Founder Animals Research Articles

Pedigree-Based Description of Danubia Alba Rabbit Breed Lines.

The diversity of livestock animal breeds is an integral part of global biodiversity and requires careful management for sustainability and future availability. Avoiding inbreeding is a crucial aspect of mating of breeding animals. Our aims were to describe the quality of the pedigree, generation interval, gene origin, inbreeding, and effective population size of Danubia Alba rabbit lines. Line "D" is the maternal, whereas lines "C" and "X" are used as the paternal lines. The pedigree information was followed back from the actual breeding rabbits up to the founder animals. The rabbits having offspring in 2023 were chosen as reference populations for each line. The complete generation equivalent (GenCom) was 17.68 for line "C", 18.32 for line "D", and 17.49 for line "X", respectively. The maximum number of generations (GenMax) was above 30 for each line. The estimated bottleneck effect is mostly the result of selection and not a real genetic loss. The Wright inbreeding coefficient (F_Wright) was the highest for the "X" line rabbits, whereas it was the lowest for the line "D". Kalinowski's decomposition of inbreeding showed that it originated mostly from the past; the current fixation of alleles was quite similar for the line "C" and "D". Based on the predicted effective population sizes, it seems that there is no problem in maintaining of Danubia Alba lines.

Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.

Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice

BackgroundThe ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route.ResultsWe present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods.ConclusionsOur results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9–4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

Abstract B029: Centuries of genome instability and evolution in soft-shell clam (Mya arenaria) transmissible cancer

Abstract Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. Using genomic and transcriptomic analyses, we investigated the evolutionary history of a recently identified transmissible cancer lineage that has spread though the soft-shell clam (Mya arenaria) population. We first assembled a chromosome-scale soft-shell clam reference genome and characterized somatic mutations in the cancer lineage, discovering a novel mutational signature that estimates the lineage to be >200 years old and observing a wide variety of mutation types indicative of an extremely unstable cancer genome. We next quantified gene expression, observing differential expression of stereotypical cancer hallmarks in addition to key pathways that may have facilitated the cancer’s ability to survive seawater transfer to new hosts and evade immune rejection. Finally, we developed genetic assays to track bivalve transmissible neoplasia in the soft-shell clam and identify a novel lineage in the Baltic clam (Macoma balthica). Taken together, this study reveals the long-term survival of an invertebrate cancer lineage and identifies adaptive mechanisms to evade physical and immune barriers to cancer transmission, which may have been facilitated in part by the adaptive potential of its unstable genome. Citation Format: Samuel F.M. Hart, Michael J. Metzger. Centuries of genome instability and evolution in soft-shell clam (Mya arenaria) transmissible cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B029.

Genetic insights to assist management of the Critically Endangered hangul Cervus hanglu hanglu in the Kashmir Himalaya

AbstractThe hangul Cervus hanglu hanglu, a Critically Endangered mountain ungulate of Jammu and Kashmir, India, faces the imminent threat of population loss and extinction. Effective management of its largest viable population in Dachigam National Park in the Kashmir Himalaya requires reliable demographic information. Using 14 microsatellite markers we identified 293 individuals (208 females and 85 males) through faecal analysis, and generated data on the genetic status and population size of the hangul in its winter habitat. The mean expected and observed heterozygosities of 0.62 and 0.59 are comparable to those of several red deer Cervus elaphus populations elsewhere. The effective population sizes were 46.3 and 93.7 when the frequencies of rare alleles were considered to be 0.050 and 0.010, respectively. The average mean kinship of the population was 0.34, and there was no evidence of a recent bottleneck event. In genetic mark–recapture analysis the best model included an effect of sex on both detection and recapture probabilities. Detection of males was highest in November, coinciding with the hangul breeding season, whereas detection of females was highest in December. Our estimate of the hangul population using genetic mark–recapture with bootstrapping was 394 individuals. To our knowledge, this is the first study to use genetic data to estimate the population of the hangul. It will guide future studies of this subspecies and also serve as an impetus for identifying founder animals for captive breeding, and for connecting the population in Dachigam National Park with the other small, isolated populations to ensure the long-term survival of this subspecies.

Production of a PSEN1 genetically engineered marmoset model of early‐onset Alzheimer’s Disease

AbstractBackgroundThe lack of an adequate animal model of Alzheimer’s disease (AD) that mirrors the natural onset and progression of the human disorder has been a substantial barrier to basic and translational research into AD. Consequently, many fundamental questions remain about the key mechanisms that initiate the disease and the factors that promote its progression. While rodents are valuable AD models, nonhuman primates (NHPs) offer many advantages and have greater translational relevance. The common marmoset (Callithrix jacchus) is a small, prolific New World NHP that is amenable to genetic engineering and is rapidly becoming a valuable model organism, especially for neuroscience research.MethodWe used CRISPR/Cas9 technology to generate marmosets with C410Y or A426P point mutations in the PSEN1 gene. In humans, these mutations lead to familial early‐onset autosomal dominant Alzheimer’s Disease. Briefly, we designed, synthesized, and injected CRISPR/Cas9 reagents into single‐cell embryos that were subsequently transferred to synchronized recipient females. One adult C410Y founder animal was bred to a wild‐type mate.ResultTo date, we have produced seven live‐born marmosets harboring the PSEN1 C410Y (3 males, 1 female) or A426P (1 male, 2 females) mutations. One male founder animal, a compound heterozygote harboring C410Y and 1bp insertion PSEN1 alleles, has successfully sired three litters and transmitted the C410Y substitution (n = 6) and the 1bp insertion (n = 5) alleles to F1 offspring. We are currently characterizing the founders and F1 offspring using fluid‐based biomarkers, non‐invasive neuroimaging, and behavioral assessment to identify early markers of disease progression.ConclusionCRISPR/Cas9 gene editing was used to successfully establish a line of genetically engineered marmosets expected to model early‐onset AD. This nonhuman primate model will bridge the rodent‐to‐human translational gap and enable detailed studies of the early‐life molecular determinants of AD pathogenesis.

45 Genome Editing of Livestock for Production Agriculture and Biomedical Applications

Abstract Continued population growth and clear evidence of climate change are placing unprecedented strain on food production and health care systems worldwide. Agricultural production goals that highlight environmental sustainability are at the forefront of social dialog, with animal agriculture routinely targeted for inefficiencies, welfare concerns and impact on climate. Genomic selection has transformed animal production and the rate of genetic gain in each generation continues to increase. Nonetheless, it is apparent that the rate of production gains will fall short of the necessary goals of increased efficiency and adaptation to the changing environment. Genetic engineering may have an important role in enhancing livestock production as well as providing new models for research in human health. The CRISPR/Cas and related systems for targeted gene editing has revolutionized livestock genetic engineering by increasing the rate of targeted mutation, thus drastically reducing the cost of producing founder animals with the desired trait. Intentional insertion of well-known mutations into genes such as myostatin have been shown to impart the expected increase in meat production, but have also resulted in undesirable associated traits affecting fertility, marbling and the skeleton. Thus, the approach to increasing meat production may require nuances such as promotor targeting and multiple gene targeting to obtain the desired production characteristics. Efforts to increase disease resistance could have dramatic impacts on animal welfare and production rates, potentially saving millions of animals from morbidity and mortality. However, confronting a single disease with each genetic alteration will be costly and inefficient. Considerations on how broad scale disease resistance could be achieved through genetic engineering will be instrumental in developing livestock with advantages to avoid infection and/or transmission of multiple pathogens. Gene editing also allows correction of mutations that arise in livestock through natural mechanisms while under intense genomic selection for increased production. Removing disease causing mutations in otherwise high value livestock will allow continued production from these lines without concern for inheriting the causative mutation. The same process can be utilized to insert genetic mutations known to phenocopy human conditions providing valuable large animal models of human genetic disease. Increased milk, meat, or eggs per animal unit will sustainably improve high quality, nutrient dense food production while large animal models of human disease will provide a more translational biomedical model for accelerated development of drugs and treatments. These goals are achievable, but require continued investment in innovation, clear regulatory framework for commercialization, education, and public engagement to ensure success.

Centuries of genome instability and evolution in soft-shell clam, Mya arenaria, bivalve transmissible neoplasia

Transmissible cancers are infectious parasitic clones that metastasize to new hosts, living past the death of the founder animal in which the cancer initiated. We investigated the evolutionary history of a cancer lineage that has spread though the soft-shell clam (Mya arenaria) population by assembling a chromosome-scale soft-shell clam reference genome and characterizing somatic mutations in transmissible cancer. We observe high mutation density, widespread copy-number gain, structural rearrangement, loss of heterozygosity, variable telomere lengths, mitochondrial genome expansion and transposable element activity, all indicative of an unstable cancer genome. We also discover a previously unreported mutational signature associated with overexpression of an error-prone polymerase and use this to estimate the lineage to be >200 years old. Our study reveals the ability for an invertebrate cancer lineage to survive for centuries while its genome continues to structurally mutate, likely contributing to the evolution of this lineage as a parasitic cancer.

Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

Hope is the last thing lost: Colombian captive-bred population of the critically endangered Orinoco crocodile (Crocodylus intermedius) is a genetic reservoir that could help to save the species from extinction

A purpose ofex-situpopulations is the preservation of genetic variation, but this is a challenging task since genetic diversity is commonly lost through each generation, and so the establishment of management guidelines should be a high priority. Fifty years ago, the National University of Colombia began a breeding program in the Roberto Franco Tropical Station (in Villavicencio, Meta) to conserve the critically endangered Orinoco crocodileCrocodylus intermedius. Despite the large number of individuals raised and kept in captivity, the Station has not been able to release individuals due to a lack of a complete genetic characterization that could determine whether the population is genetically viable. In this study we used a panel of 17 microsatellite loci to overcome this problem. We estimated from the founder animals and the live crocodiles the inbreeding, heterozygosities, the number of alleles, and their richness, and frequencies to understand the effects of managing a captive breeding program without considering genetic profiles. Our results revealed that the living population maintains much of its founder diversity with high levels of heterozygosity and low overall inbreeding, making it suitable for maintaining captive breeding and for implementing wild releases. We estimated the individual genetic diversity of the living crocodiles, as well as their relationships. This information, combined with the size, sex, and location, allowed us to propose combinations and to restructure the breeding groups. We demonstrated that molecular data could be used to improve the management ofex-situconservation programs well beyond what could be achieved with pedigree information alone.

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for “correcting” variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Wild origins and mitochondrial genetic diversity of Angolan Colobus monkeys (Colobus angolensis) in AZA-accredited zoos and its implications for ex situ population management.

Across zoo's accredited by the Association of Zoos and Aquariums(AZA), species are typically managed as a single population to retain 90% of the founding members' gene diversity. Often, little is known about the specific geographic origins of the founders or how representative the ex situ population's genetic diversity is of the wild population. This study uses mitochondrial DNA (mtDNA) sequencing to investigate haplotype diversity and geographic female founder origin of the AZA-managed Angolan colobus (Colobus angolensis) monkey population. We obtained fecal samples from individuals closely related to founder animals at five zoos and found four haplotypes among 23 individuals. Analyzed together with wild C. angolensis haplotypes, we found two haplotypes identical to those found in Tanzanian populations: one haplotype, possessed by 13 individuals (descended from three founders), matched an East Usambara Mountains haplotype, while the other, possessed by seven individuals (from four founders), matched a haplotype found in both the South Pare Mountains and Rufiji River. Two haplotypes were not detected in wild populations but were closely related to haplotypes found in the Rufiji River (one individual descended from one founder) and Shimoni, Kenya (two individuals descended from one founder) populations, suggesting nearby origins. Thus, the AZA-managed population of Angolan colobus likely originated from several localities, but all have mtDNA lineages associated with the subspecies C. a. palliatus, a Vulnerable subspecies. Examining founders' mtDNA haplotypes may be a useful addition to the zoo population management toolkit to help improve breeding recommendations by identifying individuals with rare haplotypes and revealing likely kinship among founders.

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Irregularities in Meiotic Prophase I as Prerequisites for Reproductive Isolation in Experimental Hybrids Carrying Robertsonian Translocations

The basic causes of postzygotic isolation can be elucidated if gametogenesis is studied, which is a drastically different process in males and females. As a step toward clarifying this problem, we obtained an experimental inbred lineage of the eastern mole vole Ellobius tancrei, whose founder animals were animals with identical diploid numbers 2n = 50 but with different Robertsonian translocations (Rb), namely 2Rb4.12 and 2Rb9.13 in the female and 2Rb.2.18 and 2Rb5.9 in the male. Here, we analyzed strictly inbred hybrids (F1, fertile and F10, sterile) using immunocytochemical methods in order to study spermatocytes during the meiotic prophase I. Previously, the presence of trivalents was assumed to have no significant effect on spermatogenesis and fertility in hybrids, but we demonstrated that spermatogenesis might be disturbed due to the cumulative effects of the retarded synapses of Rb bivalents as well as trivalents and their associations with XX sex bivalents. Alterations in the number of gametes due to the described processes led to a decrease in reproductive capacity up to sterility and can be examined as a mechanism for reproductive isolation, thus starting speciation.

Genetic Diversity and Trends of Ancestral and New Inbreeding in German Sheep Breeds by Pedigree Data.

In Germany, many autochthonous sheep breeds have developed, adapted to mountain, heath, moorland, or other marginal sites, but breeds imported from other countries have also contributed to the domestic breeds, particularly improving wool and meat quality. Selective breeding and the intense use of rams may risk losing genetic diversity and increasing rates of inbreeding. On the other hand, breeds with a low number of founder animals and only regional popularity may not leave their endangered status, as the number of breeders interested in the breed is limited. The objective of the present study was to determine demographic measures of genetic diversity and recent as well as ancestral trends of inbreeding in all autochthonous German sheep breeds and sheep of all breeding directions, including wool, meat, and milk. We used pedigree data from 1,435,562 sheep of 35 different breeds and a reference population of 981,093 sheep, born from 2010 to 2020. The mean number of equivalent generations, founders, effective founders, effective ancestors, and effective founder genomes were 5.77, 1669, 123.2, 63.5, and 33.0, respectively. Genetic drift accounted for 69% of the loss of genetic diversity, while loss due to unequal founder contributions was 31%. The mean inbreeding coefficient, individual rate of inbreeding (∆Fi), and realized effective population size across breeds were 0.031, 0.0074, and 91.4, respectively, with a significantly decreasing trend in ∆Fi in 11/35 breeds. New inbreeding, according to Kalinowski, contributed to 71.8% of individual inbreeding, but ancestral inbreeding coefficients showed an increasing trend in all breeds. In conclusion, in our study, all but one of the mountain-stone sheep breeds and the country sheep breed Wald were the most vulnerable populations, with Ne < 50. The next most endangered breeds are exotic, country, and heath breeds, with average Ne of 66, 83, and 89, respectively. The wool, meat, and milk breeds showed the highest genetic diversity, with average Ne of 158, 120, and 111, respectively. The results of our study should help strengthen conservation program efforts for the most endangered sheep breeds and maintain a high genetic diversity in all sheep breeds.

Genotyping Genome-Edited Founders and Subsequent Generation.

Targeted nucleases allow the production of many types of genetic mutations directly in the early embryo. However, the outcome of their activity is a repair event of unpredictable nature, and the founder animals that are produced are generally of amosaic nature. Here, we present the molecular assays and genotyping strategies that will support the screening of the first generation for potential founders and the validation of positive animals in the subsequent generation, according to the type of mutation generated.

CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Structural variants and tandem repeats in the founder individuals of four F2 pig crosses and implications to F2 GWAS results

BackgroundStructural variants and tandem repeats are relevant sources of genomic variation that are not routinely analyzed in genome wide association studies mainly due to challenging identification and genotyping. Here, we profiled these variants via state-of-the-art strategies in the founder animals of four F2 pig crosses using whole-genome sequence data (20x coverage). The variants were compared at a founder level with the commonly screened SNPs and small indels. At the F2 level, we carried out an association study using imputed structural variants and tandem repeats with four growth and carcass traits followed by a comparison with a previously conducted SNPs and small indels based association study.ResultsA total of 13,201 high confidence structural variants and 103,730 polymorphic tandem repeats (with a repeat length of 2-20 bp) were profiled in the founders. We observed a moderate to high (r from 0.48 to 0.57) level of co-localization between SNPs or small indels and structural variants or tandem repeats. In the association step 56.56% of the significant variants were not in high LD with significantly associated SNPs and small indels identified for the same traits in the earlier study and thus presumably not tagged in case of a standard association study. For the four growth and carcass traits investigated, many of the already proposed candidate genes in our previous studies were confirmed and additional ones were identified. Interestingly, a common pattern on how structural variants or tandem repeats regulate the phenotypic traits emerged. Many of the significant variants were embedded or nearby long non-coding RNAs drawing attention to their functional importance. Through which specific mechanisms the identified long non-coding RNAs and their associated structural variants or tandem repeats contribute to quantitative trait variation will need further investigation.ConclusionsThe current study provides insights into the characteristics of structural variants and tandem repeats and their role in association studies. A systematic incorporation of these variants into genome wide association studies is advised. While not of immediate interest for genomic prediction purposes, this will be particularly beneficial for elucidating biological mechanisms driving the complex trait variation.

Abstract 001: Germline Mutagenesis Induces Blood Pressure And Heart Rate Variation In Mice Uncovering Novel Insights On The Genesis Of Cardiovascular Disease Risks

Resting blood pressure (BP) and heart rate (HR) levels are independent risk factors for cardiovascular diseases and mortality. Both phenotypes are critical for lifetime maintenance of the cardiovascular homeostasis and are under the influence of multiple factors. The estimated heritability for both phenotypes is high, but their main genetic determinants remain elusive. To address this shortcoming, we used a strategy to systematically identify genes affecting BP and HR regulation through high-throughput N-ethyl-N-Nitrosurea-induced germline mutagenesis. First, the random chemical-induced mutations were identified in founder animals by whole exome G1 progenitor sequencing and their zygosity established in G2/G3 mice before phenotypic assessment. We used the noninvasive tail cuff method to assess BP and HR in conscious G3 mice distributed in pedigrees of 30-100 G1 descendants. The quantitative traits were then analyzed with Linkage Analyzer in single or multiple combined pedigrees, detecting significant linkage between individual mutations and altered phenotypic scores in heterozygous or homozygous mice. Here, we report the initial results of BP and HR candidate genes selected during the screening of 45,261 G3 mice from 878 pedigrees, testing the effects of 45,317 variant alleles within 14,575 genes, in which 23 % of all autosomal genes were severely damaged. The data indicate that ENU mutagenesis effectively identified 85 candidate genes affecting BP and 132 HR levels, including only 13 and 18 genes previously known to influence BP and HR, respectively. Moreover, we validated the influence of three and seven new genes on BP and HR control through reverse genetics that may be associated with ageing, vascular integrity and cardiac development. These findings shed light on novel underlying mechanisms associated with both phenotypes and highlight sub-systems contributing to the genesis of critical risk factors associated with cardiovascular diseases.