Andy Law

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology

Background The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay. Methodology/Principal Findings A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illuminas Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274. Conclusions/Significance Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.

The dynamics of chromosome evolution in birds and mammals.

Comparative mapping, which compares the location of homologous genes in different species, is a powerful tool for studying genome evolution. Comparative maps suggest that rates of chromosomal change in mammals can vary from one to ten rearrangements per million years. On the basis of these rates we would expect 84 to 600 conserved segments in a chicken comparison with human or mouse. Here we build comparative maps between these species and estimate that numbers of conserved segments are in the lower part of this range. We conclude that the organization of the human genome is closer to that of the chicken than the mouse and by adding comparative mapping results from a range of vertebrates, we identify three possible phases of chromosome evolution. The relative stability of genomes such as those of the chicken and human will enable the reconstruction of maps of ancestral vertebrates.

Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary

Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5 u200amm), pre-ovulatory follicles (6.0-7.0 u200amm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.

The chicken gene nomenclature committee report

Comparative genomics is an essential component of the post-genomic era. The chicken genome is the first avian genome to be sequenced and it will serve as a model for other avian species. Moreover, due to its unique evolutionary niche, the chicken genome can be used to understand evolution of functional elements and gene regulation in mammalian species. However comparative biology both within avian species and within amniotes is hampered due to the difficulty of recognising functional orthologs. This problem is compounded as different databases and sequence repositories proliferate and the names they assign to functional elements proliferate along with them. Currently, genes can be published under more than one name and one name sometimes refers to unrelated genes. Standardized gene nomenclature is necessary to facilitate communication between scientists and genomic resources. Moreover, it is important that this nomenclature be based on existing nomenclature efforts where possible to truly facilitate studies between different species. We report here the formation of the Chicken Gene Nomenclature Committee (CGNC), an international and centralized effort to provide standardized nomenclature for chicken genes. The CGNC works in conjunction with public resources such as NCBI and Ensembl and in consultation with existing nomenclature committees for human and mouse. The CGNC will develop standardized nomenclature in consultation with the research community and relies on the support of the research community to ensure that the nomenclature facilitates comparative and genomic studies.

Evaluation of approaches for identifying population informative markers from high density SNP Chips

BackgroundGenetic markers can be used to identify and verify the origin of individuals. Motivation for the inference of ancestry ranges from conservation genetics to forensic analysis. High density assays featuring Single Nucleotide Polymorphism (SNP) markers can be exploited to create a reduced panel containing the most informative markers for these purposes. The objectives of this study were to evaluate methods of marker selection and determine the minimum number of markers from the BovineSNP50 BeadChip required to verify the origin of individuals in European cattle breeds. Delta, Wrights FST, Weir & Cockerhams FST and PCA methods for population differentiation were compared. The level of informativeness of each SNP was estimated from the breed specific allele frequencies. Individual assignment analysis was performed using the ranked informative markers. Stringency levels were applied by log-likelihood ratio to assess the confidence of the assignment test.ResultsA 95% assignment success rate for the 384 individually genotyped animals was achieved with < 80, < 100, < 140 and < 200 SNP markers (with increasing stringency threshold levels) across all the examined methods for marker selection. No further gain in power of assignment was achieved by sampling in excess of 200 SNP markers. The marker selection method that required the lowest number of SNP markers to verify the animals breed origin was Wrights FST (60 to 140 SNPs depending on the chosen degree of confidence). Certain breeds required fewer markers (< 100) to achieve 100% assignment success. In contrast, closely related breeds require more markers (~200) to achieve > 95% assignment success. The power of assignment success, and therefore the number of SNP markers required, is dependent on the levels of genetic heterogeneity and pool of samples considered.ConclusionsWhile all SNP selection methods produced marker panels capable of breed identification, the power of assignment varied markedly among analysis methods. Thus, with effective exploration of available high density genetic markers, a diagnostic panel of highly informative markers can be produced.

Detection of quantitative trait loci for locomotion and osteochondrosis-related traits in Large White ✕ Meishan pigs

Data from the F 2 generation of a Large White (LW) ✕ Meishan (MS) crossbred population were analysed to detect quantitative trait loci (QTL) for leg and gait scores, osteochondrosis and physis scores. Legs, feet and gait score were assessed in 308 F 2 animals at 85 ( + 5) kg and osteochondrosis and physis scores were recorded for the right foreleg after slaughter. A genome scan was performed using 111 genetic markers chosen to span the genome that were genotyped on the F 2 animals and their F 1 parents and purebred grandparents. A QTL on chromosome 1 affecting gait score was significant at the genome-wide significance level. Additional QTL significant at the chromosomewide 5% threshold level (approx. equivalent to the genome-wide suggestive level) were detected on chromosome 1 for front feet and back legs scores, on chromosome 13 for front legs and front feet scores, on chromosome 14 for front legs, front feet and back legs scores and on chromosome 15 for back feet score. None of the QTL for osteochondrosis score exceeded the chromosome-wide suggestive level, but one chromosome-wide QTL for physis score was found on chromosome 7. On chromosome 1, gait and front feet scores mapped to the middle of the chromosome and showed additive effects in favour of the LW alleles and no dominance effects. The QTL for back legs score mapped to the distal end of the chromosome and showed a dominant effect and no additive effect. On chromosomes 14 and 15, the LW allele was again superior to the MS allele. On chromosome 13, there were both additive and dominance effects in favour of the MS allele. The MS alleles on chromosome 13 may have potential for introgression into a commercial LW population. The other putative QTLs identified may have value in marker-assisted selection in LW or MSsynthetic populations.

Decreased expression of the satiety signal receptor CCKAR is responsible for increased growth and body weight during the domestication of chickens

Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.

Enhancing the egg's natural defence against bacterial penetration by increasing cuticle deposition

The cuticle is a proteinaceous layer covering the avian egg and is believed to form a defence to microorganism ingress. In birds that lay eggs in challenging environments, the cuticle is thicker, suggesting evolutionary pressure; however, in poultry, selection pressure for this trait has been removed because of artificial incubation. This study aimed to quantify cuticle deposition and to estimate its genetic parameters and its role on trans-shell penetration of bacteria. Additionally, cuticle proteins were characterised to establish whether alleles for these genes explained variation in deposition. A novel and reliable quantification was achieved using the difference in reflectance of the egg at 650xa0nm before and after staining with a specific dye. The heritability of this novel measurement was moderate (0.27), and bacteria penetration was dependent on the natural variation in cuticle deposition. Eggs with the best cuticle were never penetrated by bacteria (Pxa0<xa00.001). The cuticle proteome consisted of six major proteins. A significant association was found between alleles of one of these protein genes, ovocleidin-116 (MEPE), and cuticle deposition (Pxa0=xa00.015) and also between alleles of estrogen receptor 1 (ESR1) gene and cuticle deposition (Pxa0=xa00.008). With the heritability observed, genetic selection should be possible to increase cuticle deposition in commercial poultry, so reducing trans-generational transmission of microorganisms and reversing the lack of selection pressure for this trait during recent domestication.

Overlap of quantitative trait loci for early growth rate, and for body weight and age at onset of sexual maturity in chickens

Critical age, weight and body composition have been suggested as necessary correlates of sexual maturity. A genome scan to identify quantitative trait loci (QTL) for age and body weight at first egg (AFE and WFE) was conducted on 912 birds from an F(2) broiler-layer cross using 106 microsatellite markers. Without a covariate, QTL for body WFE were detected on chromosomes 2, 4, 8, 27 and Z and a single QTL for AFE was detected on chromosome 2. With AFE as a covariate, additional QTL for body WFE were found on chromosomes 1 and 13, with abdominal fat pad as covariate a QTL for body WFE was found on chromosome 1. With body WFE as covariate, additional QTL for AFE were found on chromosomes 1, 3, 4, 13 and 27. The QTL generally acted additively and there was no evidence for epistasis. Consistent with the original line differences, broiler alleles had positive effects on body WFE and negative effects on AFE, whereas the phenotypic correlation between the two traits was positive. The mapped QTL for body WFE cumulatively accounted for almost half the body weight difference between the chicken lines at puberty. Overlapping QTL for body WFE and body weight to 9 weeks of age indicate that most QTL affecting growth rate also affect body WFE. The co-localisation of QTL for body weight, growth and sexual maturity suggests that body weight and growth rate are closely related to the attainment of sexual maturity and that the genetic determination of growth rate has correlated effects on puberty.

Genomic analysis reveals extensive gene duplication within the bovine TRB locus

BackgroundDiverse TR and IG repertoires are generated by V(D)J somatic recombination. Genomic studies have been pivotal in cataloguing the V, D, J and C genes present in the various TR/IG loci and describing how duplication events have expanded the number of these genes. Such studies have also provided insights into the evolution of these loci and the complex mechanisms that regulate TR/IG expression. In this study we analyze the sequence of the third bovine genome assembly to characterize the germline repertoire of bovine TRB genes and compare the organization, evolution and regulatory structure of the bovine TRB locus with that of humans and mice.ResultsThe TRB locus in the third bovine genome assembly is distributed over 5 scaffolds, extending to ~730 Kb. The available sequence contains 134 TRBV genes, assigned to 24 subgroups, and 3 clusters of DJC genes, each comprising a single TRBD gene, 5–7 TRBJ genes and a single TRBC gene. Seventy-nine of the TRBV genes are predicted to be functional. Comparison with the human and murine TRB loci shows that the gene order, as well as the sequences of non-coding elements that regulate TRB expression, are highly conserved in the bovine. Dot-plot analyses demonstrate that expansion of the genomic TRBV repertoire has occurred via a complex and extensive series of duplications, predominantly involving DNA blocks containing multiple genes. These duplication events have resulted in massive expansion of several TRBV subgroups, most notably TRBV6, 9 and 21 which contain 40, 35 and 16 members respectively. Similarly, duplication has lead to the generation of a third DJC cluster. Analyses of cDNA data confirms the diversity of the TRBV genes and, in addition, identifies a substantial number of TRBV genes, predominantly from the larger subgroups, which are still absent from the genome assembly. The observed gene duplication within the bovine TRB locus has created a repertoire of phylogenetically diverse functional TRBV genes, which is substantially larger than that described for humans and mice.ConclusionThe analyses completed in this study reveal that, although the gene content and organization of the bovine TRB locus are broadly similar to that of humans and mice, multiple duplication events have led to a marked expansion in the number of TRB genes. Similar expansions in other ruminant TR loci suggest strong evolutionary pressures in this lineage have selected for the development of enlarged sets of TR genes that can contribute to diverse TR repertoires.