Grant A. Walling

Highly Prolific Booroola Sheep Have a Mutation in the Intracellular Kinase Domain of Bone Morphogenetic Protein IB Receptor (ALK-6) That Is Expressed in Both Oocytes and Granulosa Cells

Abstract The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23–31, which is syntenic to human chromosome 4q21–25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-β (TGF-β) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-β pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.

Mapping of multiple quantitative trait loci for growth and carcass traits in a complex commercial sheep pedigree

The confirmation of the segregation of experimentally discovered quantitative trait loci (QTL) in a variety of commercial populations is required before their commercial significance can be fully realized. The use of complex pedigrees in the design of such confirmation experiments has the potential to increase the probability of the QTL segregating within the pedigree while maintaining the power to detect this segregation. Here a QTL analysis is applied across candidate chromosomes of a complex pedigree of 570 Charollais sheep from commercial flocks in the UK. This pedigree also contained a moderately sized half-sib family which was analysed separately. Two QTL significant at the 5% chromosome- wide level were detected in the half-sib analysis and seven were detected in a maximum likelihood variance component analysis of the complex pedigree using identity-by-descent values estimated by Markov chain Monte Carlo methods. The estimation of QTL effects was achieved by fitting all QTL for a specific trait simultaneously, reducing the large upward bias observed in the single QTL models. Both methods of analysis detected QTL for live weight, although these mapped to different regions, and the variance components method detected QTL for ultrasonically measured fat depth. This analysis demonstrates the viability of applying a variance component analysis to large pedigrees with the presence of considerable inbreeding.

Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds

BackgroundObesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs).ResultsBy sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4).ConclusionsSelective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals.

A comparison of bootstrap methods to construct confidence intervals in QTL mapping

Summary The determination of empirical confidence intervals for the location of quantitative trait loci (QTLs) by interval mapping was investigated using simulation. Confidence intervals were created using a non-parametric (resampling method) and parametric (resimulation method) bootstrap for a backcross population derived from inbred lines. QTLs explaining 1%, 5% and 10% of the phenotypic variance were tested in populations of 200 or 500 individuals. Results from the two methods were compared at all locations along one half of the chromosome. The non-parametric bootstrap produced results close to expectation at all non-marker locations, but confidence intervals when the QTL was located at the marker were conservative. The parametric method performed poorly; results varied from conservative confidence intervals at the location of the marker, to anti-conservative intervals midway between markers. The results were shown to be influenced by a bias in the mapping procedure and by the accumulation of type 1 errors at the location of the markers. The parametric bootstrap is not a suitable method for constructing confidence intervals in QTL mapping. The confidence intervals from the non-parametric bootstrap are accurate and suitable for practical use.

On the mapping of quantitative trait loci at marker and non-marker locations.

Previous studies have noted that the estimated positions of a large proportion of mapped quantitative trait loci (QTLs) coincide with marker locations and have suggested that this indicates a bias in the mapping methodology. In this study we predict the expected proportion of QTLs with positions estimated to be at the location of a marker and further examine the problem using simulated data. The results show that the higher proportion of putative QTLs estimated to be at marker positions compared with non-marker positions is an expected consequence of the estimation methods. The study initially focused on a single interval with no QTLs and was extended to include multiple intervals and QTLs of large effect. Further, the study demonstrated that the larger proportion of estimated QTL positions at the location of markers was not unique to linear regression mapping. Maximum likelihood produced similar results, although the accumulation of positional estimates at outermost markers was reduced when regions outside the linkage group were also considered. The bias towards marker positions is greatest under the null hypothesis of no QTLs or when QTL effects are small. This study discusses the impact the findings could have on the calculation of thresholds and confidence intervals produced by bootstrap methods.

Novel approach for deriving genome wide SNP analysis data from archived blood spots

BackgroundThe ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored.FindingsDNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way.ConclusionsDNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.

Isolation of subtelomeric sequences of porcine chromosomes for translocation screening reveals errors in the pig genome assembly

Summary Balanced chromosomal aberrations have been shown to affect fertility in most species studied, often leading to hypoprolificacy (reduced litter size) in domestic animals such as pigs. With an increasing emphasis in modern food production on the use of a small population of high quality males for artificial insemination, the potential economic and environmental costs of hypoprolific boars, bulls, rams etc. are considerable. There is therefore a need for novel tools to facilitate rapid, cost‐effective chromosome translocation screening. This has previously been achieved by standard karyotype analysis; however, this approach relies on a significant level of expertise and is limited in its ability to identify subtle, cryptic translocations. To address this problem, we developed a novel device and protocol for translocation screening using subtelomeric probes and fluorescence in situ hybridisation. Probes were designed using BACs (bacterial artificial chromosomes) from the subtelomeric region of the short (p‐arm) and long (q‐arm) of each porcine chromosome. They were directly labelled with FITC or Texas Red (p‐arm and q‐arm respectively) prior to application of a ‘Multiprobe’ device, thereby enabling simultaneous detection of each individual porcine chromosome on a single slide. Initial experiments designed to isolate BACs in subtelomeric regions led to the discovery of a series of incorrectly mapped regions in the porcine genome assembly (from a total of 82 BACs, only 45 BACs mapped correctly). Our work therefore highlights the importance of accurate physical mapping of newly sequenced genomes. The system herein described allows for robust and comprehensive analysis of the porcine karyotype, an adjunct to classical cytogenetics that provides a valuable tool to expedite efficient, cost effective food production.

Balancing selection at a premature stop mutation in the myostatin gene underlies a recessive leg weakness syndrome in pigs

Balancing selection provides a plausible explanation for the maintenance of deleterious alleles at moderate frequency in livestock, including lethal recessives exhibiting heterozygous advantage in carriers. In the current study, a leg weakness syndrome causing mortality of piglets in a commercial line showed monogenic recessive inheritance, and a region on chromosome 15 associated with the syndrome was identified by homozygosity mapping. Whole genome resequencing of cases and controls identified a mutation coding for a premature stop codon within exon 3 of the porcine Myostatin (MSTN) gene, similar to those causing a double-muscling phenotype observed in several mammalian species. The MSTN mutation was in Hardy-Weinberg equilibrium in the population at birth, but significantly distorted amongst animals still in the herd at 110 kg, due to an absence of homozygous mutant genotypes. In heterozygous form, the MSTN mutation was associated with a major increase in muscle depth and decrease in fat depth, suggesting that the deleterious allele was maintained at moderate frequency due to heterozygous advantage. Knockout of the porcine MSTN by gene editing has previously been linked to problems of low piglet survival and lameness. This MSTN mutation is an example of putative balancing selection in livestock, providing a plausible explanation for the lack of disrupting MSTN mutations in pigs despite many generations of selection for lean growth.

The production of pig preimplantation embryos in vitro: Current progress and future prospects

Human assisted reproductive technology procedures are routinely performed in clinics globally, and some of these approaches are now common in other mammals such as cattle. This is currently not the case in pigs. Given that the global population is expected to increase by over two billion people between now and 2050, the demand for meat will also undoubtedly increase. With this in mind, a more sustainable way to produce livestock; increasing productivity and implementing methods that will lead to faster genetic selection, is imperative. The establishment of routine and production scale pig embryo in vitro production could be a solution to this problem. Producers would be able to increase the overall number of offspring born, animal transportation would be more straightforward and in vitro produced embryos could be produced from the gametes of selected elite. Here we review the most recent developments in pig embryology, outline the current barriers and key challenges that exist, and outline research priorities to surmount these difficulties.

Upgrading Molecular Cytogenetics to Study Reproduction and Reproductive Isolation in Mammals, Birds, and Dinosaurs

amazing diversity of life, including over 2,000 species of vascular plants, exotic mammals such as tapirs, giant anteaters, howler monkeys, ocelots, and jaguars, in addition to hundreds of different bird species and thousands of different insects, the choice of Foz is an excellent analogy for the diverse approaches and systems chromosome biologists explore, and that will be emphasized throughout this conference. The 2016 ICC program offers seven sessions, beginning with a session on Chromosome Structure and Nuclear Architecture, highlighting the influences and interactions chromosomes have on the three-dimensional space of the nucleus. Session II will focus on Specialized Chromosomes, such as sex chromosomes and B chromosomes, whose structure and behavior are often distinguished from that of autosomal chromosomes. Population and Evolutionary Chromosome Biology, the third session, covers a synthesis of chromosome biology and The International Chromosome Conferences (ICC) originated from the Oxford Chromosome Conferences, inaugurated by C.D. Darlington and K.R. Lewis in 1964 and held subsequently in England in 1967 and 1970. The Chromosome Conference grew to an international event with its fourth meeting, held in Jerusalem, Israel in 1972, heralding the beginning of 40 years of technological advances that have expanded our understanding of chromosome biology in model and non-traditional biological systems. Having been hosted in Europe and the United States 16 times since then, this year the ICC will be held across the equator in Foz do Iguaçu, Brazil, on July 10–13, 2016. The event will bring scientists from across the globe to a biannual meeting focused on modern advances in chromosome biology, technology and theory. The Iguaçu National Park, a UNESCO World Heritage Centre, includes the Iguaçu Falls and has been chosen as one of the ‘New Natural Seven Wonders of the World’. Home to an Published online: June 2, 2016I.O. Furo a , R. Kretschmer b , R.J. Gunski c , A.D.V. Garnero c , M.A. Ferguson-Smith d , P.C.M. O ́Brien d , E.H.C. de Oliveira e, f a Programa de Pós-Graduação em Genética e Biologia Molecular, PPGBM, Universidade Federal do Pará, Belém, b PPGBM, Universidade Federal do Rio Grande do Sul, Porto Alegre, and c Programa de Pós-Graduação em Ciências Biológicas, Universidade Federal do Pampa, São Gabriel, Brazil; d Department of Veterinary Medicine, University of Cambridge, Cambridge, UK; e Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Belém, and f Laboratório de Cultura de Tecidos e Citogenética, SAMAM, Instituto Evandro Chagas, Ananindeua, Brazil