Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

Associations between prefrontal γ-aminobutyric acid concentration and the tryptophan hydroxylase isoform 2 gene, a panic disorder risk allele in women

Associations between the central serotonergic and γ-aminobutyric acid (GABA) systems play key roles in the prefrontal cortical regulation of emotion and cognition and in the pathophysiology and pharmacotherapy of highly prevalent psychiatric disorders. The goal of this study was to test the effects of common variants of the tryptophan hydroxylase isoform 2 (TPH2) gene on GABA concentration in the prefrontal cortex (PFC) using magnetic resonance spectroscopy. In this study involving 64 individuals, we examined the associations between prefrontal cortical GABA concentration and 12 single nucleotide polymorphisms (SNPs) spanning the TPH2 gene, including rs4570625 (−703 G/T SNP), a potentially functional TPH2 polymorphism that has been associated with decreased TPH2 mRNA expression and panic disorder. Our results revealed a significant association between increased GABA concentration in the PFC and the T-allele frequencies of two TPH2 SNPs, namely rs4570625 (−703 G/T) and rs2129575 (p≤0.0004) and the C-allele frequency of one TPH2 SNP, namely rs1386491 (p = 0.0003) in female subjects. We concluded that rs4570625 (−703 G/T), rs2129575 and rs1386491 play a significant role in GABAergic neurotransmission and may contribute to the sex-specific dysfunction of the GABAergic system in the PFC.

Origins and evolution of VNTR loci: The apolipoprotein B 3$\sp\prime$ VNTR

I studied the apolipoprotein (apo) B 3$\sp\prime$ variable number tandem repeat (VNTR) and did computer simulations of the stepwise mutation model to address four questions: (1) How did the apo B VNTR originate? (2) What is the mutational mechanism of repeat number change at the apo B VNTR? (3) To what extent are population and molecular level events responsible for the determination of the contemporary apo B allele frequency distribution? (4) Can VNTR allele frequency distributions be explained by a simple and conservative mutation-drift model? I used three general approaches to address these questions: (1) I characterized the apo B VNTR region in non-human primate species; (2) I constructed haplotypes of polymorphic markers flanking the apo B VNTR in a sample of individuals from Lorrain, France and studied the associations between the flanking-marker haplotypes and apo B VNTR size; (3) I did computer simulations of the one-step stepwise mutation model and compared the results to real data in terms of four allele frequency distribution characteristics.^ The results of this work have allowed me to conclude that the apo B VNTR originated after an initial duplication of a sequence which is still present as a single copy sequence in New World monkey species. I conclude that this locus did not originate by the transposition of an array of repeats from somewhere else in the genome. It is unlikely that recombination is the primary mutational mechanism. Furthermore, the clustered nature of these associations implicates a stepwise mutational mechanism. From the high frequencies of certain haplotype-allele size combinations, it is evident that population level events have also been important in the determination of the apo B VNTR allele frequency distribution. Results from computer simulations of the one-step stepwise mutation model have allowed me to conclude that bimodal and multimodal allele frequency distributions are not unexpected at loci evolving via stepwise mutation mechanisms. Short tandem repeat loci fit the stepwise mutation model best, followed by microsatellite loci. I therefore conclude that there are differences in the mutational mechanisms of VNTR loci as classed by repeat unit size. (Abstract shortened by UMI.) ^

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^

Population genetics of VNTR loci and their applications in evolutionary studies

Variable number of tandem repeats (VNTR) are genetic loci at which short sequence motifs are found repeated different numbers of times among chromosomes. To explore the potential utility of VNTR loci in evolutionary studies, I have conducted a series of studies to address the following questions: (1) What are the population genetic properties of these loci? (2) What are the mutational mechanisms of repeat number change at these loci? (3) Can DNA profiles be used to measure the relatedness between a pair of individuals? (4) Can DNA fingerprint be used to measure the relatedness between populations in evolutionary studies? (5) Can microsatellite and short tandem repeat (STR) loci which mutate stepwisely be used in evolutionary analyses?^ A large number of VNTR loci typed in many populations were studied by means of statistical methods developed recently. The results of this work indicate that there is no significant departure from Hardy-Weinberg expectation (HWE) at VNTR loci in most of the human populations examined, and the departure from HWE in some VNTR loci are not solely caused by the presence of population sub-structure.^ A statistical procedure is developed to investigate the mutational mechanisms of VNTR loci by studying the allele frequency distributions of these loci. Comparisons of frequency distribution data on several hundreds VNTR loci with the predictions of two mutation models demonstrated that there are differences among VNTR loci grouped by repeat unit sizes.^ By extending the ITO method, I derived the distribution of the number of shared bands between individuals with any kinship relationship. A maximum likelihood estimation procedure is proposed to estimate the relatedness between individuals from the observed number of shared bands between them.^ It was believed that classical measures of genetic distance are not applicable to analysis of DNA fingerprints which reveal many minisatellite loci simultaneously in the genome, because the information regarding underlying alleles and loci is not available. I proposed a new measure of genetic distance based on band sharing between individuals that is applicable to DNA fingerprint data.^ To address the concern that microsatellite and STR loci may not be useful for evolutionary studies because of the convergent nature of their mutation mechanisms, by a theoretical study as well as by computer simulation, I conclude that the possible bias caused by the convergent mutations can be corrected, and a novel measure of genetic distance that makes the correction is suggested. In summary, I conclude that hypervariable VNTR loci are useful in evolutionary studies of closely related populations or species, especially in the study of human evolution and the history of geographic dispersal of Homo sapiens. (Abstract shortened by UMI.) ^

Bayesian models for the analysis of genetic structure when populations are correlated

Motivation: Population allele frequencies are correlated when populations have a shared history or when they exchange genes. Unfortunately, most models for allele frequency and inference about population structure ignore this correlation. Recent analytical results show that among populations, correlations can be very high, which could affect estimates of population genetic structure. In this study, we propose a mixture beta model to characterize the allele frequency distribution among populations. This formulation incorporates the correlation among populations as well as extending the model to data with different clusters of populations. Results: Using simulated data, we show that in general, the mixture model provides a good approximation of the among-population allele frequency distribution and a good estimate of correlation among populations. Results from fitting the mixture model to a dataset of genotypes at 377 autosomal microsatellite loci from human populations indicate high correlation among populations, which may not be appropriate to neglect. Traditional measures of population structure tend to over-estimate the amount of genetic differentiation when correlation is neglected. Inference is performed in a Bayesian framework.

Genetic variability and the hypothalamic control of energy balance

Background. Obesity is a major health problem throughout the industrialized world. Despite numerous attempts to curtail the rapid growth of obesity, its incidence continues to rise. Therefore, it is crucial to better understand the etiology of obesity beyond the concept of energy balance.^ Aims. The first aim of this study was to first investigate the relationship between eating behaviors and body size. The second goal was to identify genetic variation associated with eating behaviors. Thirdly, this study aimed to examine the joint relationships between eating behavior, body size and genetic variation.^ Methods. This study utilized baseline data ascertained in young adults from the Training Interventions and Genetics of Exercise (TIGER) Study. Variables assessed included eating behavior (Emotional Eating Scale, Eating Attitudes Test-26, and the Block98 Food Frequency Questionnaire), body size (body mass index, waist and hip circumference, waist/hip ratio, and percent body fat), genetic variation in genes implicated related to the hypothalamic control of energy balance, and appropriate covariates (age, gender, race/ethnicity, smoking status, and physical activity. For the genetic association analyses, genotypes were collapsed by minor allele frequency, and haplotypes were estimated for each gene. Additionally, Bayesian networks were constructed in order to determine the relationships between genetic variation, eating behavior and body size.^ Results. We report that the EAT-26 score, Caloric intake, percent fat, fiber intake, HEAT index, and daily servings of vegetables, meats, grains, and fats were significantly associated with at least one body size measure. Multiple SNPs in 17 genes and haplotypes from 12 genes were tested for their association with body size. Variation within both DRD4 and HTR2A was found to be associated with EAT-26 score. In addition, variation in the ghrelin gene (GHRL) was significantly associated with daily Caloric intake. A significant interaction between daily servings of grains and the HEAT index and variation within the leptin receptor gene (LEPR) was shown to influence body size.^ Conclusion. This study has shown that there is a substantial genetic component to eating behavior and that genetic variation interacts with eating behavior to influence body size.^

Multi-year high-frequency current and water quality in-situ observations at the Guadiana Estuary

High-frequency data collected continuously over a multiyear time frame are required for investigating the various agents that drive ecological and hydrodynamic processes in estuaries. Here, we present water quality and current in-situ observations from a fixed monitoring station operating from 2008 to 2014 in the lower Guadiana Estuary, southern Portugal (37°11.30' N, 7°24.67' W). The data were recorded by a multi-parametric probe providing hourly records (temperature, salinity, chlorophyll, dissolved oxygen, turbidity, and pH) at a water depth of ~1 m, and by a bottom-mounted acoustic Doppler current profiler measuring the pressure, near-bottom temperature, and flow velocity through the water column every 15 min. The time-series data, in particular the probe ones, present substantial gaps arising from equipment failure and maintenance, which are ineluctable with this type of observations in harsh environments. However, prolonged (months-long) periods of multi-parametric observations during contrasted external forcing conditions are available. The raw data are reported together with flags indicating the quality status of each record. River discharge data from two hydrographic stations located near the estuary head are also provided to support data analysis and interpretation.

Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm

Strategies for delaying pest resistance to genetically modified crops that produce Bacillus thuringiensis (Bt) toxins are based primarily on theoretical models. One key assumption of such models is that genes conferring resistance are rare. Previous estimates for lepidopteran pests targeted by Bt crops seem to meet this assumption. We report here that the estimated frequency of a recessive allele conferring resistance to Bt toxin Cry1Ac was 0.16 (95% confidence interval = 0.05–0.26) in strains of pink bollworm (Pectinophora gossypiella) derived from 10 Arizona cotton fields during 1997. Unexpectedly, the estimated resistance allele frequency did not increase from 1997 to 1999 and Bt cotton remained extremely effective against pink bollworm. These results demonstrate that the assumptions and predictions of resistance management models must be reexamined.

A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history.

A polymorphic C-->T transition located on the human Y chromosome was found by the systematic comparative sequencing of Y-specific sequence-tagged sites by denaturing high-performance liquid chromatography. The results of genotyping representative global indigenous populations indicate that the locus is polymorphic exclusively within the Western Hemisphere. The pre-Columbian T allele occurs at > 90% frequency within the native South and Central American populations examined, while its occurrence in North America is approximately 50%. Concomitant genotyping at the polymorphic tetranucleotide microsatellite DYS19 locus revealed that the C-->T mutation displayed significant linkage disequilibrium with the 186-bp allele. The data suggest a single origin of linguistically diverse native Americans with subsequent haplotype differentiation within radiating indigenous populations as well as post-Columbian European and African gene flow. The mutation may have originated either in North America at a very early time during the expansion or before it, in the ancestral population(s) from which all Americans may have originated. The analysis of linkage of the DYS199 and the DYS19 tetranucleotide loci suggests that the C-->T mutation may have occurred around 30,000 years ago. We estimate the nucleotide diversity over 4.2 kb of the nonrecombining portion of the Y chromosome to be 0.00014. compared to autosomes, the majority of variation is due to the smaller effective population size of the Y chromosome rather than selective sweeps. There begins to emerge a pattern of pronounced geographical localization of Y-specific nucleotide substitution polymorphisms.

Do harvest refuges buffer kangaroos against evolutionary responses to selective harvesting?

There is a wealth of literature documenting a directional change of body size in heavily harvested populations. Most of this work concentrates on aquatic systems, but terrestrial populations are equally at risk. This paper explores the capacity of harvest refuges to counteract potential effects of size-selective harvesting on the allele frequency,of populations. We constructed a stochastic, individual-based model parameterized with data on red kangaroos. Because we do not know which part of individual growth would change in the course of natural selection, we explored the effects of two alternative models of individual growth in which alleles affect either the growth rate or the maximum size. The model results show that size-selective harvesting can result in significantly smaller kangaroos for a given age when the entire population is subject to harvesting. In contrast, in scenarios that include dispersal from harvest refuges, the initial allele frequency remains virtually unchanged.

Derivation of lake mixing and stratification indices from high-resolution lake buoy data

Lake Analyzer is a numerical code coupled with supporting visualization tools for determining indices of mixing and stratification that are critical to the biogeochemical cycles of lakes and reservoirs. Stability indices, including Lake Number, Wedderburn Number, Schmidt Stability, and thermocline depth are calculated according to established literature definitions and returned to the user in a time series format. The program was created for the analysis of high-frequency data collected from instrumented lake buoys, in support of the emerging field of aquatic sensor network science. Available outputs for the Lake Analyzer program are: water temperature (error-checked and/or down-sampled), wind speed (error-checked and/or down-sampled), metalimnion extent (top and bottom), thermocline depth, friction velocity, Lake Number, Wedderburn Number, Schmidt Stability, mode-1 vertical seiche period, and Brunt-Väisälä buoyancy frequency. Secondary outputs for several of these indices delineate the parent thermocline depth (seasonal thermocline) from the shallower secondary or diurnal thermocline. Lake Analyzer provides a program suite and best practices for the comparison of mixing and stratification indices in lakes across gradients of climate, hydro-physiography, and time, and enables a more detailed understanding of the resulting biogeochemical transformations at different spatial and temporal scales.

Low-frequency and common genetic variation in ischemic stroke: The METASTROKE collaboration

Erratum in: Low-frequency and common genetic variation in ischemic stroke: The METASTROKE collaboration. [Neurology. 2016]

Predictive ability of Genomic Estimated Family Values (GEFV).

Genomic selection (GS) has been used to compute genomic estimated breeding values (GEBV) of individuals; however, it has only been applied to animal and major plant crops due to high costs. Besides, breeding and selection is performed at the family level in some crops. We aimed to study the implementation of genome-wide family selection (GWFS) in two loblolly pine (Pinus taeda L.) populations: i) the breeding population CCLONES composed of 63 families (5-20 individuals per family), phenotyped for four traits (stem diameter, stem rust susceptibility, tree stiffness and lignin content) and genotyped using an Illumina Infinium assay with 4740 polymorphic SNPs, and ii) a simulated population that reproduced the same pedigree as CCLONES, 5000 polymorphic loci and two traits (oligogenic and polygenic). In both populations, phenotypic and genotypic data was pooled at the family level in silico. Phenotypes were averaged across replicates for all the individuals and allele frequency was computed for each SNP. Marker effects were estimated at the individual (GEBV) and family (GEFV) levels with Bayes-B using the package BGLR in R and models were validated using 10-fold cross validations. Predicted ability, computed by correlating phenotypes with GEBV and GEFV, was always higher for GEFV in both populations, even after standardizing GEFV predictions to be comparable to GEBV. Results revealed great potential for using GWFS in breeding programs that select families, such as most outbreeding forage species. A significant drop in genotyping costs as one sample per family is needed would allow the application of GWFS in minor crops.

Predictions of angle dependent tortuosity and elasticity effects on sound propagation in cancellous bone

The anisotropic pore structure and elasticity of cancellous bone cause wave speeds and attenuation in cancellous bone to vary with angle. Previously published predictions of the variation in wave speed with angle are reviewed. Predictions that allow tortuosity to be angle dependent but assume isotropic elasticity compare well with available data on wave speeds at large angles but less well for small angles near the normal to the trabeculae. Claims for predictions that only include angle-dependence in elasticity are found to be misleading. Audio-frequency data obtained at audio-frequencies in air-filled bone replicas are used to derive an empirical expression for the angle-and porosity-dependence of tortuosity. Predictions that allow for either angle dependent tortuosity or angle dependent elasticity or both are compared with existing data for all angles and porosities.