine the paternity and identity in the European sturgeon population. Results demonstrated that SNPs gave more accurate identification  than STRs and that the maximum resolution of STR was achieved using only 28 SNPs. These results are comparable to a study on another critically endangered species, the Iberian lynx, with very low diversity and for which a relatively reduced number of SNPs  and future breeders (F2) reduced the probability of crosses among highly related individuals. However, because most of these fish are produced from a small number of families and breeders, some of them being related, progenies are genetically closed. For this reason, it was important to select the most reliable relatedness parameter to avoid an overrepresentation of inbred individuals in the future families. This strategy has been applied in the A. sturio breeding program since 2014 based on STR markers . Interestingly, the mean relatedness over all pairs of individuals in each kin group corresponds well to the expected pedigree relatedness, but these distributions are overlapping for different kinship categories, especially for the half‐sib category  than STRs and sensitivity increases with increasing the number of markers used. Similarly, the expected correlation between inbreeding and marker heterozygosity was also almost twice for SNPs than for STRs and increased when the number of markers was increased  is to build a reliable and comprehensive studbook, which will optimize the retention of diversity and limit inbreeding in the captive and sustained population. This will be done by suggesting potential pairings among the available mature breeders or eventually selecting cryopreserved gametes. Optimum pairings will be based on the mean kinship  and by calculating inbreeding coefficients to select the most heterozygous individuals to maximize diversity for releases into the wild or for breeding individuals.In this study, we observed that SNPs performed better than STRs in estimating inbreeding in groups with known expected level of inbreeding (based on relatedness of parents). Inbreeding is another important parameter to measure in captive breeding programs because inbred individuals have lower fitness than the offspring of unrelated parents  may, however, suggest that variation at both markers may reflect genome‐wide genetic diversity. The analysis of large number of SNPs is supposed to provide a greater power and precision to quantify genomic levels of diversity and inbreeding  found for the European sturgeon may give an empirical and interesting support to the above theory.Because of the absence of a reference genome sequence for the European sturgeon, the locations of putative SNPs on chromosomes or linkage between markers could not determine and further analysis would be necessary. The significant positive correlations found between STRs and genome‐wide SNPs in this study for both individual heterozygosity and inbreeding  is low and is expected to further decrease in the future , a suitable genetic tool with high resolution was required for assessing relatedness, inbreeding, and to assess parentage. We present here a highly efficient and reliable SNP panel that could be genotyped easily with reduced cost and typing efforts, thus providing a standardized panel for the exchange of genotype data between laboratories. Regarding the conservation of small or captive populations, there is a great concern for the loss of genetic diversity through genetic drift and inbreeding. In the context of A. sturio conservation program, this assay will be useful for the genetic management of the broodstock and further restocking in France, in Germany or for the future reintroductions in other systems. This approach may also prove suitable for other case studies in which highly discriminatory genetic markers are needed  and in which the transition to SNP markers is planned.The increasing amount of genetic marker information that can be generated by new sequencing techniques should undoubtedly provide better genetic tools and better description of genome‐wide diversity, useful for the conservation of endangered species. The right selection of markers has been discussed in recent papers that have stressed the importance of the type, number, reliability, and genome representability, for an optimum choice, although detailed empirical assessments of such parameters have been scarce (Kleinman‐Ruiz et al., None declared.S.R. designed the study, performed SNP development and analysis, analyzed and interpreted data for parentage, relatedness and inbreeding assessment, and wrote the manuscript with help from all coauthors. M.L.A supervised the study, coordinated sampling collection, and got funding. E.C. and C.B. designed and performed SNP discovery experiments, analyzed, and interpreted data. M.P. performed all statistical analyses. All authors revised the manuscript critically and approved the version to be submitted. Final approval of the version to be published. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Neospora caninum is an apicomplexan parasite responsible for neosporosis, a disease causing hind limb paralysis in dogs and abortion in cattle, resulting in substantial economic losses to beef and dairy industries. Marked differences in pathogenicity exist between N. caninum strains suggesting that intrinsic genetic differences exist between them. These differences likely exist in genes expressed during the tachyzoite lifecycle stage which is responsible for the pathogenesis of neosporosis. An improved understanding of these genetic differences is essential to understanding N. caninum virulence, though such knowledge is scarce. Using a variant detection workflow we compared the tachyzoite transcriptomes of two N. caninum strains with different virulence properties: NC-Liverpool (virulent) and NC-Nowra (avirulent). This workflow identified 3130 SNPs and 6123 indels between the strains, and nine markers capturing 30 variants were Sanger sequenced for both strains. Sequencing of these loci was extended to an additional eight strains and subsequent phylogenetic analysis supported a genetic population structure comprised of two major clades with no geographical segregation. Sequence polymorphisms within coding regions of tachyzoite-associated genes were concentrated on chromosomes XI and XII, with 19 distinct tachyzoite-associated SNP hotspot regions identified within coding regions of the N. caninum nuclear genome. The variants were predominantly located in loci associated with protein binding, protein–protein interactions, transcription, and translation. Furthermore, 468 nonsynonymous SNPs identified within protein-coding genes were associated with protein kinase activity, protein binding, protein phosphorylation, and proteolysis. This work may implicate these processes and the specific proteins involved as novel effectors of N. caninum tachyzoite virulence. Neospora caninum is a cyst forming coccidian of the phylum Apicomplexa first described as the cause of a potentially fatal neurological disease of dogs , was developed by typing 25 cultured N. caninum isolates which identified 11 genotypes  proteins are released upon contact with host cells and facilitate adhesion  and NC-Nowra (avirulent in mice). Phylogenetic analysis of sequenced polymorphic markers identified in silico, revealed a population structure consisting of two major clades showing no obvious geographical segregation. Tachyzoite-associated polymorphisms were associated with kinase activity, ATP binding, protein–protein interactions, and proteolysis, implicating several proteins involved in these processes as potentially novel determinants of N. caninum virulence.The present study employed a variant detection workflow to compare the transcriptomes of two Neospora caninum strains ( strains  online wN. caninum and Neospora hughesi strain NE1 (imported from ATCC). Briefly, cells were pelleted and then extracted three times with equal volumes of phenol and chloroform and then once more with chloroform only, with thorough vortexing and centrifuging at 13,000 × g for 1 min between extraction steps. The DNA was precipitated from the final aqueous phase by isopropanol and resuspended in 100 μl of ddH2O. The DNA extracts were stored at −20 °C until required.For laboratory confirmation of the SNPs, genomic DNA was also extracted using the solvent extraction technique, from cultured tachyzoites of http://galaxyproject.org/; Last accessed June 2015). Illumina reads <15 base pairs long, and with per base quality scores <20, were discarded using the Filter FASTQ tool  using TopHat version 2.1.1 . A summary of assembly metrics assessed is contained within An in-house reference transcriptome was created for NC-Liverpool by performing a de novo transcriptome assembly. The TopHat alignment tool was first used to map the N. caninum reference genome FASTA file was uploaded to IGV (version 2.3.67), along with the sorted BAM files for each sample. Hundreds of variants were randomly selected for viewing from the SNP and indel lists produced by VarScan, and a set of high confidence variants were selected from amongst these for laboratory validation.SAMtools  was usedE-value ≤ 1E−50, Bit-score ≥ 200, and PID ≥ 90%) were subsequently cross-referenced with the contig location of each SNP, for allocation of SNPs to a chromosome. A BLASTX search was also performed querying the de novo NC-Liverpool transcriptome assembly against NC-Liverpool annotated proteins from ToxoDB , to assign SNPs to a gene ID (PID ≥ 90%). The location of SNPs along the N. caninum genome was then plotted to investigate the distribution of variants. Circos plots  hotspots. A gene region was classified as a mutational hotspot if it contained >15 SNPs within a 50 kb window. The genes within these regions and their SNP information were extracted for gene ontology analysis.The de novo transcriptome assembly generated for NC-Liverpool was queried against the published NC-Liverpool reference genome using a BLASTN search (version 2.7.1), to facilitate assignment of each transcript to a chromosome. High confidence hits . DNAPlotter (InterProScan (version 68.0) was used to assign functional information to proteins putatively encoded by the genes within each SNP hotspot . For eacAPlotter  was thenAPlotter .A de novo transcriptome was generated for NC-Nowra using Trinity, as described previously for NC-Liverpool. TransDecoder  was used2O. The temperature cycling conditions employed were as follows: 1) 95 °C for 5 min, 2) 95 °C for 1 min, 3) 57–61 °C  of 1.42. The 3,130 high confidence SNPs were distributed across 2,021 unique transcripts encoded by 1,879 genes. Additionally, the current N. caninum reference genome consists of multiple large contigs that are not assigned to one of the 14 chromosomes. There were 162 SNPs distributed across 22 such contigs, with the majority of this callset (∼78%) assigned to eight of these contigs alone. These SNPs were subsequently allocated to 34 unique protein-coding genes. It is worth noting that when blasting the NC-Liverpool transcriptome against the published N. caninum annotated proteins to assign SNPs to annotated genes, several contigs containing SNPs returned high confidence BLAST hits  to multiple genes along the genome, however not all SNPs were assigned to protein-coding genes based on the BLAST results.The NC-Nowra and the NC-Liverpool tachyzoite transcriptomes differed by 3,130 SNPs and 6,123 indels . SupplemA set of 27 variants identified in silico between NC-Liverpool and NC-Nowra were subject to PCR and sequencing analysis . No falsN. caninum genome , transcription (NCLIV_057870 and NCLIV_065940), ribosomal subunit formation , GTP binding and GTPase activity (NCLIV_057820 and NCLIV_057390), protein transport (NCLIV_057490), and kinase activity or protein phosphorylation (NCLIV_ 056620 and NCLIV_057940). The genomic location of these 73 genes contained within SNP hotspots and their annotations are tabulated in Multiple SNP hotspots were identified, distributed unevenly throughout the m genome . A largePrior to functional analysis, 23 of the 35 SNP hotspot genes were identified as hypothetical proteins or unspecified products based on their corresponding gene IDs. All but nine were assigned GO terms, protein families, domains and/or repeats by InterProScan. Protein superfamilies that appeared more than once among these 35 SNP hotspot genes included WD40 repeat containing domain superfamily (IPR036322), ARM-like helical (IPR011989), ARM-type fold (IPR016024), and P-loop-containing nucleoside triphosphate hydrolase (IPR027417). Other superfamilies of functional interest were zinc finger RING/FYVE/PHD type (IPR013083), Sec1-like superfamily (IPR036045), EF-hand domain pair (IPR011992), ABC transporter superfamily (IPR036640), and the translation initiation factor eIF-4e-like (IPR023398) superfamilies. Domains and repeats featured were AAA+ ATPase domain (IPR003593), tetratricopeptide repeat (IPR019734), subtilisin SUB1-like catalytic domain (IPR034204), and WD40-repeat-containing protein (IPR017986).Recurring Gene Ontologies (GO) for molecular function included protein binding (GO: 0005515), binding (GO: 0005488), and hydrolase activity (GO: 0016787). Regarding biological process GOs, those assigned included lipid metabolic process (GO: 0006629), translation initiation (GO: 0006413), metabolic process (GO: 0008152), proteolysis (GO: 0006508), and transmembrane transport (GO: 0055085). NCLIV_056900 and NCLIV_057550). Interestingly, two TM proteins and two signal peptide containing proteins could not be assigned any additional annotations or gene ontologies.The three bioinformatic tools employed to identify transmembrane (TM) proteins and signal peptides within the SNP hotspot list, did not present consistent results for all protein sequences explored. However, mutually reported between both Phobius and Philius, were four transmembrane proteins, and six globular proteins with signal peptides, all of which were present on either chromosome VI or XI, except for one signal peptide containing protein which was located on chromosome V. Two hotspot genes also encoded transmembrane proteins with signal peptides, both of which were located on chromosome XI , XI (FR823392), and XII (FR823393), whereas almost all the SNPs located on chromosome V (FR823386) were found to be synonymous mutations. Additionally, the aforementioned callsets included 60 nonsynonymous SNPs, and 63 synonymous SNPs part of large contigs within the N. caninum genome, which are not pictured in the Circos plots generated.When the translated transcriptomes of NC-Nowra and NC-Liverpool were compared, 652 SNPs were found to be located in open reading frames that possessed different translations between the strains, and these SNPs were distributed across 287 unique genes. There were also 470 SNPs assigned to a protein-coding gene where the translation of the respective transcript was identical between the strains . However, where the number of mismatches reported by BLASTP exceeded the number of SNPs within a contig, it was assumed that VarScan had filtered out real sequence variants between the two strains . Alternatively, in the event that there were more SNPs identified by VarScan within a contig than BLASTP mismatches, the additional SNPs were presumed to result in synonymous mutations. Therefore, it was estimated that the final VarScan SNP callset contained at least 468 nonsynonymous SNPs, and 654 synonymous SNPs. The GOs that were overrepresented in transcripts containing nonsynonymous SNPs from amongst the molecular function GO category included protein kinase activity (GO: 0004672), ATP binding (GO: 0005524), and protein binding (GO: 0005515). Recurring GOs from the biological process category included protein phosphorylation (GO: 0006468), proteolysis (GO: 0005576), and oxidation–reduction process (GO: 0055114). The protein superfamilies repeatedly featured were protein kinase-like domain superfamily (IPR011009), p-loop containing domain-like superfamily (IPR027417), WD40-repeat containing domain superfamily (IPR036322), and tetratricopeptide-like helical domain superfamily (IPR011990). Recurring protein domains of functional importance included protein kinase (IPR000719), AAA+ ATPase (IPR003593), EF-hand calcium binding (IPR018247), and PAN/Apple domain (IPR003609), as well as featured protein repeats such as WD40 repeat (IPR001680), and Ankyrin repeat (IPR002110). Also of interest as reported by InterProScan, were protein signatures such as serine/threonine protein kinase active-site signature, protein kinase ATP binding site signature, protozoan surface antigen signature (SAG1), and ABC transporters family signature.Twenty-seven of the 35 SNP hotspot genes were found to contain nonsynonymous SNPs, including proteins coding for kinesin, SUB2, an ABC transporter, a Sec1 protein, and fatty acyl-CoA desaturase.z-test confirmed that the two chromosomes encoding a significantly larger number of SNP-dense transcripts , compared with the number of contigs with a low SNP density, were chromosomes VI (FR823387) and XI (FR823392). N. caninum tachyzoites, and only a very small number of noncoding regions exist between genes. The SNP hotspots within these chromosomes seem localized to selected genomic windows. The chromosomes also encode ncRNA (noncoding RNA) molecules, including tRNAs, dispersed unevenly along the length of each chromosome. Additionally, there are clear areas where the GC content along the chromosome either peaks above average, or decreases. Some SNP hotspots on either chromosome also appear to coincide with peaks in GC content, such as that on chromosome VI between approximately 450,000 and 500,000 bases.Chromosome XI (FR823392) possessed the largest number of contigs with a high SNP density across the genome. The N. caninum strains, across nine selected loci  containing 30 of the confirmed variants, did not reveal any specific patterns of segregation . Nevertheless, JPA1, BPA1, and NC1 were more similar to NC-Liverpool, whereas NC-Nowra, NC-SweB1, NC-Beef, BPA6, and WA-K9 were more similar to each other than to the formerly mentioned isolates. The NC-Liverpool strains from different passage numbers were identical. The neighbor-joining tree presented in The Sanger data generated for eight additional N. hughesi, through the DNA extraction and PCR amplification methodology described. The sequencing results for N. hughesi revealed the presence of all 28 variants identified in silico for N. caninum, as well as the existence of an additional 28 SNPs that were unique to N. hughesi. Presented in N. hughesi and all ten N. caninum strains. These results provide further additional support for the existence of two clades of N. caninum.Seven of the nine sequenced polymorphic loci  online wT. gondii genotyping markers examined, only two SNPs were present within N. caninum orthologous genes. They were in dense granule protein (GRA7) and class I chitinase (CLP1). These results appear to be consistent with that of N. caninum strains. This included SAG1, SRS2, and GRA6, all of which are among the 12 T. gondii genotyping markers explored in this study. Due to the location of one SNP at the very beginning of the contig, PCR and sequencing analysis was only performed and confirmed for one of these SNPs, as per the last entry in Of the 12 N. caninum genome, it was observed that chromosome Ia (FR823380) had the second lowest SNP density with <100 SNPs being present, only second to chromosome Ib (FR823381).After assigning the location of each SNP to a chromosome  to examiNeospora caninum is an apicomplexan parasite, responsible for reproductive failure in cattle and neurological disease in dogs. Intraspecies diversity is known in the form of extreme differences in virulence between strains found across the globe. The genetic basis of this diversity is unknown though an improved understanding of this could help to identify novel virulence loci.N. caninum. These strains vary drastically in their pathogenic propensity, and represent extremes of N. caninum virulence  as belonging to the tetratricopeptide-like helical domain superfamily. As with members of the WD40 family, TPR containing proteins are involved in protein–protein interactions and various metabolic and regulatory processes, and thus play an important role in virulence , ATP binding (GO: 0005524), and ATPase activity coupled to transmembrane movement of substances (GO: 0042626). However, while Phobius recognized the transmembrane topology of this protein coding sequence, Philius and TMHMM did not.The ATP binding cassette (ABC) superfamily of proteins are expressed as efflux transporters in eukaryotes, that translocate a plethora of substrates including proteins, ions, toxins and amino acids across membranes . All ABCSince the data exploited in this study was generated from RNA-seq data, it was unexpected that SNPs were identified that were not located in annotated genes. This suggests that either the current gene annotation is incorrect or incomplete, or that new/novel abundantly expressed transcripts were present in the culture from which the RNA-seq data was generated. However, this study did not investigate the presence of sequence variants located within apicoplast or mitochondrial DNA, to which some of the identified SNPs may have been located within.N. caninum genome in this study, the nonsynonymous mutations identified can also contribute to the current understanding of pathogenic variability within the species. As a nonsynonymous SNP alters a protein’s sequence, their presence can cause changes in biochemical activity, protein–protein interactions, and molecular function, which can consequently establish the link between genotype and biologically significant phenotypes  and theN. caninum in repeat regions, it is expected that SNPs will replace repetitive sequences as DNA markers, due to their distribution throughout the entire genome and their low mutation rates , and nonsynonymous mutations within protein-coding genes. This work provides new insights into the molecular basis behind the marked virulence properties reported between strains of N. caninum, which is knowledge that will be pertinent to the future development of a subunit vaccine against bovine neosporosis.In addition, we present a bioinformatic workflow that identified thousands of genetic variants in loci that are transcriptionally active during the tachyzoite stage of the We would like to thank Martin Krzywinski for his assistance with the Circos software. This study was completed by L.C. in partial fulfilment of the Ph.D. degree at UTS.Supplementary DataClick here for additional data file.