Restriction site-associated DNA sequencing technologies as an alternative to low-density SNP chips for genomic selection: a simulation study in layer chickens.

BACKGROUND: To reduce the cost of genomic selection, a low-density (LD) single nucleotide polymorphism (SNP) chip can be used in combination with imputation for genotyping selection candidates instead of using a high-density (HD) SNP chip. Next-generation sequencing (NGS) techniques have been increasingly used in livestock species but remain expensive for routine use for genomic selection. An alternative and cost-efficient solution is to use restriction site-associated DNA sequencing (RADseq) techniques to sequence only a fraction of the genome using restriction enzymes. From this perspective, use of RADseq techniques followed by an imputation step on HD chip as alternatives to LD chips for genomic selection was studied in a pure layer line. RESULTS: Genome reduction and sequencing fragments were identified on reference genome using four restriction enzymes (EcoRI, TaqI, AvaII and PstI) and a double-digest RADseq (ddRADseq) method (TaqI-PstI). The SNPs contained in these fragments were detected from the 20X sequence data of the individuals in our population. Imputation accuracy on HD chip with these genotypes was assessed as the mean correlation between true and imputed genotypes. Several production traits were evaluated using single-step GBLUP methodology. The impact of imputation errors on the ranking of the selection candidates was assessed by comparing a genomic evaluation based on ancestry using true HD or imputed HD genotyping. The relative accuracy of genomic estimated breeding values (GEBVs) was investigated by considering the GEBVs estimated on offspring as a reference. With AvaII or PstI and ddRADseq with TaqI and PstI, more than 10 K SNPs were detected in common with the HD SNP chip, resulting in an imputation accuracy greater than 0.97. The impact of imputation errors on genomic evaluation of the breeders was reduced, with a Spearman correlation greater than 0.99. Finally, the relative accuracy of GEBVs was equivalent. CONCLUSIONS: RADseq approaches can be interesting alternatives to low-density SNP chips for genomic selection. With more than 10 K SNPs in common with the SNPs of the HD SNP chip, good imputation and genomic evaluation results can be obtained. However, with real data, heterogeneity between individuals with missing data must be considered.

Nest preference and laying duration traits to select against floor eggs in laying hens.

BACKGROUND: Floor eggs, which are defined as eggs that hens lay off-nest, are a major issue in cage-free layer poultry systems. They create additional work for farmers because they must be collected by hand. They are also usually soiled or broken, which results in economic losses. Nonetheless, knowledge about the genetics of nesting behavior is limited. The aim of this study was to estimate genetic parameters for traits related to nest preference for laying and to time spent in the nests used for laying (laying duration). METHODS: Two pure lines of laying hens were studied: 927 Rhode Island Red and 980 White Leghorn. Electronic nests were used to record the nesting behavior of these hens in floor pens from 24 to 64 weeks of age. Nest preference was studied based on the mean distance between nests used for laying and the percentage of nests used for laying. Laying duration was studied based on mean laying duration, mean duration in the nest before laying, and mean duration in the nest after laying. Genetic parameters were estimated for each line using a restricted maximum-likelihood method applied to a pedigree-based multi-trait animal model. RESULTS: Estimates of genetic parameters were similar for the two lines. Estimates of heritability ranged from 0.18 to 0.37 for nest preference traits and from 0.54 to 0.70 for laying duration traits. Estimates of genetic correlations of these traits with clutch number or mean oviposition time were favorable. Positive genetic correlations were estimated between nest preference and laying rate in the nests or nest acceptance for laying (+ 0.06 to + 0.37). CONCLUSIONS: These results show that genetics influences traits related to nest preference and laying duration. Selecting hens that have no preference for particular nests and spend little time laying in the nests could help optimize nest use, reduce their occupation rate, and thus decrease the incidence of floor eggs in cage-free systems. Genetic correlations of these traits with other traits of interest related to hen welfare and egg quality have yet to be estimated.

Nest acceptance, clutch, and oviposition traits are promising selection criteria to improve egg production in cage-free system.

In cage-free systems, laying hens must lay their eggs in the nests. Selecting layers based on nesting behavior would be a good strategy for improving egg production in these breeding systems. However, little is known about the genetic determinism of nest-related traits. Laying rate in the nests (LRN), clutch number (CN), oviposition traits (OT), and nest acceptance for laying (NAL) of 1,430 Rhode Island Red (RIR) hens and 1,008 White Leghorn (WL) hens were recorded in floor pens provided with individual electronic nests. Heritability and genetic and phenotypic correlations of all traits were estimated over two recording periods-the peak (24-43 weeks of age) and the middle (44-64 weeks of age) of production-by applying the restricted maximum likelihood method to an animal model. The mean oviposition time (MOT) ranged from 2 h 5 min to 3 h and from 3 h 35 min to 3 h 44 min after turning on the lights for RIR and WL hens, respectively. The mean oviposition interval ranged from 24 h 3 min to 24 h 16 min. All heritability and correlation estimates were similar for RIR and WL. Low to moderate heritability coefficients were estimated for LRN (0.04-0.25) and moderate to high heritability coefficients for CN and OT (0.27-0.68). CN and OT were negatively genetically correlated with LRN (-0.92 to -0.39) except during peak production for RIR (-0.30 to +0.43). NAL was weakly to moderately heritable (0.13-0.26). Genetic correlations between NAL and other traits were low to moderate (-0.41 to +0.44). In conclusion, CN and OT are promising selection criteria to improve egg production in cage-free systems. NAL can be also used to reduce the number of eggs laid off-nest in these breeding systems. However, variability in MOT must be maintained to limit competition for the nests.

Interest of using imputation for genomic evaluation in layer chicken.

With the availability of the 600K Affymetrix Axiom high-density (HD) single nucleotide polymorphism (SNP) chip, genomic selection has been implemented in broiler and layer chicken. However, the cost of this SNP chip is too high to genotype all selection candidates. A solution is to develop a low-density SNP chip, at a lower price, and to impute all missing markers. But to routinely implement this solution, the impact of imputation on genomic evaluation accuracy must be studied. It is also interesting to study the consequences of the use of low-density SNP chips in genomic evaluation accuracy. In this perspective, the interest of using imputation in genomic selection was studied in a pure layer line. Two low-density SNP chip designs were compared: an equidistant methodology and a methodology based on linkage disequilibrium. Egg weight, egg shell color, egg shell strength, and albumen height were evaluated with single-step genomic best linear unbiased prediction methodology. The impact of imputation errors or the absence of imputation on the ranking of the male selection candidates was assessed with a genomic evaluation based on ancestry. Thus, genomic estimated breeding values (GEBV) obtained with imputed HD genotypes or low-density genotypes were compared with GEBV obtained with the HD SNP chip. The relative accuracy of GEBV was also investigated by considering as reference GEBV estimated on the offspring. A limited reordering of the breeders, selected on a multitrait index, was observed. Spearman correlations between GEBV on HD genotypes and GEBV on low-density genotypes (with or without imputation) were always higher than 0.94 with more than 3K SNP. For the genetically closer, top 150 individuals for a specific trait, with imputation, the reordering was reduced with correlation higher than 0.94 with more than 3K SNP. Without imputation, the correlations remained lower than 0.85 with less than 3K and 16K SNP for equidistant and linkage disequilibrium methodology, respectively. The differences in GEBV correlations between both methodologies were never significant. The conclusions were the same for all studied traits.

Reliability of genomic evaluation for egg quality traits in layers.

BACKGROUND: Genomic evaluation, based on the use of thousands of genetic markers in addition to pedigree and phenotype information, has become the standard evaluation methodology in dairy cattle breeding programmes over the past several years. Despite the many differences between dairy cattle breeding and poultry breeding, genomic selection seems very promising for the avian sector, and studies are currently being conducted to optimize avian selection schemes. In this optimization perspective, one of the key parameters is to properly predict the accuracy of genomic evaluation in pure line layers. RESULTS: It was observed that genomic evaluation, whether performed on males or females, always proved more accurate than genetic evaluation. The gain was higher when phenotypic information was narrowed, and an augmentation of the size of the reference population led to an increase in accuracy prediction with regard to genomic evaluation. By taking into account the increase of selection intensity and the decrease of the generation interval induced by genomic selection, the expected annual genetic gain would be higher with ancestry-based genomic evaluation of male candidates than with genetic evaluation based on collaterals. This advantage of genomic selection over genetic selection requires more detailed further study for female candidates. CONCLUSIONS: In conclusion, in the population studied, the genomic evaluation of egg quality traits of breeding birds at birth seems to be a promising strategy, at least for the selection of males.

Design of low density SNP chips for genotype imputation in layer chicken.

BACKGROUND: The main goal of selection is to achieve genetic gain for a population by choosing the best breeders among a set of selection candidates. Since 2013, the use of a high density genotyping chip (600K Affymetrix® Axiom® HD genotyping array) for chicken has enabled the implementation of genomic selection in layer and broiler breeding, but the genotyping costs remain high for a routine use on a large number of selection candidates. It has thus been deemed interesting to develop a low density genotyping chip that would induce lower costs. In this perspective, various simulation studies have been conducted to find the best way to select a set of SNPs for low density genotyping of two laying hen lines. RESULTS: To design low density SNP chips, two methodologies, based on equidistance (EQ) or on linkage disequilibrium (LD) were compared. Imputation accuracy was assessed as the mean correlation between true and imputed genotypes. The results showed correlations more sensitive to false imputation of SNPs having low Minor Allele Frequency (MAF) when the EQ methodology was used. An increase in imputation accuracy was obtained when SNP density was increased, either through an increase in the number of selected windows on a chromosome or through the rise of the LD threshold. Moreover, the results varied depending on the type of chromosome (macro or micro-chromosome). The LD methodology enabled to optimize the number of SNPs, by reducing the SNP density on macro-chromosomes and by increasing it on micro-chromosomes. Imputation accuracy also increased when the size of the reference population was increased. Conversely, imputation accuracy decreased when the degree of kinship between reference and candidate populations was reduced. Finally, adding selection candidates' dams in the reference population, in addition to their sire, enabled to get better imputation results. CONCLUSIONS: Whichever the SNP chip, the methodology, and the scenario studied, highly accurate imputations were obtained, with mean correlations higher than 0.83. The key point to achieve good imputation results is to take into account chicken lines' LD when designing a low density SNP chip, and to include the candidates' direct parents in the reference population.

Combined GWAS and LDLA approaches to improve genome-wide quantitative trait loci detection affecting carcass and meat quality traits in pig.

Many QTL affecting meat quality and carcass traits have been reported. However, in most of the cases these QTL have been detected in non-commercial populations. Therefore, a family structured population of 457 F2 pigs issued from an inter-cross between 2 commercial sire lines was used to detect QTL affecting meat quality and carcass traits. All animals were genotyped using the Illumina PorcineSNP60 BeadChip platform. Genome-wide association studies were used in combination with linkage disequilibrium-linkage analysis to identify QTL. A total of 32 QTL were detected. Nine of these QTL exceeded the genome-wide 5% significance threshold. We detected 18 QTL affecting carcass composition traits and 16 QTL affecting meat quality traits. Using post-QTL bioinformatics analysis we highlighted 26 functional candidate genes related to fatness, muscle development, meat color and meat pH. Finally, our results shed light on the advantage of using different QTL detection methodologies to get a global overview of the QTL present in the studied population.

GWAS analyses reveal QTL in egg layers that differ in response to diet differences.

BACKGROUND: The genetic architecture of egg production and egg quality traits, i.e. the quantitative trait loci (QTL) that influence these traits, is still poorly known. To date, 33 studies have focused on the detection of QTL for laying traits in chickens, but less than 10 genes have been identified. The availability of a high-density SNP (single nucleotide polymorphism) chicken array developed by Affymetrix, i.e. the 600K Affymetrix(®) Axiom(®) HD genotyping array offers the possibility to narrow down the localization of previously detected QTL and to detect new QTL. This high-density array is also anticipated to take research beyond the classical hypothesis of additivity of QTL effects or of QTL and environmental effects. The aim of our study was to search for QTL that influence laying traits using the 600K SNP chip and to investigate whether the effects of these QTL differed between diets and age at egg collection. RESULTS: One hundred and thirty-one QTL were detected for 16 laying traits and were spread across all marked chromosomes, except chromosomes 16 and 25. The percentage of variance explained by a QTL varied from 2 to 10 % for the various traits, depending on diet and age at egg collection. Chromosomes 3, 9, 10 and Z were overrepresented, with more than eight QTL on each one. Among the 131 QTL, 60 had a significantly different effect, depending on diet or age at egg collection. For egg production traits, when the QTL × environment interaction was significant, numerous inversions of sign of the SNP effects were observed, whereas for egg quality traits, the QTL × environment interaction was mostly due to a difference of magnitude of the SNP effects. CONCLUSIONS: Our results show that numerous QTL influence egg production and egg quality traits and that the genomic regions, which are involved in shaping the ability of layer chickens to adapt to their environment for egg production, vary depending on the environmental conditions. The next question will be to address what the impact of these genotype × environment interactions is on selection.

The Longissimus and Semimembranosus muscles display marked differences in their gene expression profiles in pig.

BACKGROUND: Meat quality depends on skeletal muscle structure and metabolic properties. While most studies carried on pigs focus on the Longissimus muscle (LM) for fresh meat consumption, Semimembranosus (SM) is also of interest because of its importance for cooked ham production. Even if both muscles are classified as glycolytic muscles, they exhibit dissimilar myofiber composition and metabolic characteristics. The comparison of LM and SM transcriptome profiles undertaken in this study may thus clarify the biological events underlying their phenotypic differences which might influence several meat quality traits. METHODOLOGY/PRINCIPAL FINDINGS: Muscular transcriptome analyses were performed using a custom pig muscle microarray: the 15 K Genmascqchip. A total of 3823 genes were differentially expressed between the two muscles (Benjamini-Hochberg adjusted P value ≤0.05), out of which 1690 and 2133 were overrepresented in LM and SM respectively. The microarray data were validated using the expression level of seven differentially expressed genes quantified by real-time RT-PCR. A set of 1047 differentially expressed genes with a muscle fold change ratio above 1.5 was used for functional characterization. Functional annotation emphasized five main clusters associated to transcriptome muscle differences. These five clusters were related to energy metabolism, cell cycle, gene expression, anatomical structure development and signal transduction/immune response. CONCLUSIONS/SIGNIFICANCE: This study revealed strong transcriptome differences between LM and SM. These results suggest that skeletal muscle discrepancies might arise essentially from different post-natal myogenic activities.

QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi × Leghorn F2 cross, using a medium-density SNP panel.

BACKGROUND: Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F2 cross. The 860 F2 animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied. RESULTS: Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL. CONCLUSIONS: The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between different models and marker densities.

Genome-wide interval mapping using SNPs identifies new QTL for growth, body composition and several physiological variables in an F2 intercross between fat and lean chicken lines.

BACKGROUND: For decades, genetic improvement based on measuring growth and body composition traits has been successfully applied in the production of meat-type chickens. However, this conventional approach is hindered by antagonistic genetic correlations between some traits and the high cost of measuring body composition traits. Marker-assisted selection should overcome these problems by selecting loci that have effects on either one trait only or on more than one trait but with a favorable genetic correlation. In the present study, identification of such loci was done by genotyping an F2 intercross between fat and lean lines divergently selected for abdominal fatness genotyped with a medium-density genetic map (120 microsatellites and 1302 single nucleotide polymorphisms). Genome scan linkage analyses were performed for growth (body weight at 1, 3, 5, and 7 weeks, and shank length and diameter at 9 weeks), body composition at 9 weeks (abdominal fat weight and percentage, breast muscle weight and percentage, and thigh weight and percentage), and for several physiological measurements at 7 weeks in the fasting state, i.e. body temperature and plasma levels of IGF-I, NEFA and glucose. Interval mapping analyses were performed with the QTLMap software, including single-trait analyses with single and multiple QTL on the same chromosome. RESULTS: Sixty-seven QTL were detected, most of which had never been described before. Of these 67 QTL, 47 were detected by single-QTL analyses and 20 by multiple-QTL analyses, which underlines the importance of using different statistical models. Close analysis of the genes located in the defined intervals identified several relevant functional candidates, such as ACACA for abdominal fatness, GHSR and GAS1 for breast muscle weight, DCRX and ASPSCR1 for plasma glucose content, and ChEBP for shank diameter. CONCLUSIONS: The medium-density genetic map enabled us to genotype new regions of the chicken genome (including micro-chromosomes) that influenced the traits investigated. With this marker density, confidence intervals were sufficiently small (14 cM on average) to search for candidate genes. Altogether, this new information provides a valuable starting point for the identification of causative genes responsible for important QTL controlling growth, body composition and metabolic traits in the broiler chicken.

Graphics processing unit-accelerated quantitative trait Loci detection.

Mapping quantitative trait loci (QTL) using genetic marker information is a time-consuming analysis that has interested the mapping community in recent decades. The increasing amount of genetic marker data allows one to consider ever more precise QTL analyses while increasing the demand for computation. Part of the difficulty of detecting QTLs resides in finding appropriate critical values or threshold values, above which a QTL effect is considered significant. Different approaches exist to determine these thresholds, using either empirical methods or algebraic approximations. In this article, we present a new implementation of existing software, QTLMap, which takes advantage of the data parallel nature of the problem by offsetting heavy computations to a graphics processing unit (GPU). Developments on the GPU were implemented using Cuda technology. This new implementation performs up to 75 times faster than the previous multicore implementation, while maintaining the same results and level of precision (Double Precision) and computing both QTL values and thresholds. This speedup allows one to perform more complex analyses, such as linkage disequilibrium linkage analyses (LDLA) and multiQTL analyses, in a reasonable time frame.

XVth QTLMAS: simulated dataset.

BACKGROUND: Our aim was to simulate the data for the QTLMAS2011 workshop following a pig-type family structure under an oligogenic model, each QTL being specific. RESULTS: The population comprised 3000 individuals issued from 20 sires and 200 dams. Within each family, 10 progenies belonged to the experimental population and were assigned phenotypes and marker genotypes and 5 belonged to the selection population, only known on their marker genotypes. A total of 10,000 SNPs carried by 5 chromosomes of 1 Morgan each were simulated. Eight QTL were created (1 quadri-allelic, 2 linked in phase, 2 linked in repulsion, 1 imprinted and 2 epistatic). Random noise was added giving an heritability of 0.30. The marker density, LD and MAF were similar to real life parameters.

Comparison of the analyses of the XVth QTLMAS common dataset II: QTL analysis.

BACKGROUND: The QTLMAS XVth dataset consisted of the pedigrees, marker genotypes and quantitative trait performances of 2,000 phenotyped animals with a half-sib family structure. The trait was regulated by 8 QTL which display additive, imprinting or epistatic effects. This paper aims at comparing the QTL mapping results obtained by six participants of the workshop. METHODS: Different regression, GBLUP, LASSO and Bayesian methods were applied for QTL detection. The results of these methods are compared based on the number of correctly mapped QTL, the number of false positives, the accuracy of the QTL location and the estimation of the QTL effect. RESULTS: All the simulated QTL, except the interacting QTL on Chr5, were identified by the participants. Depending on the method, 3 to 7 out of the 8 QTL were identified. The distance to the real location and the accuracy of the QTL effect varied to a large extent depending on the methods and complexity of the simulated QTL. CONCLUSIONS: While all methods were fairly efficient in detecting QTL with additive effects, it was clear that for non-additive situations, such as parent-of-origin effects or interactions, the BayesC method gave the best results by detecting 7 out of the 8 simulated QTL, with only two false positives and a good precision (less than 1 cM away on average). Indeed, if LASSO could detect QTL even in complex situations, it was associated with too many false positive results to allow for efficient GWAS. GENMIX, a method based on the phylogenies of local haplotypes, also appeared as a promising approach, which however showed a few more false positives when compared with the BayesC method.

Comparison of analyses of the XVth QTLMAS common dataset III: Genomic Estimations of Breeding Values.

BACKGROUND: The QTLMAS XVth dataset consisted of pedigree, marker genotypes and quantitative trait performances of animals with a sib family structure. Pedigree and genotypes concerned 3,000 progenies among those 2,000 were phenotyped. The trait was regulated by 8 QTLs which displayed additive, imprinting or epistatic effects. The 1,000 unphenotyped progenies were considered as candidates to selection and their Genomic Estimated Breeding Values (GEBV) were evaluated by participants of the XVth QTLMAS workshop. This paper aims at comparing the GEBV estimation results obtained by seven participants to the workshop. METHODS: From the known QTL genotypes of each candidate, two "true" genomic values (TV) were estimated by organizers: the genotypic value of the candidate (TGV) and the expectation of its progeny genotypic values (TBV). GEBV were computed by the participants following different statistical methods: random linear models (including BLUP and Ridge Regression), selection variable techniques (LASSO, Elastic Net) and Bayesian methods. Accuracy was evaluated by the correlation between TV (TGV or TBV) and GEBV presented by participants. Rank correlation of the best 10% of individuals and error in predictions were also evaluated. Bias was tested by regression of TV on GEBV. RESULTS: Large differences between methods were found for all criteria and type of genetic values (TGV, TBV). In general, the criteria ranked consistently methods belonging to the same family. CONCLUSIONS: Bayesian methods - A

Statistical properties of interval mapping methods on quantitative trait loci location: impact on QTL/eQTL analyses.

BACKGROUND: Quantitative trait loci (QTL) detection on a huge amount of phenotypes, like eQTL detection on transcriptomic data, can be dramatically impaired by the statistical properties of interval mapping methods. One of these major outcomes is the high number of QTL detected at marker locations. The present study aims at identifying and specifying the sources of this bias, in particular in the case of analysis of data issued from outbred populations. Analytical developments were carried out in a backcross situation in order to specify the bias and to propose an algorithm to control it. The outbred population context was studied through simulated data sets in a wide range of situations.The likelihood ratio test was firstly analyzed under the "one QTL" hypothesis in a backcross population. Designs of sib families were then simulated and analyzed using the QTL Map software. On the basis of the theoretical results in backcross, parameters such as the population size, the density of the genetic map, the QTL effect and the true location of the QTL, were taken into account under the "no QTL" and the "one QTL" hypotheses. A combination of two non parametric tests - the Kolmogorov-Smirnov test and the Mann-Whitney-Wilcoxon test - was used in order to identify the parameters that affected the bias and to specify how much they influenced the estimation of QTL location. RESULTS: A theoretical expression of the bias of the estimated QTL location was obtained for a backcross type population. We demonstrated a common source of bias under the "no QTL" and the "one QTL" hypotheses and qualified the possible influence of several parameters. Simulation studies confirmed that the bias exists in outbred populations under both the hypotheses of "no QTL" and "one QTL" on a linkage group. The QTL location was systematically closer to marker locations than expected, particularly in the case of low QTL effect, small population size or low density of markers, i.e. designs with low power. Practical recommendations for experimental designs for QTL detection in outbred populations are given on the basis of this bias quantification. Furthermore, an original algorithm is proposed to adjust the location of a QTL, obtained with interval mapping, which co located with a marker. CONCLUSIONS: Therefore, one should be attentive when one QTL is mapped at the location of one marker, especially under low power conditions.

Genetic variability of transcript abundance in pig peri-mortem skeletal muscle: eQTL localized genes involved in stress response, cell death, muscle disorders and metabolism.

BACKGROUND: The genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering. RESULTS: QTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative cis-drivers of gene expression programs in skeletal muscle as COQ4 (SSC1), LOC100513192 (SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests cis-causing genetic polymorphims affecting gene expression levels, with (e.g. COQ4) or without (e.g. LOC100513192) potential pleiotropic effects that affect the expression of other genes (cluster of trans-eQTLs). CONCLUSION: Genetic analysis of transcription levels revealed dependence among molecular phenotypes as being affected by variation at the same loci. We observed the genetic variation of molecular phenotypes in a specific situation of cellular stress thus contributing to a better description of muscle physiologic response. In turn, this suggests that large amounts of genetic variation, mediated through transcriptional networks, can drive transient cell response phenotypes and contribute to organismal adaptative potential.

Complex trait subtypes identification using transcriptome profiling reveals an interaction between two QTL affecting adiposity in chicken.

BACKGROUND: Integrative genomics approaches that combine genotyping and transcriptome profiling in segregating populations have been developed to dissect complex traits. The most common approach is to identify genes whose eQTL colocalize with QTL of interest, providing new functional hypothesis about the causative mutation. Another approach includes defining subtypes for a complex trait using transcriptome profiles and then performing QTL mapping using some of these subtypes. This approach can refine some QTL and reveal new ones.In this paper we introduce Factor Analysis for Multiple Testing (FAMT) to define subtypes more accurately and reveal interaction between QTL affecting the same trait. The data used concern hepatic transcriptome profiles for 45 half sib male chicken of a sire known to be heterozygous for a QTL affecting abdominal fatness (AF) on chromosome 5 distal region around 168 cM. RESULTS: Using this methodology which accounts for hidden dependence structure among phenotypes, we identified 688 genes that are significantly correlated to the AF trait and we distinguished 5 subtypes for AF trait, which are not observed with gene lists obtained by classical approaches. After exclusion of one of the two lean bird subtypes, linkage analysis revealed a previously undetected QTL on chromosome 5 around 100 cM. Interestingly, the animals of this subtype presented the same q paternal haplotype at the 168 cM QTL. This result strongly suggests that the two QTL are in interaction. In other words, the "q configuration" at the 168 cM QTL could hide the QTL existence in the proximal region at 100 cM. We further show that the proximal QTL interacts with the previous one detected on the chromosome 5 distal region. CONCLUSION: Our results demonstrate that stratifying genetic population by molecular phenotypes followed by QTL analysis on various subtypes can lead to identification of novel and interacting QTL.

Joint analysis of quantitative trait loci and major-effect causative mutations affecting meat quality and carcass composition traits in pigs.

BACKGROUND: Detection of quantitative trait loci (QTLs) affecting meat quality traits in pigs is crucial for the design of efficient marker-assisted selection programs and to initiate efforts toward the identification of underlying polymorphisms. The RYR1 and PRKAG3 causative mutations, originally identified from major effects on meat characteristics, can be used both as controls for an overall QTL detection strategy for diversely affected traits and as a scale for detected QTL effects. We report on a microsatellite-based QTL detection scan including all autosomes for pig meat quality and carcass composition traits in an F2 population of 1,000 females and barrows resulting from an intercross between a Pietrain and a Large White-Hampshire-Duroc synthetic sire line. Our QTL detection design allowed side-by-side comparison of the RYR1 and PRKAG3 mutation effects seen as QTLs when segregating at low frequencies (0.03-0.08), with independent QTL effects detected from most of the same population, excluding any carrier of these mutations. RESULTS: Large QTL effects were detected in the absence of the RYR1 and PRKGA3 mutations, accounting for 12.7% of phenotypic variation in loin colour redness CIE-a* on SSC6 and 15% of phenotypic variation in glycolytic potential on SSC1. We detected 8 significant QTLs with effects on meat quality traits and 20 significant QTLs for carcass composition and growth traits under these conditions. In control analyses including mutation carriers, RYR1 and PRKAG3 mutations were detected as QTLs, from highly significant to suggestive, and explained 53% to 5% of the phenotypic variance according to the trait. CONCLUSIONS: Our results suggest that part of muscle development and backfat thickness effects commonly attributed to the RYR1 mutation may be a consequence of linkage with independent QTLs affecting those traits. The proportion of variation explained by the most significant QTLs detected in this work is close to the influence of major-effect mutations on the least affected traits, but is one order of magnitude lower than effect on variance of traits primarily affected by these causative mutations. This suggests that uncovering physiological traits directly affected by genetic polymorphisms would be an appropriate approach for further characterization of QTLs.

Detection of QTL with effects on osmoregulation capacities in the rainbow trout (Oncorhynchus mykiss).

BACKGROUND: There is increasing evidence that the ability to adapt to seawater in teleost fish is modulated by genetic factors. Most studies have involved the comparison of species or strains and little is known about the genetic architecture of the trait. To address this question, we searched for QTL affecting osmoregulation capacities after transfer to saline water in a nonmigratory captive-bred population of rainbow trout. RESULTS: A QTL design (5 full-sib families, about 200 F2 progeny each) was produced from a cross between F0 grand-parents previously selected during two generations for a high or a low cortisol response after a standardized confinement stress. When fish were about 18 months old (near 204 g body weight), individual progeny were submitted to two successive hyper-osmotic challenges (30 ppt salinity) 14 days apart. Plasma chloride and sodium concentrations were recorded 24 h after each transfer. After the second challenge, fish were sacrificed and a gill index (weight of total gill arches corrected for body weight) was recorded. The genome scan was performed with 196 microsatellites and 85 SNP markers. Unitrait and multiple-trait QTL analyses were carried out on the whole dataset (5 families) through interval mapping methods with the QTLMap software. For post-challenge plasma ion concentrations, significant QTL (P < 0.05) were found on six different linkage groups and highly suggestive ones (P < 0.10) on two additional linkage groups. Most QTL affected concentrations of both chloride and sodium during both challenges, but some were specific to either chloride (2 QTL) or sodium (1 QTL) concentrations. Six QTL (4 significant, 2 suggestive) affecting gill index were discovered. Two were specific to the trait, while the others were also identified as QTL for post-challenge ion concentrations. Altogether, allelic effects were consistent for QTL affecting chloride and sodium concentrations but inconsistent for QTL affecting ion concentrations and gill morphology. There was no systematic lineage effect (grand-parental origin of QTL alleles) on the recorded traits. CONCLUSIONS: For the first time, genomic loci associated with effects on major physiological components of osmotic adaptation to seawater in a nonmigratory fish were revealed. The results pave the way for further deciphering of the complex regulatory mechanisms underlying seawater adaptation and genes involved in osmoregulatory physiology in rainbow trout and other euryhaline fishes.