Keywords: gene expression, indirect selection, loin meat quality, longissimus muscle, Sus scrofa, transcriptome analysis

ABSTRACT: A family structured population of 325 pigs (females and barrows) was produced as an intercross between 2 commercial sire lines and was subjected to a systematic transcriptome analysis of LM samples obtained shortly after slaughter. Additionally, measurements of meat quality traits of fresh and cooked loin were gathered from the same animals. The transcriptome analysis was achieved by microarray hybridization, using a custom repertoire of 15,000 6mer DNA probes targeting transcripts expressed in growing pig skeletal muscle. These data allowed us to estimate the heritability of expression abundance for each of the quantified RNA species. The abundance of 9,765 RNA was estimated as heritable with a false discovery rate of 5%, from which 1,174 were deemed as highly heritable (h2 > 0.50). We also observed a large number of transcripts whose LM expression abundance is genetically correlated with 4 meat quality traits: the loin pH measured at 45 min postmortem (pH45), 253 transcripts; the loin cooking loss (CL), 134 transcripts; the cooked loin shear force (SFc), 184 transcripts; and the loin color redness (a*) value, 190 transcripts. Heritable and meat quality genetically correlated transcripts showed an over-representation of biological processes involved in the induction of apoptosis (genetically correlated with CL), complement activation (genetically correlated with SFc), glucose metabolism (genetically correlated with a*), and cation channel activity (genetically correlated with pH45). Overall, the biological functions highlighted in the highly heritable transcripts and the lack of transcript that would be genetically correlated with LM glycolytic potential suggest that the genetic variability of the LM postmortem transcriptome is focused on muscle tissue response to postmortem ischemia and reflects more distantly the antemortem muscle physiology. Because of the contrasting distributions of the genetic correlations between LM RNA concentrations and the different meat quality traits studied, indirect selection strategies of meat quality traits based on measurements of selected LM RNA species could be only proposed for a subset of the analyzed meat characteristics (pH45, SFc, a*, CL). A substantial improvement in the efficiency of selection for these meat quality traits could result from measuring muscle RNA concentrations on selection candidates, if the same genetic parameters can be verified using in vivo-sampled muscles.

INTRODUCTION

Quantification of each of the RNA species found in animal cells or transcriptome analysis developed as a follow-up of genome sequencing and array technology. Transcriptome analysis initially allowed an in-depth biological description of cells or tissues that were exposed to well-defined treatments, using differential expression experiments with a limited number of samples. Further improvements in technology standardization and throughput now allow for a large number of samples to be reliably processed. Population-scale transcriptome analysis can, therefore, be used to assess the full extent of the variation in transcript concentrations in a given cell type or tissue among individuals in a population.

Variations in transcript abundance can drive or accompany variations in livestock production phenotypes, as mediating responses to environmental challenges or the translation of genetic variability. Meat qualitative traits are contributed by the physiology of corresponding skeletal muscles, from development [START_REF] Wimmers | Associations of functional candidate genes derived from gene-expression files of prenatal porcine muscle tissue with meat quality and muscle deposition[END_REF] until conversion from muscle to meat [START_REF] Davoli | Molecular approaches in pig breeding to improve meat quality[END_REF][START_REF] Ponsuksili | Expression profiling of muscle reveals transcripts differentially expressed in muscle that affect water-holding capacity of pork[END_REF]. Transcriptome analysis of skeletal muscle thus has the potential to uncover variations in the abundance of selected transcripts, which could be seen and used as intermediate outcomes of the genetic variation that contributes to meat quality traits. Indirect selection strategies based on these intermediate molecular phenotypes might prove valuable for the genetic improvement of meat quality traits. Opportunities for using those molecular phenotypes in breeding applications ultimately depends on the genetic parameters for these traits, defined as skeletal muscle RNA concentrations, heritabilities (h 2 ), and genetic correlations (r G ) with meat quality traits of economic value. Here, we report a population-scale analysis of transcript abundance in pig skeletal muscle, which enabled the estimation of genetic parameters for these molecular phenotypes.

MATERIALS AND METHODS

Procedures and slaughtering facilities were approved by the French Veterinary Services.

Experimental Population

The 325 pigs (females and barrows) used in the gene expression analyses are part of a larger F 2 resource population of 1,000 animals which was setup within the framework of a QTL detection program. These animals were produced as a second generation intercross between 2 commercial sire lines (FH016, Pietrain type line, and FH019, synthetic line from Hampshire, Duroc, and Large-White founders, France-Hybrides, St Jean de Braye, France).

The 325 animals considered for LM transcriptome analysis were produced as 4 half-sib families, using 4 F 1 males and 15 F 1 females, where each F 1 male was mated with the same group of 3 to 4 full-sib F 1 females for 2 to 3 successive litters, thus purposely generating largesize full-sib families. All F 2 animals were genotyped as homozygous wild type genotypes NN and rn+rn+ with regard to the HAL and RN loci, respectively [START_REF] Otsu | Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia[END_REF][START_REF] Milan | A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle[END_REF].

All F 2 animals were raised on the same farm and slaughtered in the same abattoir. All pigs were killed at 110 kg of BW according to standard procedures for commercial slaughtering (Orleans Viandes, Fleury-les-Aubrais, France). Half-carcasses were chilled at -12°C for 4 h postmortem, then stored at 3°C, and cut at 24 h postmortem.

Meat Quality Measurements

Loin meat quality measurements are briefly described because they have been already detailed previously [START_REF] Laville | Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat[END_REF]. The summary statistics of the meat quality traits as recorded in the whole resource population are provided in Table 1.

Loin Processing and Measurements. The loins were sliced at 30 h postmortem, chops were stored at 4°C for 2 d, then grilled in a dry oven at 240°C for 30 min. Drip loss (DL) was calculated as 100 × (1 -weight after storage/weight before storage). Cooking loss (CL) of the LM was calculated as 100 × (1 -weight after cooking/weight after storage).

Warner-Bratzler Shear Force Measurements. For both the raw (SFr) and the cooked (SFc) meat shear force measurements, 10 cylinders of 1 cm in diameter were sheared in a Warner-Bratzler cell. The maximal force recorded during shearing (at rupture time) was used as the cylinder shear force value and expressed in Newtons.

Loin Color Components and pH. Indicators of lightness (L*), redness (a*), and yellowness (b*) were recorded from each rib eye surface within 2 h after loin slicing.

The pH at 45 min in LM (pH45) was measured in a 1:10 (wt/wt) dispersion of LM in a 5 mM sodium io- Intramuscular Fat Content. Total lipids were extracted according to the method outlined by [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF]. The lipid content of fresh tissue (g/100 g) was obtained by considering DM content determined from the weight of minced LM tissues.

Glycolytic Potential. One gram of LM was homogenized into 10 mL of 0.55 M perchloric acid. The glycolytic potential (GP) was then calculated according to [START_REF] Monin | Pork of low technological quality with a normal rate of muscle pH fall in the immediate postmortem period: The case of the Hampshire breed[END_REF]

Microarray Design and Annotation

A specific repertoire of 15,198 6mer DNA probes was set up to maximize the information content and gene coverage in analysis of pig skeletal muscle transcriptome at slaughter [START_REF] Damon | Characterization of a pig skeletal muscle microarray to study pork quality: The GenmascqChip 15K[END_REF]. Briefly, we used consensus contig sequences from SIGENAE pig transcript assembly (SIGENAE v8, http://www.sigenae.fr/) selected from previous analyses of LM transcriptome [START_REF] Lobjois | A muscle transcriptome analysis identifies positional candidate genes for a complex trait in pig[END_REF]. Thirty-three thousand contig sequences were submitted to eARRAY online software v4.5 (Agilent Technologies, http://earray.chem.agilent. com/earray) to design one to two 60-mer oligo probes per contig sequence (average of 1.33 probe per contig). Analysis of test LM RNA samples using all designed probes allowed for the final selection of 15,198 informative probes, while maximizing gene coverage when analyzing LM samples. Microarrays were manufactured by in situ synthesis on glass slides using an Agilent 15K (8-plex) microarray format (Agilent Technologies France, Massy, France). This repertoire was deposited in NCBI GEO online resource (http://www.ncbi.nlm. nih.gov/geo/) under reference GPL11016.

The identification of transcripts targeted by each of the 60-mer probes was conducted from probe and contig sequence homology searches using known pig transcripts (Ensembl Sscrofa9, http://www.ensembl.org/ Sus_scrofa/; NCBI Sus Scrofa RefSeq, http://www. ncbi.nlm.nih.gov/RefSeq/) or the transcriptomes of related species (Bos taurus, Equus caballus, Homo sapiens). Annotation was based on similarity and quality criteria established by [START_REF] Casel | SigReannot: An oligo-set re-annotation pipeline of Ensembl transcripts and Unigene clusters[END_REF]i.e., at least 18 consecutive base pairs within 60-mer probe sequence and 85% of homology). Overall, we validated at least 1 reference annotation for 12,939 probes (85%) corresponding to 9,169 unique genes.

RNA Extraction and Microarray Hybridization

Ten-gram samples from the LM were collected 20 min after stunning and bleeding and snap frozen in liquid N.

Total RNA was extracted by crushing the frozen tissue in Trizol reagent (Invitrogen, Cergy-Pontoise, France).

Total RNA (350 ng) from each animal was labeled individually with Cy3 using the Low RNA Input Linear Amplification Kit PLUS, Agilent Technologies France). A single 1-color labeled sample was hybridized (65°C, 17 h) per array in Agilent's SureHyb Hybridization Chambers containing 300 ng of Cy3-labeled cRNA sample. Microarrays were scanned at 5 µm/pixel resolution using the Agilent DNA Microarray Scanner G2505B, and images were analyzed with Agilent Feature Extraction Software (Version 9.5), using the GE2-v5_95_Feb07 FE extraction protocol. The raw intensities from the 15,198 probes were logtransformed and then centered within sample by subtraction of the sample median value across all probes. The microarray data have been deposited in the NCBI GEO and are accessible through GEO Series accession number GSE28714 (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE28714).

Functional Analysis

The functional analysis of transcript lists of interest was undertaken by the analysis of the enrichment for specific Gene Ontology (GO) terms using the Database for Annotation, Visualization and Integrated Discovery (DAVID; Huang et al., 2009a,b) as implemented in the online software (http://david.abcc.ncifcrf.gov/). The annotated probes for both the background reference and the lists of interest were matched to their human orthologs using official human Entrez gene ID. Analyses were performed using the GO-FAT terms of DAVID. The background included 12,063 probes, representing 8,638 ortholog human genes. The functional annotation clustering of the enriched GO terms was performed with a similarity score (kappa) of 0.75 and a multiple linkage threshold of 0.50. Cluster enrichment scores are geometrical means of P-values for each GO term included in the cluster and expressed on a negative logarithmic scale. Clusters were selected using enrichment score cutoffs of 1.30 and 1.10 for highly heritable transcripts and meat quality trait correlated transcripts, respectively.

Genetic Parameters Estimation

For each hybridization probe, we conducted a univariate variance analysis and independent bivariate analyses combining RNA quantification measurement with each of the meat quality traits under study. We analyzed the data with a mixed model of variance including a random polygenic effect fitted under an animal model, a fixed effect for slaughter batch, and a fixed effect for sex (intramuscular fat model). The chop weight was used as a covariate for the CL and SFc models. A fixed effect fitting hybridization batch (15 levels) was used for RNA quantification traits. A REML methodology was applied to estimate the variance components, corresponding genetic parameters, and their SE, using the software package ASREML 2.0 [START_REF] Gilmour | ASReml User Guide Reference 2.0[END_REF]. The pedigree used to set up the animal model included 3 generation of ancestors in addition to the phenotyped animals. The meat quality phenotypes from all NN rn+rn+ genotype animals of the entire F 2 resource population were used in bivariate analyses when available (731 to 1,023 records per trait, as reported in Table 1). Variance component estimates were considered in the results only when model loglikelihood converged.

Proportion of Heritable Transcripts

The probability of estimating a nonzero heritability in the absence of genetic variability was determined empirically from the distribution of 3,000 estimates produced using 3,000 independent phenotype data sets of 325 records simulated without genetic variation. A false discovery rate (FDR) was calculated using the software package R/qvalue (http://cran.r-project.org/ web/packages/qvalue/index.html) according to the methodology proposed by [START_REF] Storey | Statistical significance for genomewide studies[END_REF].

RESULTS

Estimates of Heritability for RNA Quantification Abundance

From univariate analyses, we estimated narrow sense heritability values as significantly different from zero with a FDR of 5%, corresponding to a threshold of 0.051 when applied to this result set, for 9,765 probes over 15,198 probes analyzed. The median value of these significant estimates of additive genetic variation is 0.236 (the median of SE estimates is 0.211), including heritability estimates greater than 0.50 for 1,174 probes targeting 1,146 contigs. The distribution of the 9,765 significant heritability estimates is presented in Figure 1. Those 9,765 probes represent 65% of probes, 9,219 unique design contig sequences, and were annotated as products from 6,000 unique genes. We have verified that the heritability estimates presented here were not substantially correlated with either the average expression level (r = -0.11) or the expression level SD (r = 0.09), suggesting that our observations are not the result of a scaling effect.

Biological Processes Represented in Highly Heritable LM Transcripts

To study genes associated with highly heritable probe signals into clusters of related functional terms, a functional annotation clustering was performed with 1,174 probes, corresponding to 911 annotated transcripts that were transcribed from 843 unique genes. Nine clusters were found to be significant with an enrichment score greater than 1.30 (Table 2). The lists of genes included within each cluster are listed in supplemental file 1 (available in the online version of this paper). The highlighted GO terms show an over-representation of GO terms associated with the regulation of Rho protein signal transduction (cluster 1), the induction, regulation, and process of apoptosis (clusters 2, 4, and 8), calcium transport and metal ion binding (clusters 3 and 6), focal adhesion and adherent cell junctions (clusters 5 and 7) and glucose metabolism (cluster 9).

Genetic Correlations Between RNA Concentrations and Meat Quality Traits

Estimation of the genetic correlation (r G ) between expression and meat phenotype was determined only for the 9,765 probes that showed a significant genetic variability (h 2 > 0.051). The limited number of animals available per molecular trait (n = 325) makes it possible to only broadly estimate the genetic correlations between RNA concentrations and meat quality traits, with the SE of r G estimates ranging from a median SE of 0.48 (a*) to a median SE of 0.60 (pH45; Table 3).

The uncertainty of those estimates is reduced for the most heritable molecular traits, ranging from a me- dian SE of 0.37 (a*) to a median SE of 0.44 (pH45), when considering only those probes where h 2 in bivariate analyses is greater than 0.50 (929 to 1,509 probes, Table 3). We summarized in Table 3 the properties of the distribution of the r G estimates for these highly heritable transcript levels (interquartile range and median value), further restricted to the r G estimates with an SE less than 0.50 to focus on the most interpretable results. We illustrated contrasting examples of the distributions of these sets of genetic correlation estimates for 3 different traits (pH45, GP, a*) in Figure 2. Larger numbers of genetically correlated transcripts (|r G | > 0.70) were observed for the measurements of pH45, SFc, a*, and CL than regarding other meat quality traits. In contrast, the measurements of DL, GP, and pH24 show a substantial genetic correlation with very few muscle transcript levels.

Functional Analysis of Transcripts Genetically Correlated with Meat Quality Traits

The list of genes assigned to transcripts that were quantified as heritable traits (h 2 > 0.50) and were genetically correlated (|r G | > 0.70) with pH45, SFc, a*, and CL were characterized for functional enrichment in GO terms using the microarray repertoire as background. The clusters of GO terms enriched in the correlated gene lists for each of these 4 traits established using an enrichment scores cutoff of 1.10 are presented in Table 4. For each cluster, the most specific GO term explicit definition and the enrichment P-value associated with this term are shown. The lists of genes included within each cluster are listed in supplemental file 2 (available in the online version of this paper). For CL, 3 clusters highlight the genetic variability of transcripts involved in the induction of apoptosis and regulation of fatty acid oxidation. Transcript levels from genes known to be involved in complement activation and protein polymerization were observed as genetically correlated with SFc. The clusters associated with transcripts genetically correlated with a* are focused on glucose metabolism and neoglucogenesis. The transcripts that are genetically correlated with pH45 highlight 2-cell compartments, the lumen of intracellular organelles and the membrane of cytoplasmic vesicles, whereas the voltage-dependent cation channel activity is highlighted as a biological process.

DISCUSSION

Genetic Variability of Transcript Abundance in Pig Postmortem LM

Our results document that 65% of the postmortem LM RNA concentration measurements, as assayed by hybridization on a muscle-specific array, are genetically determined because essentially all probes were informative [START_REF] Damon | Characterization of a pig skeletal muscle microarray to study pork quality: The GenmascqChip 15K[END_REF]. The limited number of full-and half-sib families included in this experimental population may generate a sampling variance in estimates. Nonetheless, the SE of the heritability estimates for these molecular traits do support our interpretation of the actual genetic variability of LM transcriptome, even if the individual transcript heritability values were only broadly assessed.

The observed genetic variability in LM transcript concentration is consistent with other reports of genetic variability in transcript abundance in a variety of organisms. Studies in yeast have shown that more than 60% of the transcripts whose abundance was measured in segregating populations could be characterized with heritabilities greater than 0.69 [START_REF] Brem | The landscape of genetic complexity across 5,700 gene expression traits in yeast[END_REF]. The available data from human tissues show that the relative concentrations of respectively 59 and 71% of transcripts, as measured in total blood or adipose tissue respectively, are significantly heritable, with an average heritability estimate of 0.30 for those heritable transcripts [START_REF] Emilsson | Genetics of gene expression and its effect on disease[END_REF]. Data from studies performed in mice document a median value of 0.14 for heritability estimated among all of the possible transcripts quantification levels measured in adipose tissues [START_REF] Petretto | Heritability and tissue specificity of expression quantitative trait loci[END_REF].

Functional Interpretation of Transcriptome Genetic Variability

Apoptosis induction has been proposed as an essential mechanism involved in the transformation of muscle in meat, contributing to the tenderization of meat through an early triggering activity of caspases [START_REF] Herrera-Mendez | Meat ageing: Reconsideration of the current concept[END_REF][START_REF] Ouali | Revisiting the conversion of muscle into meat and the underlying mechanisms[END_REF]. Variations in the regulation of apoptosis have been further suggested as an underlying cause of differential tenderness in bovine meat by the observation of differences in the concentrations of proteins from the inner mitochondrial membranes of muscle samples taken as early as 10 min postexsanguination [START_REF] Laville | Proteome changes during meat aging in tough and tender beef suggest the importance of apoptosis and protein solubility for beef aging and tenderization[END_REF]. Therefore, the observation of large individual and genetic variation in the induction of the apoptosis processes in postmortem LM samples can be seen as a consequence of the exsanguination-induced muscle ischemia. The selection of a muscle sampling point at 20 min after stunning may have highlighted individual variation in the early and unbuffered response to a sudden challenge.

Proportion of Transcripts Genetically Correlated with Meat Quality Traits

The genetic correlation between 2 traits is thought to reflect either genetic linkage between loci affecting each trait or pleiotropic effects of the same loci on both traits. Our population design maximizes long-range genetic linkage effects in the F 2 generation. Therefore, this mechanism may have substantially inflated some of our estimates of the genetic correlations between molecular and meat quality traits in this population. The accuracy of the genetic correlation estimates proved to be severely limiting for interpretation because of the limited size of our data set. However, we propose a cautious interpretation of the entire distribution of the genetic correlations estimates for each meat quality trait that we restricted to the most heritable RNA concentrations.

The large differences we observed between the distributions of the genetic correlation estimates among the different meat quality traits was not expected, considering the genome-wide relevance of the repertoire of probes used and the existence of genetic variability in all of the meat quality traits studied. A naïve assumption may have been to identify, among the large number of heritable transcripts, some transcripts that would be genetically correlated with each of the meat quality traits. This would be expected if postmortem muscle transcriptome was patterning the whole muscle physiology. In this respect, the lack of RNA species showing substantial genetic correlations with the meat pH24 and the glycolytic potential (traits nonetheless determined by a sizable genetic variation, h 2 of GP = 0.22) brings into question the dynamics of phenotype construction and the persistence of its footprint on gene expression profiles over time. To explain this observation, one may simply hypothesize that the expression of genes involved in the genetic control of muscle glycogen stores depletion and recovery during antemortem period does not persist, or at least does not dominate the patterns of postmortem gene expression that we analyzed. But this hypothesis does not account for the extent of our observation that all of the genes contributing to the control of glycogen storage and mobilization are well represented in the array used for transcriptome analysis, are transcribed in LM postmortem, and some even showed substantial genetic variation in LM mRNA content (as those reported for their genetic correlation with meat redness a*). An alternative hypothesis, therefore, would be that the gene expression levels are essentially constrained by homeostatic regulatory mechanisms, which over time are buffering all sources of variation, including the genetic variability, in this case between the antemortem and postmortem time points. Gene expression buffering has been recognized as an essential control mechanism to dampen the consequences of transcription noise [START_REF] Fraser | Noise minimization in eukaryotic gene expression[END_REF][START_REF] Raser | Control of stochasticity in eukaryotic gene expression[END_REF] and proposed as an essential function of microRNA [START_REF] Wu | Evolution under canalization and the dual roles of microRNAs: A hypothesis[END_REF]. This latter hypothesis would support a relative independence between the genetic variability of antemortem muscle transcription (the genome expression profile that one may suppose is responsible for mediating or accompanying variation in the control of antemortem glycogen stores) and the genetic variability of postmortem transcription profiles that we evaluated in this work, reflecting muscle reaction to ischemia. Finally, this independence would support the observed disconnection between the genetic variability of the muscle glycolytic potential or the ultimate pH, and the genetic variability of postmortem transcript abundance. We observed that the genetic variability of the postmortem LM transcriptome is linked with the genetic variability of a postmortem muscle physiology measurement (pH45) and distantly assessed measurements of meat color (a*) and loin cooking (SFc, CL). The transcription profiles may subsequently better reflect muscle physiology contemporary of the RNA sampling point, beside its translation into remote consequences that affect meat consumption. A recent study reported significant phenotypic associations between postmortem LM transcriptome analysis and loin color redness a* [START_REF] Te Pas | Longissimus muscle transcrip-tome profiles related to carcass and meat quality traits in fresh meat Pietrain carcasses[END_REF]. Our data suggest that the associations reported by these authors may result from a genetic determinism with pleiotropic consequences on the transcriptome and loin redness.

Functional Interpretation of Transcripts Genetically Correlated with Meat Quality Traits

Cooking Loss. The functional analysis of tran- scripts that show a substantial genetic correlation with loin cooking losses highlights apoptosis as an overrepresented cell function in this group of genes. The first steps of apoptosis involve the condensation of the cytoplasm, cell shrinking, and result in the expansion of extracellular space [START_REF] Bortner | Cell shrinkage and monovalent cation fluxes: Role in apoptosis[END_REF]. The genetic correlations that we observed may thus result from a genetic variability that drives the dynamics of apoptosis induction postmortem and subsequently affects the water-holding capacity of meat during cooking.

Cooked Loin Shear Force. Complement activation has been described as an essential component of skeletal muscle ischemia-reperfusion injury [START_REF] Weiser | Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement[END_REF] that is thought to be mediated through the action of membrane-complement attack complexes [START_REF] Kyriakides | Skeletal muscle reperfusion injury is mediated by neutrophils and the complement membrane attack complex[END_REF]. More specifically, the identified transcripts of C1S and C1R complement subunits are encoding essential proteases of this complex [START_REF] Davis | Biological activities of C1 inhibitor[END_REF], and genetic correlation of their expression levels with meat shear force would be consistent with the role generally accepted for postmortem proteases activity in meat tenderness variation in pigs [START_REF] Melody | Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles[END_REF].

Loin Color a*. Loin color redness principally results from oxymyoglobin concentration and oxidative status [START_REF] Gorelik | Oxymyoglobin oxidation and membrane lipid peroxidation initiated by iron redox cycle: Preven-tion of oxidation by enzymic and nonenzymic antioxidants[END_REF]. We identified the transcript concentration of glycolysis enzymes as genetically correlated with loin color redness a*, which suggests that the regulation of glycolysis kinetics or its extent may drive the muscle oxidative capacity with regard to oxymyoglobin.

pH45. The voltage-gated cation channel activity and cation channel complex cell component point to the excitation-contraction coupling mechanism, which operates through the release of free Ca 2+ from sarcoplasmic reticulum stores. Contraction mobilizes the available energy and results in increased glycolysis in the case of hypoxic tissues, leading to acidosis where resulting pyruvate is metabolized into lactate. The RYR1 mutation [START_REF] Fujii | Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia[END_REF] does constitute, by itself, an example of genetic variability with documented pleiotropic effects on both calcium channel activity in skeletal muscle and pH45 [START_REF] Larzul | Effect of the halothane genotype (NN,Nn,nn) on growth, carcass and meat quality traits of pigs slaughtered at 95 kg or 125 kg live weight[END_REF][START_REF] Sellier | Genetics of meat and carcass traits[END_REF]. However, the genetic variability observed here in pH45 and transcription is independent of the RYR1 polymorphism because the animals used in the present study were not carrying the RYR1 mutated allele (see methods). This observation illustrates that the same set of functions can be affected by different polymorphisms leading to similar effects, and that the genetic variability for the pH45 trait in a HAL NN (wild-type) population could be mediated nonetheless through similar biological pathways as those affected by the RYR1 mutation. Malignant hyperthermia susceptibility (MHS) is a genetic condition in human and pig resulting from the same or additional mutations in the RYR1 gene [START_REF] Fujii | Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia[END_REF][START_REF] Manning | Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation[END_REF]. Malignant hyperthermia susceptibility in humans can also result from a mutation in the α 1S subunit of the voltage-gated calcium channel CACNA1S [START_REF] Monnier | Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle[END_REF]. We identified as genetically correlated with pH45, the LM concentration of the CACNA1A and CACNA1G transcripts, encoding 2 paralogs of CACNA1S.

Use of RNA Measurements for Genetic Improvement of Meat Quality

Alternative selection criteria are to be considered relative to their heritability and genetic correlation with traits belonging to breeding goals. For measurements taken on the same set of animals relative to selection candidates, an alternative selection criteria with an heritability h′ 2 and a genetic correlation r G with a selected trait of heritability h 2 would have to verify r G * h′ > h to support a potential benefit in increased accuracy of breeding value estimation [START_REF] Searle | The value of indirect selection: I. Mass selection[END_REF]. In the case of selection for improvement of a meat quality trait with a typical heritability of 0.25, an illustration of a competitive alternative selection criterion could be the quantification of a specific RNA species in a given tissue sample associated with an heritability of at least 0.50 and a genetic correlation of at least 0.70 with the selected meat quality trait. These conditions are met by a large number of LM transcript levels for pH45, a*, SFc, and CL. These traits would therefore be candidates for the application of indirect selection strategies based on measurements of RNA concentrations in postmortem LM.

A substantial improvement in the accuracy of breeding values estimation may be at hand if informative RNA species such as those identified in this work for selected meat quality traits could be measured in skeletal muscle from selection candidates themselves. Compared with the measurements of the same trait in relatives, measurements on selection candidates would allow an improvement in the selection response by 50% when compared with the systematic measurement of 2 full-sibs, or 100% when compared with the systematic measurement of 4 half-sibs, for a typical meat quality trait with a heritability of 0.25. Improvement in the selection response would result from increased accuracy of breeding values estimated from an animal itself vs. phenotypes of relatives [START_REF] Falconer | Selection: III. Information from relatives[END_REF].

Taken together, our data support the likelihood of heritable LM transcripts that are genetically correlated with some of the pig meat quality traits. It would be of significant practical interest for applications in breeding programs to re-estimate these genetic parameters using RNA quantification measurements acquired from in vivo-sampled muscles.

Figure 1 .

 1 Figure 1. Distribution of heritability estimates for transcript abundance in postmortem LM, as measured using 9,765 probes (h 2 > 0 under a false discovery rate of 5%).

Figure 2 .

 2 Figure 2. Distribution of genetic correlation estimates between heritable transcripts (h 2 > 0.50, SE r G < 0.50) and LM pH 45 min postmortem (pH45), LM glycolytic potential (GP), and loin color redness (a*). r G = genetic correlation.

Genetic variability of transcript abundance in pig skeletal muscle at slaughter: Relationships with meat quality traits 1

  Hendrix Genetics RTC, F-45808 St. Jean de Braye, France; †INRA, UMR0598 Génétique Animale, F-35042 Rennes, France; ‡INRA, UMR 1079 SENAH, F-35590 Saint Gilles, France; and §Agrocampus Ouest, UMR1079 SENAH, F-35000 Rennes, France

P. Cherel,* F. Herault, † A. Vincent, ‡ § P. Le Roy, † and M. Damon ‡ § 2 *

Table 1 .

 1 Summary statistics for loin meat quality traits1

	Loin quality trait	Abbreviation	Unit	n	µ	σ	Range
	pH 45-min	pH45	pH unit	840	6.55	0.18	5.45 to 7.40
	pH 24-h	pH24	pH unit	1,023	5.71	0.20	5.37 to 6.73
	Glycolytic potential	GP	µmol/g	805	159.40	24.97	70.30 to 276.80
	Intramuscular fat	IMF	g/100 g	804	2.24	0.66	0.79 to 4.92
	Color CIE L*	L*	CIE u	852	49.63	3.05	37.92 to 57.54
	Color CIE a*	a*	CIE u	852	7.86	1.22	4.64 to 13.00
	Color CIE b*	b*	CIE u	852	5.12	1.30	0.48 to 9.30
	Shear force, cooked	SFc	N	753	33.65	1.16	17.94 to 55.48
	Shear force, raw	SFr	N	753	35.34	1.19	17.92 to 61.81
	Cook loss	CL	%	739	26.46	2.58	15.01 to 38.52
	Drip loss	DL	%	731	1.72	0.95	0.06 to 5.63
	1 L* = lightness; a* = redness; b* = yellowness.

Table 2 .

 2 Functional annotation clustering of highly heritable transcripts (h 2 > 0.50)

	Cluster	ES 1	n GO	2	Cluster-specific GO term 3

5

Modified Fisher's exact test P-value.

Table 3 .

 3 Distribution and SE of genetic correlation (r G ) estimates between LM transcript concentrations and meat quality traits

			0 < SE r G < 1				h 2 [RNA] > 0.5, SE r G < 0.5
			All r G values		All r G values		|r G | > 0.7	|r G | > 0.9
	MQ trait 1	h 2 2	n 3	SE r G 4	n 3	IQR r G	5	Q2 r G 5	SE r G 4	n 3	SE r G 4	n 3
	pH45	0.12	7,489	0.60	929	1.07		-0.11	0.43	253	0.39	55
	pH24	0.21	8,775	0.51	1,140	0.37		-0.07	0.39	4	0.27	0
	GP	0.22	8,987	0.52	1,164	0.24		0.02	0.40	0	-	0
	IMF	0.11	7,386	0.59	1,001	0.32		-0.04	0.44	31	0.41	6
	L*	0.20	7,760	0.54	1,139	0.51		-0.11	0.41	66	0.35	9
	a*	0.44	7,303	0.48	1,171	0.56		0.22	0.37	190	0.25	44
	b*	0.24	8,608	0.55	1,171	0.41		0.03	0.43	44	0.31	7
	SFc	0.26	8,175	0.56	1,381	0.58		0.25	0.44	184	0.32	81
	SFr	0.43	8,311	0.49	1,509	0.60		0.05	0.38	36	0.28	3
	DL	0.22	8,346	0.55	1,452	0.28		0.04	0.43	12	0.34	1
	CL	0.21	8,030	0.53	1,428	0.67		0.13	0.42	134	0.35	14

Table 4 .

 4 Functional analysis of heritable transcripts that were genetically correlated with meat quality traits Identity of the lowest hierarchical level and most specific GO term. NADP = NAD phosphate.

	Cluster	ES 1	n GO	2	Cluster-specific GO term 3

3 4 Modified Fisher's exact test P-value.

at INRA Institut National de la Recherche Agronomique on January 13, 2014 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on January 13, 2014 www.journalofanimalscience.org Downloaded from

Corresponding author: Marie.Damon@rennes.inra.fr Received April 22, 2011. Accepted October 5, 2011.

at INRA Institut National de la Recherche Agronomique on January 13, 2014 www.journalofanimalscience.org Downloaded from

Cherel et al. at INRA Institut National de la Recherche Agronomique on January 13, 2014 www.journalofanimalscience.org Downloaded from

Meat quality (MQ) traits are LM pH 45 min postmortem (pH45); loin

24-h pH (pH24); glycolytic potential (GP); intramuscular fat (IMF); loin color variables L* (lightness), a* (redness) and b* (yellowness); shear force on cooked (SFc) or raw (SFr) loin; loin drip loss (DL); and loin cooking loss (CL).2 Median value of the meat quality trait heritability estimate in bivariate analyses.3 Number of probes meeting heading-specified conditions on h 2 , SE r G , and r G , reported for all converged bivariate analyses of heritable transcripts levels.4 Median value of the genetic correlation estimates SE.5 Interquartile range (IQR) and median value (Q2) of genetic correlation estimates (r G ) distribution.

 [START_REF] Davis | Biological activities of C1 inhibitor[END_REF]This work was carried out with financial support from the ANR-Agence Nationale de la Recherche-The French National Research Agency under the Programme National de Recherche en Alimentation, project ARN-PNRA-2006-25, GENMASCQ. The authors thank the staff from the Orleans Viandes abattoir, Fleury-Les-Aubrais, France, for their assistance in tracking carcasses and primary cuts, and J. Glénisson, L. Letelu, and J. Pires (Hendrix Genetics RTC, St. Jean de Braye, France), and M. Fillaut (INRA, UMR 1079 SENAH, Saint Gilles, France) for their assistance in the meat quality measurements.


Laurent Penet
email: laurent.penet@gmail.com

Anne Bonis

IMPACT OF CAPITULUM STRUCTURE ON REPRODUCTIVE SUCCESS IN THE DECLINING SPECIES CENTAUREA CYANUS (ASTERACEAE): SMALL TO SELF AND BIG TO FLIRT?

Keywords: Asteraceae, capitulum structure, Centaurea cyanus, ray and disc florets, reproductive success, self-incompatibility

Attracting pollinators and achieving successful reproduction is essential to flowering plant species, which evolved different strategies to cope with unpredictable pollination service. The ability of selfing is most widespread and represents a reproductive insurance under varying conditions. In this study, we investigated reproductive success in Centaurea cyanus, a self-incompatible declining Asteraceae species. We measured seed set under outcrossing and autonomous selfing and assessed the impact of capitulum structure (i.e., the number of disc florets) on reproductive success. We report that the incompatibility system is either flexible or evolving a breakdown in this species, since autonomous selfing often resulted in production of few seeds. We also show that capitulum structure has a strong impact on reproduction, with smaller inflorescences presenting a better ability to self than larger ones, while larger inflorescences performed better than smaller ones when cross-pollinated. Variable capitulum structure in this Asteraceae species may therefore represent a reproductive strategy to achieve efficient reproduction under diverse pollination environments. Our results also suggest that this declining species might be disrupting its auto-incompatibility system in response to reduced habitats and declining population sizes.

INTRODUCTION

Plant reproduction has fascinated scientists since ages [START_REF] Darwin | The effects of cross-and self-fertilisation in the vegetable kingdom[END_REF]. The phenotypic diversity evolved to achieve sexual reproduction in Angiosperms is simply dramatic and resulted, for instance, in extreme specialization of flowers into pollination syndromes [START_REF] Hermann | The genetic architecture of natural variation in flower morphology[END_REF] and sophisticated mechanisms of selfing (Fenster and Martén-Rodríguez 2007). At the same time, many species evolved various strategies to avoid selfing: selfincompatibility [START_REF] Levin | The evolutionary significance of pseudo-selffertility[END_REF], dichogamy [START_REF] Freeman | Sexual specialization and inbreeding avoidance in the evolution of dioecy[END_REF]) and separation of sexes on or between individual plants [START_REF] Narbona | Linking selfincompatibility, dichogamy, and flowering synchrony in two Euphorbia species: alternative mechanisms for avoiding selffertilization?[END_REF], because selfing influences the probability of unmasking mutation loads in progeny and usually reduces individual plant fitness [START_REF] Goodwillie | The evolutionary enigma of mixed mating systems in plants: Occurrence, theoretical explanations, and empirical evidence[END_REF]. Self-pollen deposition may, however, be unavoidable and for self-incompatible species this often results into a decrease in seed production (e.g., [START_REF] Kameyama | Flowering phenology influences seed production and outcrossing rate in populations of an alpine snowbed shrub, Phyllodoce aleutica : effects of pollinators and self-incompatibility[END_REF]. The effect of self-pollen deposition is two-fold: first it represents a loss of pollen available to outcrossing (i.e., pollen discounting; [START_REF] Busch | The relative importance of reproductive assurance and automatic selection as hypotheses for the evolution of self-fertilization[END_REF], and second it interferes with outcross-pollen on stigmas [START_REF] Dai | Do dichogamy and herkogamy reduce sexual interference in a self-incompatible species?[END_REF]. These effects may be enhanced in case of pollen limitation and in small populations [START_REF] Busch | The evolution of self-incompatibility when mates are limiting[END_REF]. Plant mating system is mainly a pollinator driven process, though plant characteristics (e.g., asexual reproduction; [START_REF] Navascués | Genetic diversity and fitness in small populations of partially asexual, self-incompatible plants[END_REF] or other ecological interactions also play a role (e.g., nutriment availability; [START_REF] Helenurm | Genetic load, nutrient limitation, and seed production in Lupinus texensis (Fabaceae)[END_REF], interactions with herbivores [START_REF] Ivey | Effects of herbivory and inbreeding on the pollinators and mating system of Mimulus guttatus (Phrymaceae)[END_REF] or florivores [START_REF] Ashman | Direct and indirect effects of a sexbiased antagonist on male and female fertility: Consequences for reproductive trait evolution in a gender-dimorphic plant[END_REF][START_REF] Penet | Florivory increases selfing: an experimental study in the wild strawberry, Fragaria virginiana[END_REF]. All these interactions are potentially threatened by the effects of climate change [START_REF] Schweiger | Multiple stressors on biotic interactions: how climate change and alien species interact to affect pollination[END_REF], such as loss of diversity [START_REF] Sander | Fine-scale patterns of species and phylogenetic turnover in a global biodiversity hotspot: Implications for climate change vulnerability[END_REF] and changes in pollinator specialization [START_REF] Fontaine | Generalist foraging of pollinators: diet expansion at high density[END_REF]), and we may face a dramatic change in pollination service in the near future [START_REF] Potts | Developing European conservation and mitigation tools for pollination services: approaches of the STEP (Status and Trends of European Pollinators) project[END_REF]. It is therefore crucial to increase our knowledge about pollination and reproduction of plant species [START_REF] Mayer | Pollination ecology in the 21st Century: Key questions for future research[END_REF], and particularly for rare or invasive species [START_REF] Powell | Comparing the reproductive success and pollination biology of an invasive plant to its rare and common native congeners: a case study in the genus Cirsium (Asteraceae)[END_REF] to allow for a sensible management.

In rare or declining plant species, an understanding of mating system and pollination biology in addition to causes of decline is crucial to help in implementing conservation guidelines [START_REF] Biesmeijer | How pollination ecology research can help answer important questions[END_REF]. Indeed, assessing selfing rates and plants tolerance to selfing is a first step toward managing viable reintroductions or reinforcement [START_REF] Leducq | An experimental study of the S-Allee effect in the self-incompatible plant Biscutella neustriaca[END_REF], because it will bring light to density and relatedness effects in small populations (i.e., impacts of inbreeding depression and Allee effects; [START_REF] Ågren | Population size, pollinator limitation, and seed set in the self-incompatible herb Lythrum salicaria[END_REF][START_REF] Leducq | An experimental study of the S-Allee effect in the self-incompatible plant Biscutella neustriaca[END_REF]. In self-incompatible species, these effects are altered, because direct selfing is reduced [START_REF] Busch | Does mate limitation in selfincompatible species promote the evolution of selfing? The case of Leavenworthia alabamica[END_REF]), but consanguinity may still impact fitness via bi-parental inbreeding [START_REF] Uyenoyama | Inbreeding and the cost of meiosis: The evolution of selfing in populations practicing biparental inbreeding[END_REF][START_REF] Elam | Population size and relatedness affect fitness of a self-incompatible invasive plant[END_REF]). Many selfincompatible species have actually somehow a plastic expression of incompatibility and incompatibility breakdown has been reported in several families [START_REF] Igic | Loss of self-incompatibility and its evolutionary consequences[END_REF][START_REF] Busch | Insights gained from 50 years of studying the evolution of self-compatibility in Leavenworthia (Brassicaceae)[END_REF]. Ability to self in incompatible species is indeed associated with repeated extinction and colonization demographic events and it is often greater in small populations [START_REF] Reinartz | Bottleneck-induced dissolution of self-incompatibility and breeding system consequences in Aster furcatus (Asteraceae)[END_REF]. Here we chose to investigate reproductive success in Centaurea cyanus, a selfincompatible species experiencing a dramatic decline in its native range since a few decades (e.g., [START_REF] Sutcliffe | Changes in the arable flora of central southern England since the 1960s[END_REF][START_REF] Pausic | Weed diversity in cottage building material used in 19th century: Past and present of the plant occurence[END_REF][START_REF] Ulber | Using selective herbicides to manage beneficial and rare weed species in winter wheat[END_REF], and considered as an invasive outside its native range [START_REF] Muth | Implementation of a novel framework for assessing species plasticity in biological invasions: responses of Centaurea and Crepis to phosphorus and water availability[END_REF][START_REF] Jursik | Biology and control of another important weeds of the Czech Republic: Cornflower (Centaurea cyanus L.)[END_REF].

Like other species of Asteraceae, Centaurea cyanus expresses variation in capitulum size and structure [START_REF] Jursik | Biology and control of another important weeds of the Czech Republic: Cornflower (Centaurea cyanus L.)[END_REF]. Its capitula are structured with deep blue sterile ray florets advertising for pollinators and less showy fertile disc florets [START_REF] Boršic | Centaurea sect. Cyanus : nuclear phylogeny, biogeography, and life-form evolution[END_REF]). Variation in capitulum structure would reflect alternative investments into pollinator attraction vs. seed production, and we were interested in investigating the effect of reproductive efforts on reproductive success. In this study, we investigated reproductive success under controlled conditions and we asked the following specific questions: 1) how variable is capitulum composition in ray and disc florets, hereafter capitulum structure? 2) Is C. cyanus able of autonomous self-pollination and seed production? 3) Does capitulum structure variation impact seed set differently under outcross-and self-pollination? We were particularly interested in determining if variation in capitulum structure may represent a reproductive strategy in this species.

MATERIAL AND METHODS

Plant species

Centaurea cyanus (cornflower) is an annual plant from the Asteraceae family (= Compositae, Angiosperm Phylogeny Group 2003) with a wide distribution. The species originated in Caucasus [START_REF] Boršic | Centaurea sect. Cyanus : nuclear phylogeny, biogeography, and life-form evolution[END_REF]) and dispersed as a weed species associated with cereal crops since prehistorical times [START_REF] Rösch | The history of crops and crop weeds in southwestern Germany from the Neolithic period to modern times, as shown by archaeobotanical evidence[END_REF]. Though initially not native to Europe, the species is now well naturalized and part of the flora. It may thus not be considered an alien anymore, given its ancient colonization in Europe.

Centaurea cyanus is generally plastic with regard to many traits [START_REF] Muth | Implementation of a novel framework for assessing species plasticity in biological invasions: responses of Centaurea and Crepis to phosphorus and water availability[END_REF] and grows either in crop fields (especially associated to wheat or canola) or along field margins, where it is expressing a usually high competitive ability [START_REF] Wassmuth | Spatial aggregation facilitates coexistence and diversity of wild plant species in field margins[END_REF]. Germination occurs in fall [START_REF] Stilma | Recruitment and attrition of associated plants under a shading crop canopy: Model selection and calibration[END_REF]. Flowering occurs from June to mid-summer and each plant produces lose inflorescences bearing many capitula typical of Asteraceae. Sterile peripheral ray florets are deep blue and surround several whorls of fertile tubular disc florets, each with an ovary of a single ovule. Centaurea cyanus is pollinated by diverse species of insects, though mostly bees [START_REF] Carreck | Food for insect pollinators on farmland: insect visits to flowers of annual seed mixtures[END_REF] and it is reported as self-incompatible as a vast majority of species in Asteraceae [START_REF] Charlesworth | Distribution of dioecy and selfincompatibility in angiosperms[END_REF].

Pollination treatments

Seeds from many (30 -40) individuals of wild C. cyanus were sampled from discontinuous patches from three adjacent fields in Macon (Upper Normandy) during summer 2009 and bulked for storage at +4 C. We sowed seeds in autumn at the greenhouse facility of ECOBIO (University of Rennes 1) and transplanted young plantlets into pots. We started the experiment in spring 2010 when experimental individuals were in bloom (n = 17 unrelated plants). We recorded the number of ray and disc florets of each experimental capitulum at opening. Capitula were individually marked and assigned to either manual crosspollination or autonomous self-pollination. Each experimental plant received both treatments.

For cross-pollination, we collected pollen from several unrelated pollen donors (6 -10 non-experimental plants). We used a gentle brush to cover receptive stigmas emerging from disc florets with the mix of pollen, thus outnumbering self-pollen grains already present on stigmas due to protandry (L. Penet, personal observation). In the selfing treatment, capitula were left without further manipulation until harvest. We collected achenes before their release from the capitulum. Each achene contains a single seed; we therefore estimated and counted the number of plump fertile seeds. Unfortunately, part of the capitula dried before maturation due to unexpected high temperatures in the greenhouse. For these, we therefore differentiated successfully fertilized ovules based on size and we considered those as seeds in further analyses. This maturation issue is unlikely to have biased our results since a similar number of capitula were damaged in both treatments and no difference in seed production was found between mature and damaged capitula within pollination treatments (data not shown). We collected 5 -11 capitula per plant resulting in 64 cross-and 68 self-pollinated capitula.

Statistical analyses

We calculated reproductive success as the proportion of seeds per capitulum (i.e., seed set) and tested for a difference between cross-and self-pollination treatments. We conducted a two-way full-factorial analysis of covariance of seed set with pollination treatment as fixed factor and the number of disc florets (i.e., capitulum structure) as covariate. We added individual plant to account for maternal effects; the Ancova assumptions of homoscedasticity and normality of residuals were met. We included an interaction between pollination treatment and capitulum structure because variation in number of disc florets is expected to influence local pollen availability for autonomous selfing and probability of self-pollen transfer. We found that the interaction was significant. To correlate the number of disc florets and seed set while taking into account maternal effects, we conducted separate analyses of variance of seed set for outcross-and self-pollination treatments, with individual plants as sole factor, and extracted the residuals. We then calculated the correlation between the number of disc florets and the residuals for seed set for both pollination treatments. All analyses were conducted with R statistical package (R Development Core Team 2011).

RESULTS

Capitulum structure was highly variable both within and among Centaurea cyanus plants. The total number of florets per capitulum varied from 13 to 40 (mean ± se: 23.92 ± 0.42, n = 132) and the average number of florets at the plant level varied from 19 to 29 with a strong maternal effect (F 16,115 = 2.72, P = 0.001, n = 132; Fig. 1). Overall, we found that capitula bore a significantly greater number of fertile disc florets than sterile ray florets (grand-means ± se: 15.19 ± 0.54 and 8.49 ± 0.13, respectively, n = 17; paired t -test, t = 13.11, P < 0.0001) and that variance was also greater for the number of disc florets than for the number ray ones (Levene test, F 1,32 = 18.23, P = 0.0002). The ratio of disc to ray florets at the plant level varied continuously (Fig. 1).

The proportion of disc florets that set a seed was significantly affected by pollination treatment (Table 1). Despite a reported self-incompatibility system, most autonomously self-pollinated capitula (93%) set a few seeds though significantly fewer than outcross-pollinated capitula (mean ± se seed set: 0.21 ± 0.02, and 0.66 ± 0.02, respectively). Interestingly, we found that the actual number of disc florets on the capitulum influenced seed set differently between pollination treatments (significant interaction in Table 1). Seed set increased with number of disc florets following cross pollination whereas it decreased with increasing number of disc florets in autonomously selfpollinated capitula (Fig. 2). Both correlations were statistically significant taking into account the maternal plant effect (r = 0.36, P = 0.004, and r = -0.30, P = 0.013, for outcrossing and selfing treatments, respectively). We found a significant maternal effect on seed set (Table 1), and high variation in pollination treatment effect among plants (after controlling for variance in number of disc florets, Fig. 3).

DISCUSSION

Centaurea cyanus is classified as a self-incompatible species, but we document here that most capitula produced seeds following autonomous selfing under controlled conditions. Seed set was nevertheless low, and significantly smaller than in the outcross pollination treatment with high variation in ability to self among plants (Fig. 3). Most surprisingly, seed set was strongly influenced by capitulum structure though differentially between pollination treatments: small capitula with few disc florets performed better than large ones under selfing, and the reverse was found for large capitula with many disc florets (Fig. 2). This suggests that variation in capitulum structure might reflect a reproductive strategy to ensure reproduction. We discuss these findings in the light of conservation biology for this declining species. Seed set following autonomous selfing was low but non negligible since on average 21% of available ovules were fertilized and up to 50% of seed may be achieved. Thus, even if the plant would receive no visit from pollinators during its reproductive life, it would certainly ensure the production of enough seeds to allow for persistence in the field (at plant level, the average of 21% seed-set under selfing translate into several hundred seeds). Selfincompatible pollination systems are already known to be flexible and to vary depending on environmental factors [START_REF] Reinartz | Bottleneck-induced dissolution of self-incompatibility and breeding system consequences in Aster furcatus (Asteraceae)[END_REF], and many allegedly selfincompatible species are often reported as able to self (e.g., Mena-Ali and Stephenson 2007). Most interestingly, stigmas bend outward after they emerge from the stamen crown in C. cyanus, thus loading themselves with self pollen. This mode of stigma growth might have been selected for because it improves pollen receipt when pollinators visit the capitulum, but it may also be considered a striking pre-adaptation to selfing because it dramatically increases the odds of receiving self-pollen if anthers are still loaded (e.g., [START_REF] Penet | Florivory increases selfing: an experimental study in the wild strawberry, Fragaria virginiana[END_REF]). Moreover, a species experiencing population local declinelike cornflower -may be expected to evolve toward increased selfing ability, especially if pollination service becomes unpredictable [START_REF] Goodwillie | The evolutionary enigma of mixed mating systems in plants: Occurrence, theoretical explanations, and empirical evidence[END_REF]. The extent of inbreeding depression (i.e., the lower fitness of selfed progeny compared to outcrossed ones) is an important prospect of study to explore the consequences of evolving self-compatibility in this species.

Capitulum structure had a strong impact on seed set and capitula with fewer disc florets performed better than larger ones under autonomous selfing, while the opposite was found under outcrossing (Fig. 2, larger capitula performed better than smaller ones). In the outcross pollination treatment, we would have expected the reverse, since pollen loads were probably higher in smaller inflorescences, and it is easier to leave a flower un-visited when brushing inflorescences with pollen experimentally in a larger capitulum. This may reflect negative consequences of high competition for fertilization on stigmas if pollen loads were indeed greater in smaller capitula, or that stigmas in smaller capitula were less efficient in pollen receipt. Indeed, the number of disc florets, or inflorescence size, may also reflect individual variation in flower allometry, which is known to translate into reproductive differences between flowers (i.e., herkogamy, differential stigma size; [START_REF] Nishihiro | Post-pollination process in a partially self-compatible distylous plant, Primula sieboldii (Primulaceae)[END_REF], and into decreased pollen receipt and fertilization efficiency. To our knowledge, this is the first time that differences in capitulum structure are directly related to mating efficiency in Asteraceae, usually, an increase in inflorescence or flower size results in increased geitonogamy (i.e., pollinator-mediated selfing; e.g., [START_REF] Klinkhamer | Plant size and pollinator visitation in Cynoglossum officinale[END_REF]) or an increase in seed predation [START_REF] Fenner | Relationship between capitulum size and pre-dispersal seed predation by insect larvae in common Asteraceae[END_REF]. This variation might thus reflect different reproductive strategies: small capitula, which are also probably less attractive to pollinators [START_REF] Fenner | Relationship between capitulum size and pre-dispersal seed predation by insect larvae in common Asteraceae[END_REF], would still ensure reproduction via selfing, when pollination service is insufficient or uncertain. On the other hand, the larger and more attractive capitula would result in efficient outcross reproduction under good pollination conditions.

At the individual plant level, capitulum structure as a reproductive strategy appears more strongly than at population level: investment in reproduction (disc florets) compared to advertising (ray florets) showed a continuum in experimental plants (Fig. 1), and was essentially due to variation in disc flower number. Plants with lower values of disc:ray floret ratio ( ratio < 1.6 ) may thus be considered as better "selfers" and those with higher values ( ratio > 1.9 ) as mostly outcrossing. Nevertheless, individual plants showed high inter-capitulum variation in number of disc flowers (Fig. 1), even when they tended to have larger capitula, so that plants do not entirely rely on a single reproductive strategy (selfing or allogamy). When accounting for the influence of capitulum structure, plants showed differences in their response to selfing or outcrossing pollination treatments (Fig. 3), with some individuals being more self-incompatible than others. This gives further evidence that self-incompatibility is probably evolving a disruption and it opens the door to selection on other reproductive modes, like reproductive insurance via autonomous selfing, in this species [START_REF] Reinartz | Bottleneck-induced dissolution of self-incompatibility and breeding system consequences in Aster furcatus (Asteraceae)[END_REF].

This study documents a link between capitulum structure and reproductive strategies in an Asteraceae species probably experiencing a breakdown in its self-incompatibility system. Stigma bending during female phase is possibly preadaptive and facilitates evolution towards increased selfing ability. Autonomous selfing may be selected for in small populations, especially if individuals are related and consanguinity interferes with incompatibility [START_REF] Busch | The evolution of self-incompatibility when mates are limiting[END_REF]. Since Centaurea cyanus is declining throughout its range due to massive use of herbicides, and even considered as locally endangered [START_REF] Sutcliffe | Changes in the arable flora of central southern England since the 1960s[END_REF][START_REF] Pausic | Weed diversity in cottage building material used in 19th century: Past and present of the plant occurence[END_REF], these results open several pathways for further investigation. It would be of interest to evaluate levels of inbreeding depression experienced by selfed progenies, and whether it varies among lineages with differences in self-compatibility (e.g., [START_REF] Collin | Early inbreeding depression in the sexually polymorphic plant Dianthus sylvestris (Caryophyllaceae): effects of selfing and biparental inbreeding among sex morphs[END_REF]. Then, the extent and generality of an incompatibility breakdown within the species' range may drive local persistence of populations, especially if selfing ability is correlated to population size.

Finally, self-incompatibility breakdown and selfingability could be influenced by:

(1) Visits of different pollinator species and their relative efficiency to cross-pollinate (e.g., [START_REF] Ostevik | Pollination potential of male bumble bees (Bombus impatiens ): Movement patterns and pollen-transfer efficiency[END_REF]); (2) inflorescence size and competition for pollination; (3) how visits translate into seed-set in natural conditions (e.g., as in [START_REF] Nienhuis | Effectiveness of native bumblebees as pollinators of the alien invasive plant Impatiens glandulifera (Balsaminaceae) in Ireland[END_REF] and under which conditions a newly evolving selfing ability would perform better than outcross. Indeed, natural Centaurea cyanus seed set may be lower in natura than seed sets for our cross pollination treatment. Investigating these interactions between plants and pollinators would inform us as to how the ecology of pollination could drive adaptations to new modes of reproduction.

FIGURE 1 .

 1 FIGURE 1. Variation in disc to ray florets ratio and number of floret types per inflorescence in the experimental Centaurea cyanus plants. Plants are ranked from lowest to largest individual mean disc/ray florets ratio (left y-axis); horizontal lines delineate lower and upper values segregating population in thirds. Mean (± SE) number of ray florets, open bars, and disc florets, filled bars, per plant are presented (right y-axis).

FIGURE

  FIGURE 2. Correlation between seed set and number of disc florets per inflorescence following outcrossing and selfing in Centaurea cyanus. Pollination treatments: outcrossing (dark diamonds and continuous line), and autonomous selfing (white triangles and dotted line).

FIGURE 3 .

 3 FIGURE 3. Individual plants' response to cross-pollination and autonomous selfing in Centaurea cyanus.

TABLE 1 .

 1 Analysis of covariance of seed set per capitulum. Pollination treatments were manual outcrossing and autonomous selfing and the number of disc florets per capitulum was used as covariate.

	Source of variation	df Mean Square F-value	P-value
	Pollination treatment	1	6.21	265.8 < 0.0001 < 0.0001 < 0.0001 < 0.0001
	Number disc florets	1	0.04	1.82	0.180
	Treatment * Nb discs	1	0.33	14.22	0.0002 0.0002 0.0002 0.0002
	Experimental plant	16	0.05	2.01	0.018 0.018 0.018 0.018
	Residuals	112	0.02

ACKNOWLEDGEMENTS

The authors thank Carine L. Collin and two anonymous referees for comments on early versions of the manuscript; and Thierry Fontaine and Fouad Nassur for help in the greenhouse facility. This study is dedicated to Jean-Marie Penet (1950Penet ( -2010)), who made early suggestions about investigating mating consequences of variation in capitulum structure.


Georgios Z Papadopoulos

Fabrice Theoleyre
email: theoleyre@unistra.fr

Xavi Vilajosana

Industrial Internet of Things: Specificities and Challenges

More than one decade ago emerged the Internet of Things (IoT) paradigm, which enabled new applications to be incorporated in our everyday life. We have now a large variety of radio technologies (e.g., IEEE Std 802.15.4-2015, Bluetooth, WiFi, LoRa, Z-Wave) to connect more and more smart objects. Wireless technologies have evolved to address best effort scenarios in which reliability is traded-off by bandwidth and data rate.

The specific requirements imposed by industrial applications are not comparable to mass-market needs driven by the demand of broadband and massive connectivity oriented to end user services and applications. Latency, reliability and energy consumption requirements did not consider the critical operation of industrial assets, which have been left aside and operated by wired buses for the last 30 years. This has shaped the way industries are today and limited solutions in which mobility or the lack of wires would have facilitated production processes. Yet, and understanding that industries equipment is built to last for more than 30 years, wireless could have been seen as a perfect tool to extend and instrument legacy equipment. However, the objectives of the standardization bodies and manufacturers aimed to address other more relevant areas, in which reliability was a secondary objective.

We face to a huge variety of potential applications in the industrial space, including System Automation, Wireless Control, Predictive maintenance and anomaly detection among others 1 . Considering the requirements in terms of reliability, bounded jitter and delay and efficiency, research and standardization are still required to overcome current technological barriers. We need to tackle the problems of adaptability and compatibility to legacy technologies, the standardization barriers and the lack of trust to digital technologies from the industrial sector 2 . Therefore the development of the Industrial IoT is tighten to the advances in ICT technologies in directions that diverge from those developed during the IoT and WSN hypes, opening new research and development opportunities this time, driven by already existing applications whose functionality needs to be broadly extended.

KEY CHARACTERISTICS OF WIRELESS INDUSTRIAL NETWORKS

Wireless transmissions create specific challenges to tackle. Indeed, we need to redesign the algorithms and protocols that were invented to work properly in wired networks where, everything is finely controlled.

Low Power Networks

The wide application ecosystem in the industry opens a large landscape for wireless networking research. Some critical applications may rely on battery-powered devices, e.g., human operators may employ smart remote controls, to control the production line in real-time. In a general manner, wearable devices are often energy constrained [START_REF] Syberfeldt | Augmented Reality Smart Glasses in the Smart Factory: Product Evaluation Guidelines and Review of Available Products[END_REF] .

However, it has to be well understood that machinery is often mains powered and the limits are not imposed by energy availability but rather performance may be the key. Performance can be understood from different angles, including high bandwidth, low jitter/delay or reliability. Understanding this trade off is crucial in order to provide meaningful solutions [START_REF] Kritsis | A Tutorial on Performance Evaluation and Validation Methodology for Low-Power and Lossy Networks[END_REF] . It is important, therefore, to analyze the requirements and differentiate them to those that motivated the research in the early WSN and IoT fields.

Lossy Networks

Wireless transmissions are known to be lossy by nature. A packet may not be decodable by the receiver due to e.g., external interference, a low signal strength. Acknowledgments and Automatic Repeat ReQuests (ARQ) may help to improve the reliability, but they also mechanically increase the number of transmissions, and increase also the latency. Unfortunately, asymmetrical links are common in indoor environments [START_REF] Papagiannaki | Experimental Characterization of Home Wireless Networks and Design Implications[END_REF] and jeopardize these solutions: both directions for a specific radio link may exhibit very different Packet Error Rates.

Even worse, the link characteristics are very time-dependent [START_REF] Pavkovic | Experimental Analysis and Characterization of a Wireless Sensor Network Environment[END_REF] . More precisely, we may face to a very bursty behavior: a sequence of packets may be dropped consecutively, even for medium quality links [START_REF] Brown | BurstProbe: Debugging Time-Critical Data Delivery in Wireless Sensor Networks[END_REF] . Unfortunately, critical applications suffer particularly from this situation if the infrastructure cannot respect a maximum end-to-end latency.

Shared Access

Traditional networks have shifted from a shared access to contention-free Medium Access Control: the physical bandwidth is fully dedicated to a pair of network devices. Wireless networks are based intrinsically on broadcast: any device close to the transmitter is prone to decode the packet if it maintained its radio chipset awake during the whole transmission. This shared access create two antagonistic effects: collisions (negative): two transmitters must share the radio bandwidth. Even worse, collisions may be particularly challenging to tackle in complex topologies, where hidden terminals, and asymmetrical situations may arise;

diffusion: overhearing consists in maintaining several receivers awake to receive a packet. This positive effect may be exploited to improve the reliability while limiting the number of transmissions [START_REF] Koutsiamanis | Meet the PAREO Functions: Towards Reliable and Available Wireless Networks[END_REF] .

For instance, a control packet can be received (and decoded) by any neighbor, limiting also the radio bandwidth used by the transmitter. Similarly, high reliability may be achieved with anycast routing: several receivers are selected for a single transmission, to reduce the probability of packet losses [START_REF] Huynh | On the interest of opportunistic anycast scheduling for wireless low power lossy networks[END_REF] .

Last but not least, this effect tends to be exacerbated by the fact that wireless networks often provide a low bandwidth, several orders of magnitude lower than for traditional wired networks.

OPEN CHALLENGES AND OPPORTUNITIES

Creating added value in the industrial application landscape requires the development of tailored technologies that address the important challenges of the sector. In the following we review some of them:

Interoperability with Industrial Standards and Architectures

Industrial standards and ICT standards are driven by non-connected committees causing the evolution of the fields to diverge while sometimes addressing the same sort of problems. A question now that ICT technologies and industrial application need to meet is how these worlds will interact and converge and what architectures will be developed so technologies that have been designed by the industry can now be integrated to digital systems and vice versa, of course, without having to duplicate the efforts. Research directions towards enabling digital plug and play technologies in industrial settings need to be investigated, also the development of measures to support the fast evolution of ICT technologies, a very counter-intuitive fact for industrialist which have protocols that are widely used for decades.

Mobility Support

One of the key challenges when instrumenting industrial scenarios is the support to moving objects. Today, mechanical solutions are developed in order to connect wires to moving objects such as robotic arms. Yet those impose important mobility limitations and become real maintenance problems. Dealing with mobility is therefore a challenge to be addressed by wireless technologies fostering its applicability in the industrial space. Among other it seems important to address mobility while still ensuring deterministic properties in the wireless links. This, per se, may require a combination of different techniques, being reactive to topology changes or network configuration while still meeting the stringent performance requirements in terms of latency or bounded packet losses.

Wireless for positioning

One of the most researched technologies in the last 15 years is indoors positioning. Industries are the potentially major adopters of such technologies as identifying the position of assets and workers is a critical information to improve work efficiency and safety. Wireless networks are key to fulfil the vision in which industrial objects are precisely located in the production lines, but yet, and due to the nature of industrial settings, it is still open to understand if the performance of such techniques is suitable for the scenarios. Approaches based on angle of arrival, time of flight and ultra-wide band pulses may now be challenged against industrial conditions.

High performance wireless

Derived from the WSN and IoT requirements, wireless networks with potential application in the Industrial field have been developed with low power as a key constraint. This has imposed many restrictions to the performance of that technologies leaving aside properties that may be valuable in industrial fields. Automation and control applications for example rely in control loops with very significant timing constraints. Common control loops in industrial buses have periods of 1 to 10ms. Wireless networks with that capabilities need to trade of performance to resource utilization which has a direct mapping to energy consumption. Research directions to address these requirements are therefore relevant now that wireless may enable new ways of building machinery, with less mechanical restrictions but always conditioned to address reliably this high performance needs.

Reproducibility

The behavior of a wireless infrastructure has to be reproducible, leading to the same results, when facing to the exactly same conditions [START_REF] Papadopoulos | Thorough IoT testbed Characterization: from Proof-of-concept to Repeatable Experimentations[END_REF] . Unfortunately, we do not have yet the keys to predict how exactly a wireless infrastructure will behave before a real deployment. In particular, how can we explore the network capacity limits? With battery-powered nodes, could we predict accurately their lifetime?

Consequently, we need to more accurately explore the reproducibility concept, and to construct benchmarks. To our mind, reproducibility requires, i) Exhaustive description of the setup so that we can reproduce the same situation; ii) Scenario Replay, where everything is scripts and automatized; iii) A raw dataset is provided by the benchmark; iv) A public repository should collect the whole dataset, so that any researcher or engineer can analyze the results, and verify that the solution fits well the requirements he/she identified.

Tragedy of the Commons

We envision a wide adoption of the IoT technologies for a large set of innovant services. Typically, several networks may be deployed in a smart factory, possibly using different wireless technologies. In this context, each deployed network must carefully handle external interference, which is exhibits a local pattern [START_REF] Kotsiou | Is Local Blacklisting Relevant in Slow Channel Hopping Low-Power Wireless Networks?[END_REF] : a radio channel may perform very poorly for some radio links, while providing a very high reliability for other ones. Thus, blacklisting techniques try to identify the good radio channels to use only the most efficient ones [START_REF] Kotsiou | Blacklisting-Based Channel Hopping Approaches in Low-Power and Lossy Networks[END_REF] . However, less selfish approaches should also be investigate. Indeed, we should avoid the tragedy of the commons that very dense deployments will for sure occasion [START_REF] Feeney | Avoiding an IoT 'Tragedy of the Commons[END_REF] . The different networks and technologies have to cooperate to share fairly and efficiently the bandwidth. It is not acceptable that e.g., Wi-Fi networks may preempt the medium compared with low power networks. Explicit resynchronization 14 among different networks helps to reduce the number of collisions. However, implicit, technology specific mechanisms have still to be proposed.

THIS SPECIAL ISSUE

This Special Issue comes really timely for relevant research communities, and provides a valuable contribution to this emerging field. Via a thorough review process, five papers have ultimately been selected in this special issue. Since we selected the papers based on their scientific quality and the strength of their contributions, we cannot argue that they cover all the topics of the domain. However, we are delighted to present a consistent special issue, covering a large set of problems in the Industrial Internet of Things.

The first paper "Operations, Administration and Maintenance (OAM) features for Reliable and Available Wireless (RAW) Networks" details the mechanisms to set-up to maintain a wireless industrial network fully efficient. Indeed, the wireless infrastructure must respect strictly some Service Level Objectives by using wireless transmissions, which are stochastic by nature. Thus, we need mechanisms to supervise, detect faults, and reconfigure the network to keep operating smoothly without any end-to-end failure. By exploiting the redundant nature of the wireless topology, it is expected to maintain high reliability and to respect upper bounded end-to-end latency.

The second paper "Impact of Interference Aware Metrics on Iterative Multipath Routing for Industrial WSN" focuses on the routing problem. Indeed, multipath routing seems required to provide high reliability: by exploiting multiple paths in parallel, the wireless infrastructure can be fault tolerant. However, we have to select the most efficient set of paths, i.e., the paths which minimize mutual interference. This paper tries to quantify the accuracy of the routing metrics to capture this independency metric.

6TiSCH is a very promising stack for industrial networks based on the popular IEEE802.15.4-TSCH standard. Two papers focus on this specific technology. The paper "6TiSCH Minimal Scheduling Function: Performance Evaluation" focuses on the scheduling problem. More precisely, the scheduling function is in charge of determining how many transmission opportunities should be reserved by each radio link. While this approach is fully distributed, the minimum amount of bandwidth has to be reserved to respect the SLO while minimizing both the energy consumption and the radio resource reserved for a given flow.

The paper "Trace-Based Simulation for 6TiSCH" tries to respond to the reproducibility problem. It is extremely time consuming to conduct experiments, challenging to interpret the results, and complicated to provide reproducible results. Thus, exploiting a dataset seems a promising way to mix the advantages of experiments and simulations, as a first step before realworld evaluations. This paper presents a very practical tool for the research community, to provide a benchmarking solution for simulations in wireless industrial networks that use the 6TiSCH stack.

Finally, the paper "TSCH-over-LoRA: Long Range and Reliable IPv6 Multi-hop Networks for the Internet of Things" explores how the TSCH concepts may be adapted to another wireless technology. LoRa enables long range communications, but is not deterministic: it is very challenging to provide both high reliability and a bounded latency. Thus, scheduling the transmissions on top of a LoRa radio chipset allows the network to control finely the latency and to avoid collisions. This paper represents a pioneering piece of work to explore how any wireless technology may be transformed to support real-time flows.

ACKNOWLEDGEMENTS

The guest editors are thankful to the reviewers for their effort in reviewing the manuscripts. A special thank you to Editor-in-Chief Prof. Luigi Alfredo Grieco for his supportive guidance throughout the process.