We report a complete rodent telomere-to-telomere genome assembly from the brown rat, Rattus norvegicus. Annotation was enriched with multi-tissue long-read RNA sequencing and uncovered numerous novel genes. Assembly of both sex chromosomes reveals the absence of gene coding in the presumed pseudo-autosomal regions and the presence of centromeric satellite repeats on distal chromosome Y (chrY). We provide evidence of meiotic conjunction between Xp and Yq. The genome assembly reveals several expanded autosomal regions enriched for testis-expressed genes. Finally, we have generated a pangenome from recent high-quality assemblies of 8 distinct inbred rat strain genomes. This allows the strain-specific distribution of structural variation to be examined. Non-allelic homologous recombination has produced multiple copies of several genes with evidence of transcription from duplicated copies.

The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analysed host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We found that polygenic host genetic effects were consistent across environments. We identified three replicated microbiome-associated loci, one of which involved a sialyltransferase gene and Paraprevotella. We found a similar association in a human cohort, between ST6GAL1 and Paraprevotella, both of which have been linked with immune and infectious diseases. Moreover, we found evidence of indirect genetic effects on microbiome phenotypes, which substantially increased their total genetic variance. Finally, we identified a novel mechanism whereby indirect genetic effects can contribute to "missing heritability".

The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analyse host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We show that polygenic host genetic effects are consistent across cohort environments. We identify three replicated microbiome-associated loci, one of which involves the sialyltransferase gene St6galnac1 and Paraprevotella. We find a similar association in a human cohort, between ST6GAL1 and Paraprevotella, both of which have been linked with immune and infectious diseases. Moreover, we find indirect (i.e. social) genetic effects on microbiome phenotypes, which substantially increase the total genetic variance. Finally, we identify a novel mechanism whereby indirect genetic effects can contribute to “missing heritability”. The intestinal microbiome is shaped by genetics and environment. Here, the authors show in rats that host genetic effects, including indirect social effects, influence microbiome composition, identify replicated loci, and reveal mechanisms contributing to microbiome heritability.

The seventh iteration of the reference genome assembly for Rattus norvegicus—mRatBN7.2—corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

The rat, Rattus norvegicus, has provided an important model for investigation of a range of characteristics of biomedical importance. Here we survey the origins of this species, its introduction into laboratory research, and the emergence of genetic and genomic methods that utilize this model organism. Genomic studies have yielded important progress and provided new insight into several biologically important traits. However, some studies have been impeded by the lack of a complete and accurate reference genome for this species. New sequencing and genome assembly methods applied to the rat have resulted in a new reference genome assembly, GRCr8, which is a near telomere-to-telomere assembly of high base-level accuracy that incorporates several elements not captured in prior assemblies. As genome assembly methods continue to advance and production costs become a less significant obstacle, genome assemblies for multiple inbred rat strains are emerging. These assemblies will allow a rat pangenome assembly to be constructed that captures all the genetic variations in strains selected for their utility in research and will overcome reference bias, a limitation associated with reliance on a single reference assembly. By this means, the full utility of this model organism to genomic studies will begin to be revealed.

We report the construction and analysis of a new reference genome assembly for Rattus norvegicus, the laboratory rat, a widely used experimental animal model organism. The assembly has been adopted as the rat reference assembly by the Genome Reference Consortium and is named GRCr8. The assembly has employed 40× Pacific Biosciences (PacBio) HiFi sequencing coverage and scaffolding using optical mapping and Hi-C. We used genomic DNA from a male BN/NHsdMcwi (BN) rat of the same strain and from the same colony as the prior reference assembly, mRatBN7.2. The assembly is at chromosome level with 98.7% of the sequence assigned to chromosomes. All chromosomes have increased in size compared with the prior assembly and k-mer analysis indicates that the subject animal is fully inbred and that the genome is represented as a single haploid assembly. Notable increases are observed in Chromosomes 3, 11, and 12 in the prospective rDNA regions. In addition, Chr Y has increased threefold in size and is more consistent with the rat karyotype than previous assemblies. Several other chromosomes have grown by the incorporation of sizable discrete new blocks. These contain highly repetitive sequences and encode numerous previously unannotated genes. In addition, centromeric sequences are incorporated in most chromosomes. Genome annotation has been performed by NCBI RefSeq, which confirms improvement in assembly quality and adds more than 1100 new protein coding genes. PacBio Iso-Seq data have been acquired from multiple tissues of the subject animal and are released concurrently with the new assembly to aid further analyses.

During development of the spontaneously hypertensive rat (SHR), several distinct but closely related lines were generated. Most lines are resistant to hypertensive renal disease. However, the SHR-A3 line (stroke-prone SHR) experiences end-organ injury (EOI) and provides a model of injury susceptibility that can be used to uncover genetic causation. In the present study, we generated a congenic line in which three distinct disease loci in SHR-A3 are concurrently replaced with homologous loci from an injury-resistant SHR line (SHR-B2). Verification that all three loci were homozygously replaced in this triple congenic line [SHR-A3(Trip B2)] while the genetic background of SHR-A3 was fully retained was obtained by whole genome sequencing. Congenic genome substitution was without effect on systolic blood pressure [198.9 ± 3.34 mmHg, mean ± SE, SHR-A3(Trip B2) = 194.7 ± 2.55 mmHg]. Measures of renal injury (albuminuria, histological injury scores, and urinary biomarker levels) were reduced in SHR-A3(Trip B2) animals, even though only 4.5 Mbases of the 2.8 Gbases of the SHR-B2 genome (0.16% of the genome) was transferred into the congenic line. The gene content of the three congenic loci and the functional effects of gene polymorphism within suggest a role of immunoglobulin in EOI pathogenesis. To prove the role of antibodies in EOI in SHR-A3, we generated an SHR-A3 line in which expression from the immunoglobulin heavy chain gene was knocked out (SHR-A3-IGHKO). Animals in the SHR-A3-IGHKO line lack B cells and immunoglobulin, but the hypertensive phenotype is not affected. Renal injury, however, was reduced in this line, confirming a pathogenic role for immunoglobulin in hypertensive EOI in this model of heritable risk.NEW & NOTEWORTHY Here, we used a polygenic animal model of hypertensive renal disease to show that genetic variation affecting antibody formation underlies hypertensive renal disease. We proved the genetic thesis by generating an immunoglobulin knockout in the susceptible animal model.

Clinical use of the combination therapy of the neprilysin inhibitor sacubitril and angiotensin II type 1 receptor blocker valsartan is known to be associated with albuminuria. Albuminuria is both a risk factor for and an indicator of kidney injury. Earlier work from our laboratory reported that the agonist of angiotensin II type 2 receptor Compound 21 (C21) prevents proteinuria, albuminuria, and is reno-protective in obese Zucker rats fed high salt diet (HSD). Thus, we hypothesized that sacubitril/C21 combination provides superior reno-protection compared to sacubitril/valsartan. Male obese Zucker rats 10–11 weeks old were treated daily with vehicle, sacubitril + C21, or sacubitril + valsartan while fed HSD for 16 days. HSD-feeding caused kidney dysfunction, evident by significant increases in urinary protein, osteopontin, and cystatin C. HSD-feeding lowered plasma cystatin C and creatinine concentrations suggestive of hyperfiltration, which was not affected by either treatment. Unlike sacubitril/valsartan, sacubitril/C21 treatment significantly decreases proteinuria, albuminuria, the expression of nephrin, and kidney weight, independent of hyperfiltration, compared with HSD alone. Moreover, sacubitril/valsartan therapy increased plasma renin and did not prevent HSD-induced increases in renal angiotensin II, while sacubitril/C21 completely prevented these changes. Together, this study suggests that sacubitril/C21 afforded superior reno-protection compared to sacubitril/valsartan therapy in high salt-fed obese Zucker rats.

Rat genomic tools have been slower to emerge than for those of humans and mice and have remained less thorough and comprehensive. The arrival of a new and improved rat reference genome, mRatBN7.2, in late 2020 is a welcome event. This assembly, like predecessor rat reference assemblies, is derived from an inbred Brown Norway rat. In this “user” survey we hope to provide other users of this assembly some insight into its characteristics and some assessment of its improvements as well as a few caveats that arise from the unique aspects of this assembly. mRatBN7.2 was generated by the Wellcome Sanger Institute as part of the large Vertebrate Genomes Project. This rat assembly has now joined human, mouse, chicken, and zebrafish in the National Center for Biotechnology Information (NCBI)’s Genome Reference Consortium, which provides ongoing curation of the assembly. Here we examine the technical procedures by which the assembly was created and assess how this assembly constitutes an improvement over its predecessor. We also indicate the technical limitations affecting the assembly, providing illustrations of how these limitations arise and the impact that results for this reference assembly.

Background: We report the creation and evaluation of a de novo assembly of the genome of the spontaneously hypertensive rat, the most widely used model of human cardiovascular disease. Methods: The genome is assembled from long read sequencing (PacBio HiFi and continuous long read data [CLR]) and scaffolded with long-range structural information obtained from Bionano optical maps and proximity ligation sequencing proximity analysis of the genome. The genome assembly was polished with Illumina short reads. Completeness of the assembly was investigated using Benchmarking Universal Single Copy Orthologs analysis. The genome assembly was also evaluated with the rat reference gene set, using NCBI automated protocols. We also generated orthogonal single molecule transcript sequence reads (Iso-Seq) from 8 tissues and used them to validate the coding assembly, to annotate the assembly with RNA transcripts representing unique full length transcript isoforms for each gene and to determine whether divergences between RefSeq sequences and the assembly were attributable to assembly errors or polymorphisms. Results: The assembly analysis indicates that this assembly is comparable in contiguity and completeness to the current rat reference assembly, while the use of HiFi sequencing yields an assembly that is more correct at the single base level. Synteny analysis was performed to uncover the extent of synteny and the presence and distribution of chromosomal rearrangements between the reference and this assembly. Conclusion: The resulting genome assembly is reference quality and captures significant structural variation.

Background: The spontaneously hypertensive rat (SHR) is extensively used to study hypertension. Gut microbiota dysbiosis is a notable feature in SHR for reasons unknown. Immunoglobulin A (IgA) is a major host factor required for gut microbiota homeostasis. We hypothesized that inadequate IgA contributes to gut microbiota dysbiosis in SHR. methods: IgA was measured in feces, cecum, serum, liver, gut-associated lymphoid tissue, and milk from SHR and Wistar Kyoto rats. IgA regulatory factors like IgM, IgG, and pIgR (polymeric immunoglobulin receptor) were analyzed. IgA and IgG antibodies and blood pressure (BP) were measured before and after administrating a bacterial antigen (ie, flagellin). Results: Compared with Wistar Kyoto rats, SHR displayed remarkably near-deficient IgA levels accompanied by compensatory increases in serum IgM and IgG and gut-liver pIgR expression. Inadequate milk IgA in SHR emphasized this immune defect stemmed from the neonatal stage. Reduced IgA(+) B cells in circulation and Peyer patches indicated a possible reason for the lower IgA in SHR. Noteworthy, a genetic insufficiency was unlikely because administering flagellin to SHR induced anti-flagellin IgA antibodies. This immune response surprisingly accelerated hypertension development in SHR, suggesting IgA quiescence may help maintain lower BP. Conclusions: This study is the first to reveal IgA deficiency in SHR as one host factor associated with gut microbiota dysbiosis and invigorates future research to determine the pathophysiological role of IgA in hypertension.

The pathogenic links between elevated blood pressure and chronic kidney disease remain obscure. This article examines progress in population genetics and in animal models of hypertension and chronic kidney disease. It also provides a critique of the application of genome-wide association studies to understanding the heritability of renal function. Emerging themes identified indicate that heritable risk of chronic kidney disease in hypertension can arise from genetic variation in (1) glomerular and tubular protein handling mechanisms; (2) autoregulatory capacity of the renal vasculature; and (3) innate and adaptive immune mechanisms. Increased prevalence of hypertension-associated chronic kidney disease that occurs with aging may reflect amplification of heritable risks by normal aging processes affecting immunity and autoregulation.

Chronic cardiovascular diseases are associated with inflammatory responses within the blood vessels and end organs. The origin of this inflammation has not been certain, and neither is its relationship to disease clear. There is a need to determine whether this association is causal or coincidental to the processes leading to cardiovascular disease. These processes are themselves complex: many cardiovascular diseases arise in conjunction with the presence of sustained elevation of blood pressure. Inflammatory processes have been linked to hypertension, and causality has been suggested. Evidence of causality poses the difficult challenge of linking the integrated and multifaceted biology of blood pressure regulation with vascular function and complex elements of immune system function. These include both, innate and adaptive immunity, as well as interactions between the host immune system and the omnipresent microorganisms that are encountered in the environment and that colonize and exist in commensal relationship with the host. Progress has been made in this task and has drawn on experimental approaches in animals, much of which have focused on hypertension occurring with prolonged infusion of angiotensin II. These laboratory studies are complemented by studies that seek to inform disease mechanism by examining the genomic basis of heritable disease susceptibility in human populations. In this realm too, evidence has emerged that implicates genetic variation affecting immunity in disease pathogenesis. In this article, we survey the genetic and genomic evidence linking high blood pressure and its end-organ injuries to immune system function and examine evidence that genomic factors can influence disease risk. © 2021 American Physiological Society. Compr Physiol 11:1-22, 2021.

Background Spontaneously hypertensive rats of the stroke‐prone line (SHR‐A3) develop hypertensive renal disease as a result of naturally occurring genetic variation. Our prior work identified a single‐nucleotide polymorphism unique to SHR‐A3 that results in truncation of the carboxy terminus of STIM1. The SHR‐B2 line, which is also hypertensive but resists hypertensive renal injury, expresses the wild‐type STIM1. STIM1 plays a central role in lymphocyte calcium signaling that directs immune effector responses. Here we show that major defects in lymphocyte function affecting calcium signaling, nuclear factor of activated T cells activation, cytokine production, proliferation, apoptosis, and regulatory T‐cell development are present in SHR‐A3 and attributable to STIM1. Methods and Results To assess the role of Stim1 variation in susceptibility to hypertensive renal injury, we created a Stim1 congenic line, SHR‐A3(Stim1‐B2), and STIM1 function was rescued in SHR‐A3. We found that Stim1 gene rescue restores disturbed lymphocyte function in SHR‐A3. Hypertensive renal injury was compared in SHR‐A3 and the SHR‐A3(Stim1‐B2) congenic line. Histologically assessed renal injury was markedly reduced in SHR‐A3(Stim1‐B2), as were renal injury biomarker levels measured in urine. Stim1 deficiency has been linked to the emergence of antibody‐mediated autoimmunity. Renal glomerular immunoglobulin deposition was greater in SHR‐A3 than SHR‐B2 and was reduced by Stim1 congenic substitution. Serum anti–double‐stranded DNA antibody titers in SHR‐A3 were elevated compared with SHR‐B2 and were reduced in SHR‐A3(Stim1‐B2). Conclusions Stim1 deficiency in lymphocyte function originating from Stim1 truncation in SHR‐A3 combines with hypertension to create end organ disease and may do so as a result of antibody formation.

Similar to humans, the risk of cerebrovascular disease in stroke-prone spontaneously hypertensive rats (SHR-A3/SHRSP) arises from naturally occurring genetic variation. In the present study, we show the involvement of genetic variation affecting the store-operated calcium signaling gene, Stim1, in the pathogenesis of stroke in SHR. Stim1 is a key lymphocyte activation signaling molecule and contains functional variation in SHR-A3 that diverges from stroke-resistant SHR-B2. We created a SHR-A3 congenic line in which Stim1 was substituted with the corresponding genomic segment from SHR-B2. Compared with SHR-A3 rats, Stim1 congenic SHR-A3 (SHR-A3(Stim1-B2)) have reduced cerebrovascular disease in response to salt loading including lower neurological deficit scores and cerebral edema. Microbleeds and major hemorrhages occurred in over half of SHR-A3 rats. These lesions were absent in SHR-A3(Stim1-B2) rats. Loss of Stim1 function in mice and humans is associated with antibody-mediated autoimmunity due to defects in T lymphocyte helper function to B cells. We investigated autoantibody formation using a high-density protein array to detect the presence of IgG and IgM autoantibodies in SHR-A3. Autoantibodies to key cerebrovascular stress proteins were detected that were reduced in the congenic line.

The risk of cerebrovascular disease in stroke-prone spontaneously hypertensive rats (SHR-A3/SHRSP) arises from naturally occurring genetic variation. In the present study we show the involvement of SHR genetic variation that affects antibody formation and function in the pathogenesis of stroke. We have tested the involvement in susceptibility to stroke of genetic variation in IgH, the gene encoding the immunoglobulin heavy chain by congenic substitution. This gene contains functional natural variation in SHR-A3 that diverges from stroke-resistant SHR-B2. We created a SHR-A3 congenic line in which the IgH gene was substituted with the corresponding haplotype from SHR-B2. Compared with SHR-A3 rats, congenic substitution of the IgH locus [SHR-A3(IgH-B2)] markedly reduced cerebrovascular disease. Given the role in antibody formation of the IgH gene, we investigated the presence of IgG and IgM autoantibodies and their targets using a high-density protein array containing ~20,000 recombinant proteins. High titers of autoantibodies to key cerebrovascular stress proteins were detected, including FABP4, HSP70, and Wnt signaling proteins. Serum levels of these autoantibodies were reduced in the SHR-A3(IgH-B2) congenic line.

“Congenic genotypes” This table shows sequenom generated SNP genotypes across the genomes of SHR-A3 and the SHR-A3(IgH-B2) congenic lines. There are two columns of genotypes for two individual SHR-A3(IgH-B2) congenics. These are both progeny of the same founders. All their genotypes outside of the IgHlocus are identical to SHR-A3. The list includes two genotypes targeting the proximal and distal ends of the IgHlocus. These were typed by PCR and gel analysis. Both are SHR-B2 alleles.

Stroke-prone spontaneously hypertensive rats (SHR-A3/SHRSP) develop cerebrovascular disease as a result of naturally occurring genetic variation. We recently identified a novel truncating mutation in the SHR-A3 line affecting the C-terminus of STIM1, a protein involved in the store-operated Ca 2+ entry (SOCE) pathway. The SHR-B2 line, which is also hypertensive but resists end organ injury, expresses the ‘wild type’ Stim1 . Here, we test whether the emergence of cerebrovascular disease in SHR-A3 is prevented by gene rescue of Stim1 . We created a Stim1 congenic line (SHR-A3( Stim1 -B2)), in which the functional Stim1 allele was transferred from the SHR-B2 line into the stroke-prone SHR-A3 genome. SHR-A3 and SHR-A3( Stim1 -B2) rats were salt loaded (1% NaCl in drinking water) for 8 weeks starting at 20 weeks of age to induce strokes. Baseline BP measured by telemetry before salt loading was not different between SHR-A3 and SHR-A3( Stim1 -B2) rats (199.5±6.49 vs 196.26±2.431 mmHg, ns). Salt loading resulted in a progressive increase in BP in SHR-A3 rats, but was blunted in SHR-A3( Stim1 -B2) rats. Compared to SHR-A3 rats, Stim1 -rescue congenic rats had improved survival (% survival: 100 (9 of 9) vs 22.2% (2 of 9) at the end of 8 weeks) and lower neurological deficit scores (2.45±0.412 vs 1±0.00, p<0.01). Salt loading resulted in significant cerebral edema in SHR-A3 rats but not in the SHR-A3( Stim1 -B2) rats (% brain wt/body wt: 0.805±0.045 vs 0.617±0.009, p<0.01). Gross morphology of the brain revealed microbleeds and hemorrhages in 5 of 9 SHR-A3 rats. These lesions were absent in SHR-A3( Stim1 -B2) rats. Stim1 gene rescue in the congenic line was also associated with decreased susceptibility to renal injury assessed histologically at 40 weeks of age (glomerular injury: 1.915±0.086 vs 1.355±0.071, p<0.001; tubulointerstitial injury: 3.2±0.102 vs 1.69±0.112, p<0.001). Our findings identify Stim1 as a major candidate gene that promotes susceptibility to strokes as well as renal injury in spontaneously hypertensive rats.