猪最长肌（骨骼肌的一种）总脂肪和脂肪酸含量的检测

智能文字提取功能测试中

骨骼肌的转录组学分析揭示了在葡萄牙当地猪品种中影响肌肉生长和相关脂质组成的候选基因Transcriptomic Profiling of Skeletal Muscle Reveals Candidate Genes Influencing Muscle Growth and Associated Lipid Composition in Portuguese Local Pig Breedsanimals 2 of 21Animals 2021， 11，1423 Article 骨骼肌的转录组学分析揭示了在葡萄牙当地猪品种中影响肌肉生长和相关脂质组成的候选基因 Transcriptomic Profiling of Skeletal Muscle Reveals Candidate Genes Influencing Muscle Growth and Associated Lipid Composition in Portuguese Local Pig Breeds Andre Albuquerque 1*D ， Cristina Ovilo 2D， Yolanda Nufez 2， Rita Benitez 2D， Adrián Lopez-Garcia 2²iD Fabián Garcia 2， Maria do Rosario Felix 3， Marta Laranjo 1D， Rui Charneca 4D and Jose Manuel Martins 5，*i D 葡萄牙埃库特大学地中海农业、环境与发展研究所 西班牙国家农业和食品研究技术研究所(INIA)动物遗传改 良室 check for updates 10.3390/ ani11051423 Academic Editor： J uan V i cente Delgado Bermejo Received： 26 March 2021 Accepted： 12 May 2021 Published： 16 Mav 2021 Publisher's Note： MDPI stays neutra l with regard to jurisdictional claims in published maps and institutional affil -iations. Copyright： C 2021 by the authors.Licensee MDPI ， Basel， Switzerland.This article is an open access article distributed u un n d d e e r r t t h he e terms and conditions of the Creative Commons Attribution (CC BY) license (https：//creativecommons.org/licenses/by/4.0/). 1 MED-Mediterranean I nstitute for Agriculture， Environment and Development， Instituto de Investigacao e Formacao Avangada & Universidade de Evora， Polo da Mitra， Ap. 94，7006-554 Evora， Portugal；mlaranjo@uevora.pt 2 Departamento de Mejora Genetica Animal， I ns t ituto Naciona l de I nvest i gacion y Tecnologia Agraria y Alimentaria (INIA)， 28040 Madrid， Spain； ovilo@inia.es (C.O.)； nunez.yolanda@inia.es (Y .N.)；rmbenitez@inia.es (R.B.)； adrian.lopez@inia.es (A.L.-G.)； fabian.garcia@inia.es (F.G.) 3 MED & Departamento de Fitotecnia， Escola de Ciencias e Tecnologia， Universidade de Evora， Polo da Mitra，Ap. 94， 7006-554 Evora， Portugal； mrff@uevora.pt 4 MED & Depar t amento de Medicina Veterinária， Escola de Ciencias e Tecnologia， Universidade de Evora， Polo da Mitra， Ap. 94， 7006-554 Evora， Portugal ； rmcc@uevora.pt 5 MED & Departamento de Zootecnia， Escola de Ciencias e Tecnologia， Universidade de Evora ， Polo da Mitra，Ap. 94， 7006-554 Evora，Portugal Correspondence： andrealb@uevora.pt (A.A.)； jmar t ins@uevora.pt (J .M.M.) Simple Summary： Screening and interpretation of differential l y expressed genes and associated bio-logical pathways was conducted among experimental groups wi t h divergent phenotypes providing valuable information about the metabolic events occurring and identification of candidate genes with major regulation roles. This comparative transcriptomic analysis includes the first RNA-seq analysis of the Longi s simus lumborum muscle tissue from two Portuguese autochthonous pig breeds with different genetic backgrounds， Alentejano and Bisaro. Moreover， a complementary candidate gene approach was employed to analyse， by real t i me qPCR， the expression profile of r elevant genes involved in lipid metabolism， and therefore with potential impacts on meat composition. This study contributes to explaining the biological basis of phenotypical differences occurring between breeds，particularly the ones related to meat quality traits that affect consumer interest. Abstract： Gene expression is one of the main factors to influence meat quality by modulating fatty acid metabolism， composition， and deposition rates in muscle t i ssue. This study aimed to explore the transcriptomics of t he Longissimus l umborum muscle in two local pig breeds with distinct genetic background using next-generation sequencing technology and Real-Time qPCR. RNA-seq yielded 49 differentially expressed genes between breeds， 34 overexpressed in the Alentejano (AL) and 15in the Bisaro (BI) breed. Specific slow type myosin heavy chain components were associated with AL (MYH7) and BI (MYH3) pigs， whi l e an overexpression of MAP3K14 in AL may be associated with their lower loin proportion， induced insulin resistance， and increased inflammatory response via NFkB ac t ivation. Overexpression of RUFY1 in AL pigs may explain the higher i ntramuscular (IMF) content via higher GLUT4 recruitment and consequently higher glucose uptake that can be stored as fat . Several candidate genes for l ipid metabolism， excluded in the RNA-seq analysis due to low counts， such as ACLY， ADIPOQ，ELOVL6，LEP and ME1 were identified by qPCR as main gene factors de fi ning t he processes that i nfluence mea t composition and quality. These results agree with the fatter profile of the AL pig breed and adiponectin resistance can be postulated as responsible for the overexpression of MAP3K14's coding product NIK， failing to restore insulin sensitivity. Keywords： Alentejano pig； Bisaro pig； t ranscriptome； skeletal musc l e； meat quality； intramuscular fat；MYH3；MYH7；MAP3K14 1. Introduction Alentejano (AL) and Bisaro (BI) are the prevail i ng autochthonous pig breeds in Por-tugal. AL is reared i n the southern region [1] and shares a genetic closeness with the Iberian pig [2]， showing g s S low growth rates (excluding when under the late "montanheira"fattening regime) and an early and high adipogenic activity [3，4]. BI pigs， on the other hand， common to the northern region， belong to the Celtic line and share ancestors with leaner and highly productive breeds [5]. Furthermore， BI have a lower predisposi t ion for (monounsaturated) fa t accumulation when compared to AL pigs， but higher than most commercial lean breeds [6]. These two breeds are well-adapted to the environment， and their production chains provide high quality meat and fermented and dry-cured meat products [3，6]. Fatty acid content and composition are two of the most important factors that influence overall meat quality and consumer preference. A low saturated fatty acid (SFA) content is often desired because increases in this content have been found associated with raising blood cholesterol levels， particularly low-density lipoprotein cholesterol (LDL-c)， increasing the risk of heart diseases [7]. On the other hand， increased monounsaturated fatty acid (MUFA) and essential polyunsaturated fatty acid (PUFA) contents are useful i n decreasing LDL-c levels while increasing high density lipoprotein-cholesterol， and therefore reducing the risk of heart diseases [8]. Meanwhile， today's consumers are more aware of the specific nutritional value associated with meat， and that increased fat content contributes to better meat fl avour while improving tenderness and juiciness， particularly when it occurs as intramuscular fat (IMF) at l evels higher than 2.5%[9-11]. These fat stores can appear associated to intramu mvc scular adipocytes or as droplets in the myofiber cytoplasm and can hold excess phospholipids， triacylglycerols， and cholesterol [12，13]. Although IMF content in AL pigs is generally higher than that in BI or other leaner highly productive breeds，their levels tend to fluctuate among several studies， ranging from 3 to 8%， mainly due to feeding and rearing conditions [3，14]. IMF content of the Longissimus lumborum muscle (LL)is determined and regulated by multiple metabolic pathways and is associated with the expression of genes involved in carbohydrate and lipid metabolism， cell communication，binding， response to stimulus， cell assembly， and organisation [15，16]. When compared to those of highly productive breeds， AL carcasses present a lower lean meat content (ranging from 37.5 to 51%) due to the above mentioned higher adipogenic activity and l ipid deposition [3，14] while BI carcasses yield a moderate lean meat content (from 46 to 51%) [6]. In the Longissimus lumborum muscle， AL pigs present a high MUFA level (48-58%)， particularly rich in oleic acid， and a low SFA content (35-44%) [3，17]. Studies regarding BI fat composition are scarce but show that MUFA levels (44-47%) [6，18]are lower and SFA levels (33-40%) similar or slightly lower than in AL pigs. Therefore， and when we consider the effects that unsaturated and saturated fatty acids have on consumers'health， BI pigs seem to display a slightly better balance of the unsaturated to saturated fatty acids ratio than AL pigs. On the other hand， higher PUFA levels are found in BI when compared to AL or improved genotypes， attaining 10% or higher [3，6，19]. RNA-seq experiments take advantage of nex t -generation sequencing developments to enable a quantitative screening for distinct gene expression patterns in individuals at the transcriptome level . Interpretation of differentially expressed genes (DEGs) and associated biological pathways provide valuable information about the metabolic events occurring and identification of candidate genes with major regulation roles. This comparative tran-scriptomic analysis includes the first RNA-seq analysis of the Longissimus lumborum muscle tissue from these two autochthonous pig breeds. Moreover， a complementary candidate gene approach was employed to analyse， by real time qPCR， the expression profile of rele-vant genes involved in lipid metabolism. This study can contribute to explain the biological basis of the phenotypical differences occurring between t hese breeds， particularly the ones related to meat quality traits that affect consumer interest. 2. Materials and Methods The experiment was conducted in accordance with the regulations and ethical guide-lines of t he Portuguese Animal Nutrition and Welfare Commission (DGAV， Lisbon， Portu-gal) following the 2010/63/EU Directive. Staff members of the team involved in animal trials were certified for conducting l ive animal experiments by the Directorate of Animal Protection (DSPA， DGAV， Lisbon，Portugal). 2.1. Sampling and FA Profiling 最长肌肌肉样品 Ten purebred male castrated AL and BI pigs (n =5 for each breed) were reared in a traditional free ranged system and individually fed commercial diets at estimated ad libitum consumption from ~65 kg body weight until slaughter (~150 kg)， as previously described [14]. Longissimus lumborum muscle samples were obtained at slaughter， snap frozen in l iquid nitrogen and maintained at -80 °C until further use. Total lipids were obtained using a Soxtherm automatic apparatus (S206 MK， Gerhardt).The respective FA profile was determined from the lipid extract， according to a previously described method [20]. After methylation [21]， the FA samples were identified by GC-MS QP2010 Plus (Shimadzu Corporation， Kyoto， Japan) and a 60 m ×0.25 mm x 0.2 um fused silica capillary column (Supelco SPTM 2380， Baltimore， PA， USA). The chromatographic conditions were as follows ： injector and detector temperatures were set at 250 and 280°C，respectively； helium was used as the carrier gas at 1 mL/min constant flow； the initial oven temperature of 140 °C was held for 5 min， increased at 4°C/min to 240 ℃ and held for 10 min. MS ion source temperature was set at 200 °C and interface temperature at 220°C.Identification of FAMEs was based on the retention time of reference compounds. 2.2. RNA Extraction and Sequencing Total RNA was isolated from 50-100 mg samples of LL following Ambion RiboPureTM Ki t (Thermo Fisher Scienti f ic， Waltham， MA， USA) instructions. Total extracted quantity obtained was measured using NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific， Waltham，MA， USA)， while RNA quality was assessed with an Agilent 2100BioanalyzerTM (Agilent Technologies， Santa Clara， CA， USA) following Agilent RNA 6000Nano Kit instructions， along with NanoDropTM 1000 260/280 and 260/230 coefficients. RIN values ranged from 7.8 to 9.0 with an average of 8.42. The total RNA was diluted into a concentration of 100 ng/uL and 3 ug were sent for stranded paired-end mRNA-seq sequencing in Centro Nacional de Andlisis Genomico (CNAG-CRG， Barcelona， Spain) on a HiSeq2000 sequence analyser (Ilumina， Inc.， San Diego， CA， USA). Currently， several RNA-seq experiments are performed at a low replication level and severa l publications suggest that a minimum of 2-3 replicates can be considered [22-24]. 2.3. Quality Control， Mapping and Assembly Generated Fastq f iles were analysed using FastQC (version 0.11.8) for quality con-firmation [25] and Trim Galore (version 0.5.0) [26] was used to trim sequence reads for adapters， poli-A and poli-T tails. Low quality nucleotides (Phred Score， Q<20) as well as short length reads (<40) were also removed， and the remaining reads were mapped to the reference pig genome version Sscrofa11.1 (Ensembl release 94) using HISAT2 version 2.1.0 [27]. Samtools-1.9 [28] was used to convert the obtained SAM files to BAM. Read counting and merging was performed with HTSeq-count version 0.11.1[29]. 2.4. Differential Expression Analysis The obtained Gcount files were analysed using the R package DESeq2 [30]， which estimates gene expression levels by counting total exon reads for the statistical analysis.Normalised counts were fi l tered for a minimum of 50 reads per group， and genes were considered as differently expressed when featuring an FDR lower than 0.05. The raw data shown and interpreted in this publication have been deposited in NCBI's Gene Expression Ombibus [31] and are available through the GEO Series accession number GSE159817 (https：//www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159817， accessed on 1 March 21) 2.5. Enrichment Analysis An enrichment analysis based on t he functional annotation of the differentially ex-pressed genes was performed using the I ngenuity Pathways Analysis software (IPA；QIA-GEN， Redwood City， CA， USA) to better understand their biological implications within the muscle tissue context . The list of DEGs (q<0.05， log2FC≤-0.7Vlog2FC≥0.7)was uploaded into the software and converged with IPA’s own l ibrary (Ingenuity Path-way Knowledge Base) to determine biologically pertinent i nformation such as activated pathways， functions and regulators [32]. 2.6. Real Time qPCR Real T ime quantitative PCR was performed to validate the data obtained by RNA sequencing (MYH3，MYH7，TNNT1，MAP3K14， WDR91，FBXO32 and FASN) and explore other meat quality candidate genes outside the RNA-seq output (ACACA， ACLY，ADIPOQ，ELOVL6， LEP，ME1 and SCD). Additional information on the selected primers can be found in the Supplementary Table S1. MAP3K14，MYH3， TNNT1 and WDR91 primers were designed using Primer3 version 0.4.0. Previously extracted total RNA from the experimental animals was reverse transcribed in 20 uL reactions using Maxima" First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific， Waltham，MA， USA) following manufac t urer's instructions. The reaction mixes containing 12.5 uL of NZY qPCR Green Master Mix (2×) (NZYtech，Lisbon， Portugal)， 0.3 uM of each sense primer and 12.5 ng of cDNA per sample were prepared in 96-well plates and run in a LineGene9600 Plus system (BIOER， Hangzhou，China). Standard PCRs were performed to confirm amplicon sizes， no-cDNA negative controls were performed within every plate and three replicates were performed per target sample. Cycling conditions included an initial 10 min holding denaturation stage at 95℃，followed by 40 ampli f ication cycles of 15 s denaturation at 95 °C and 50 s at 60°C. To test PCR specificity a dissociation curve was added as a final step to the program comprising a single cycle at 95 °℃(15s) followed by 60°C(60s)，and a ramp-up0.2 °C/s to 95 °℃ for 15 s with acquired f luorescence. Moreover， PCR efficiency was predicted by standard curve calculation using five points of cDNA dilutions (1：4； 1：8； 1：16；1：32；1：64). ACTB， HSPCB，RPL19 and TOP2B were the most stable genes tested using the geNorm software [33] and were， therefore， chosen as endogenous control genes for target normalisation (m<1.5).Cycle Threshold values were regressed on the log of the previously constructed template cDNA curve. 2.7. Statistical Analysis Results are presented as the mean ± SE. All data were tested for normal i ty using the Shapiro-Wilk test. Individual data of growth， plasma， carcass and meat quality traits were analysed by one-way ANOVA with genotype as the main effect. For the carcass data，hot carcass weight was included as a covariate in the model. The SPSS Statistics software (IBM SPSS Statistics for Windows，v24.0； IBM Corp.， Armonk， NY， USA) was used for data analysis. Mean differences were considered significant when p < 0.05， and values between 0.05 and 0.10 were considered trends. To determine i f the gene expression values were significantly different between the ex-perimental groups， a student’s t-test was executed using IBM SPSS Statistics software (IBM SPSS Statistics for Windows， Version 24.0. Armonk， NY： IBM Corp.) with an established significance l evel of p < 0.05. Equal variances of the samples were checked with Levene’s Test for Equality of Variances with values lower 0.05 not considered as equal variances and another Independent Samples Test was performed assuming no equal variances. Equal variances were not assumed for WDR91 (F =7.643； p=0.024) and LEP (F=8.929；p=0.017).Pearson correlation coefficients and associated p-values were also estimated. 3. Results and Discussion 3.1. Productivity of Alentejano and Bisaro Breeds -Summary of Carcass Traits and FA Proportion In previous studies， we assessed data of AL， BI and their reciprocal crosses， regarding their blood parameters， as well as their productive and meat quality traits， including the Longissimus lumborum. Briefly， BI showed significantly better carcass traits than AL and intermediate values were obtained in the crossed pigs (Table 1) [14]. Table 1. Zootechnical traits and physical-chemical parameters from muscle Longissimus lumborum of Alentejano (AL) and Bisaro (BI) pigs slaughtered at~150 kg LW. Trait AL (n=5) BI (n=5) Significance Mean SE Mean SE Days on trial 150.6 5.5 135.2 11.9 0.273 Average daily gain (g/day) 582.4 18.1 656.3 63.8 0.297 Feed conversion ratio (kg/kg) 5.45 0.21 0.47 0.039 Average backfat thickness (cm) * 7.9 0.4 43 0.3 <0.0001 Longissimus lumborum (%) 3.63 0.26 5.14 0.52 0.030 Moisture (g/100 g) 70.6 72.3 0.008 Total protein (g/100g) 23.7 23.4 0.5 0.561 Total intramuscular fat (g/100 g) 7.3 5.7 0.2 0.001 Myoglobin content(mg/g) 0.83 0.12 0.43 0.04 0.014 Total collagen (mg/gDM) 13.7 0.6 16.3 0.7 0.025 pH (24 h post-mortem) 5.76 0.03 5.50 0.04 0.001 Drip loss (g/100 g) 0.55 0.07 2.25 0.21 <0.0001 Note： * Average of measurements taken between last cervical and f i rst thoracic vertebrae (f irst rib)， and last thoracic and first -lumbar vertebrae (last rib). In this study， five randomly selected individuals from pure AL and BI breeds were chosen for transcriptome analysis through RNA-seq. AL pigs averaged a total of 150.6 days on trial with an average daily gain (ADG) of 582 g/d while BI averaged 135.2 days on t rial (p=0.273) and an ADG of 656 g/d (p =0.297). At 150 kg and when compared to BI pigs， AL showed higher plasma total protein (69.8 vs. 64.4 g/L，p<0.05)， urea (6.9 vs. 5.6 mmol/L，p < 0.05) and total cholesterol (2.66 vs. 2.23 mmol/L，p <0.05). Average backfat thickness was also significantly higher in AL (7.9 vs. 4.3 cm， p < 0.001) when compared to BI pigs.Regarding the LL muscle， AL pigs presented a lower loin proportion (3.63 vs. 5.14%，p < 0.05) but higher total IMF (7.3 vs. 5.7 g/100 g， p <0.01) when compared to BI pigs.Several blood parameters from these pigs are l isted in Tables S2 and S3. The FA analysis identified oleic acid (C18：1 n-9) as the most represented fatty acid in the LL muscle of both breeds， with AL showing higher values when compared to BI (35.00 vs. 29.91%， respectively， p<0.05). The total MUFA (C16：1 n-7， C18：1 n-7， C18：1n-9， and C20：1 n-11) proportion was also higher in AL pigs (45.42 vs. 39.47%， p<0.05).Regarding SFAs， palmitic acid (C16：0) was the most represented， with similar proportion in both breeds (22.28 vs. 21.22%，p=0.139) while stearic acid (C18：0) was lower in AL pigs (10.77 vs. 12.38%，p<0.01) when compared to BI . Tota l SFA (C10， C14， C16， C17， C18，and C20) proportion was also comparable between breeds (34.68 vs. 35.24%，p=0.587). Finally，in respect to polyunsaturated fatty acids (PUFA)， li noleic acid (C18：2 n-6) was the one most represented in both breeds， but a lower proportion was found in AL (12.06 vs. 15.17%，p<0.01). Total PUFA (C18：2 n-6， C18：3 n-3， C18：3 n-6， C20：2 n-6， C20：3 n-6， C20：4 n-6，C20：5 n-3， C22：4 n-6， C22：5 n-3， and C22：5 n-6) proportion was also lower in AL when compared to BI pigs (19.90 vs. 25.29%，p<0.01). 3.2. Mapping and Annotation More than 750 million reads were i ni t ially obtained with an average number of sequenced reads over 39 million per sample. Read length was 76 base pairs. Al l samples shared an average associated qual i ty score proximate to 40 and tota l GC content ranged from 53 to 56%. HISAT2 mapping of treated sequences was successfully achieved with an overall 97% of alignment rate， similar to a previous study conducted by our team with adipose tissue [34] but slightly higher than several previous pig transcriptome studies [35-40]. 3.3. Differential Gene Expression with DESeq2 and qPCR Comparison More than 25 k genes were firstly detected in the muscle tissue with around 3.6 k coding genes obtained after the initial f iltering. The overall top f ive most expressed genes included rabaptin (RAB GTPase Binding Effector Protein 2， RABEP2)， septin 1(SEPTIN1)， a non-identified coding gene (ENSSSCG00000036235)， cathepsin F (CTSF)and SplA/ryanodine receptor domain and SOCS box containing 2 (SPSB2). These genes averaged a total read count per LL sample between 340 k (RABEP2) and 89 k (SPSB2). A total of 49 genes were found to be differentially expressed (DE)， with 34 being overexpressed in AL (log2 FC ≥0.7， q<0.05) and 15 i n BI (log2 FC≤-0.7，q<0.05). In our LL samples， the most overexpressed gene in AL was the Cyclin and CBS domain divalent meta l cation transport mediator 3 (CNNM3) (log2 FC=4.487，q<0.01)， while in BI the most overexpressed gene was Stathmin 3 (STMN3) (log2 FC= -4.033，q<0.01). The full detailed list of DEG's can be found in Table S4. Log2 Fold Change 口 RNAseq 口 Real Time qPCR Figure 1. Gene expression comparison of MYH3 (Myosin heavy chain 3)， MYH7 (Myosin heavy chain 7)， TNNT1 (Troponin T1)， MAP3K14 (Mitogen-activated protein kinase kinase kinase 14)， FBXO32 (F-Box Protein 32)， WDR91 (WD Repeat Domain 91) and FASN (fatty acid synthase) with RNA-seq and Rea l Time qPCR of the Longissimus lumborum of Alentejano (AL) and Bisaro (BI) pigs (n=10). Positive values indicate overexpression in AL and negative values overexpression in BI . Pearson corre l ation values f luctuated between 0.93 (MYH3) and 0.58 (FASN). Significance of the correlation： *** p<0.001， **p<0.01，*p<0.05. Surprisingly， a gene encoding for another myosin heavy chain component， MYH7，showed a trend towards AL (log2 FC=0.921，q=0.076)， a difference boosted by qPCR (log2 FC =1.025，p<0.01). MYH7 is also a molecular marker for slow/type 1/oxidative muscle fibres but is generally associated with the heavy chain subunit of cardiac myosin，although it can also be found expressed in skeletal muscle tissue [47]. Another important factor regulat i ng muscle contraction i s troponin T1 (TNNT1)， a subuni t of troponin which， as MYH7， tended to be overexpressed in AL either in RNA-seq (log2 FC=0.764，q=0.091) as in qPCR (log2 FC=0.738，p=0.070). TNNT1 expression is also specific of slow skeletal muscle f ibres in vertebrates and regulates muscle contraction through the troponin complex which is mediated by calcium concentration in the cells [48]. In our results， we associate both MYH7 as well as TNNT1 signalling in AL with an increase in the differentiation of slow muscle fibres， and to a stronger extent to what occurs i n BI pigs t hrough MYH3 signalling. No markers for fast skeletal muscle fibres were detected in the p <0.05 or either p < 0.1 significance range suggesting identical presence of this fibre type in both breeds. On t he other hand， red f ibre type presence is suggested to be l inked to MYH3 signalling in BI pigs， and MYH7 and TNNT1 signalling in AL pigs. Consequently， a higher presence of slow muscle fibres in AL pigs is associated with inducing reddish meat with increased IMF content and overal l meat quality. The SET and MYND Domain-Containing Protein 5 (SMYD5) is a member of the SMYD family of methyltransferases which play a crucial role in manipulating gene expression through post translational modifications on unique histone residues and other proteins [49].SMYD proteins have been recognised as key regulators in skeletal and cardiac musc l e devel-opment and function， though litt l e i s known about t he specific roles l inked to SMYD5 [50].Some studies also suggest a role of SMYD5 in stimulating an anti-inflammatory response in Drosophila [51] and regulation of hematopoiesis in zebrafish [52]. In our data， SMYD5 was found significantly overexpressed in BI pigs (log2 FC=-2.171，q<0.01) which agree with the higher ability of this breed to increase muscle tissue when compared to AL pigs， as well as with our previously proposed obesity-induced chronic inflammation state， particularly in AL pigs [34]. Another determining factor of histone modification and transcriptional regulation found in our results was the gene encoding for lysine demethylase 2B (KDM2B)， found overexpressed in AL pigs (log2 FC=1.074，q<0.01). KDM2B i s generally associated with the demethylation of histones H3K4， H3K36， and H3K79， and repression of transcription.Recently， KDM2B was suggested to demethylate the non-histone target serum response fac-tor (SRF )， consequently inhibiting skeletal muscle cell differentiation and myogenesis [53].On the other hand， KDM2B has also been heavily linked with an increased inflammatory response through epigenetic regulation of interleukin 6 [54]. Overexpression of KDM2B in AL pigs supports our previous hypotheses of lower muscle deposition and higher inflammatory state occurring i n AL pigs when compared to BI pigs. The lymphocyte specific protein 1 (LSP1) was found to be significantly overexpressed in BI pigs (log2 FC=-1.052，q<0.01). LSP1 encodes for an intracellular F-actin binding protein that participates in functions such as cell migration and signalling， particularly by regulating the recruitment of circulating leukocytes to inflamed sites [55]. In 2013，Ehrlich [56] first suggested the role of LSP1 in influencing skeletal muscle development.Results from his work consistently associated an exceptional myogenic differential methyla-tion in various subregions of the LSP1 gene through binding to the myogenic transcription factor MYOD， particularly in the murine region of the gene. Consequently， DNA methyla-tion status of LSP1 may prove key for alternative promoter usage and stimulating highly specific myogenic f actors in BI pigs. WD Repeat Domain 91 (WDR91) was found to be significantly overexpressed in AL pigs t hrough RNAseq (log2 FC=2.818，q<0.01) as well as qPCR (log2 FC=1.197，p<0.05).This gene encodes for a protein t hat is known to negatively regulate a core subunit of the phosphoinositide 3-kinase (PI3K) complex. This complex is involved in the regulation of several functions including cell growth， proliferation， and differentiation. PI3K signals a network that ultimately leads to mTOR activation [57，58]. Continuous inhibition of this pathway in AL pigs suggests a decrease in overall protein synthesis which agrees with the lower muscle deposition when compared with BI pigs. Furthermore， PI3K activity can act as a molecular switch to regulate correct insulin signalling and activation [59].Downregulation of this pathway through WDR91 agrees with the proposed lower i nsulin sensitivity in AL pigs via PI3K inhibition. WDR91 has also been previously associated as a potential epigenetic regulator of skeletal muscle stem cells in adult mice， which are crucial for the maintenance and regeneration of adult skeletal muscles [60]. The role of epigenetics in t he modulation of myogenesis is a current topic of increas-ing scientific interest， particularly due to the development of new methods that prof i le methylation. Mammalian DNA methylation is known to regulate the expression of specific target genes through silencing or upregulation， controlling the direction of major metabolic pathways [56]. Our results suggest a solid presence and influence of these mechanisms，particularly through ac t ivation of SMYD5 and LSP1 in BI and KDM2B and possibly WDR91in AL pigs， in stimulating BI muscle growth and inhibiting skeletal muscle differentiation in AL pigs， respectively. consequently， l imit new muscle growth when compared to BI pigs， justifying the lower loin weight in AL pigs. The mitogen-activated protein kinase kinase kinase 14 (MAP3K14) was to be found significantly overexpressed in AL with RNA-seq (log2 FC= 1.829，q<0.01)， a result also confirmed with qPCR (log2 FC =0.983，p<0.05). MAP3K14 is a gene encoding for a serine/t hreonine protein-kinase (NF-xB-inducing kinase， NIK) that binds and transcription-ally regulates t he expression of a number of molecules such as proinflammatory cytokines and chemokines [65]. High levels of MAP3K14 have previous l y been associated with skele-tal muscle catabolism and atrophy [66]， t hrough increased expression of myostatin and decreased MyoD， which can be associated with the reduced loin proportion in the carcass of AL pigs when compared to that observed in BI (3.63 vs. 5.14%， p<0.05). Several studies have also previously proposed the linkage of NIK overexpression with induced skeletal muscle insulin resistance and chronic inflammation development [67，68]， in agreement with our previous suggestion of lower insulin sensitivity of the AL breed [34]. MAP3K14has also been previously reported as a candidate gene to control feed efficiency in Duroc pigs [69] and its overexpression in AL agrees with the higher feed conversion ratios esti-mated in our AL and BI pigs (5.45 vs. 4.30 kg/kg，p<0.05). Furthermore， in the hepatic tissue of obese mice，MAP3K14 has been also shown to reduce l ipid oxidation via inhibition of peroxisome proliferator-activated receptor alpha (PPARA) [70]. While effects on the muscle tissue remain unclear in the current literature， a similar outcome occurring in the skeletal muscle of our pigs would agree with the fatter profile of AL’s meat. The ATPase H+ transporting V1 subunit C1(ATP6V1C1) encodes for a component of vacuolar-type proton-translocating ATPase (V-ATPase) which is responsible for mediating the acidification of numerous intracellular components and was found to be overexpressed in AL pigs (log2 FC=0.760，q=0.046). This subunit C1 of V-ATPase i s highly expressed in osteoclasts which participate in the breakdown of bone tissue [71] and may contribute to the higher bone mass found in BI when compared to AL pigs [72]. On the other hand，V-ATPase activity upregulated by ATP6V1C1 can activate the mTOR pathway which i s involved in the regulation of multiple processes that lead to protein synthesis and muscle development [73]. Fibroblast growth factor (FGF) signalling can produce numerous beneficial effects on metabolic associated morbidi t ies. FGFs are key for skeletal muscle regeneration and higher abundance is also associated with a higher presence of connective tissue [74，75]. FGFs s S i 1gnal via FGF receptors， requiring the binding of specific klotho proteins. Klotho Beta (KLB) is a ce l l-surface protein coding gene that was found overexpressed in AL pigs (log2FC=1.232，q<0.01). The KLB product is suggested to be mandatory in the activation of several endocrine FGFs including FGF21， FGF19 and FGF15 [76]. FGF15 and 19 in particular are known to downregulate lipogenesis， bile acid metabolism and feeding response， while promoting cell proliferation [77]. On the other hand， FGF21 promotes insulin sensitivity，energy usage， and consequently weight loss [78]. In view of this， overexpression of KLB in AL pigs is， t herefore， an intriguing result since AL pigs are proposed to have l ower insulin sensitivity and higher li pid deposition than BI pigs. Nevertheless， other regulatory mechanisms might be influencing this pathway， particularly FGF receptor expression，which may play an important role in limiting FGF21，FGF19 and FGF15 signaling in the muscle tissue of AL pigs. Surprisingly， the gene encoding for leiomodin 1 (LMOD1) which is associated with smooth muscle differentiation and contraction， has been found overexpressed in BI pigs (log2 FC= -0.714， q<0.05) when compared to AL pigs. In vertebrates， LMOD2 and LMOD3 isoforms are preferably more expressed in skeletal muscle cells than LMOD1 [79].Higher expression levels of the latter may be linked to hyperthyroidism while low levels have been linked to atherosclerosis in humans [80]. In our data， Casein kinase 1 delta (CSNK1D) was found overexpressed in BI pigs (log2FC=-0.674，q<0.05) when compared to AL pigs. CSNK1D participates in the regulation of several processes through phosphorylation of many distinct protein substrates involved in cell proliferation and differentiation [81]. Additionally， it has also been demonstrated that CSNK1D is decisive in maintaining the accuracy of circadian rhythms in mammals [82].The circadian clock is known to i nfluence several canonical pathways i ncluding mTORC1activity [83]. Regarding l ipid metabolism related genes， prostaglandin E synthase 2 (PTGES2) was found to be signi f icantly overexpressed in the fatter AL pigs (log2 FC=1.551，q<0.05).Prostaglandin E2 participates in several biological activities， including smooth muscle func-tion， body temperature regulation， pain i nduction and stimulation of bone resorption [84].PUFAs can affect prostaglandins by serving as substrates and competitive inhibitors for their synthesis [85]. PTGES2 mediates the synthesis of prostaglandin E2 from arachidonic acid (C20：4 n-6) which may explain the numerically higher proportion of this PUFA in Bl pigs. Prolyl 4-hydroxylase subunit beta (P4HB) was found overexpressed i n AL pigs (log2FC=1.171，q<0.05). P4HB encodes for a highly abundant and multifunctional protein involved in the catalysis of the formation， breakage， and rearrangement of disulphide bonds. Additionally， expression of P4HB has been l inked with the biology of cytosolic l ipid droplets in specific human enterocytes [86]. The gene encoding for 5'-aminolevulinate synthase 1 (ALAS1) was found significantly overexpressed in AL (log2 FC= 2.322，q<0.05). The mitochondrial enzyme produced primarily catalyses the rate-limiting step in heme biosynthesis and i t s deficiency has previ-ously been associated with acute hepatic porphyrias [87]. ALAS1 i s ubiquitously expressed，commonly regarded as a housekeeping gene， since every nucleated cell must synthesise a heme group for respiratory cytochromes [87]. More recently， ALAS 1 has been associated with lipid metabolism regulation through peroxisome proliferator-activated receptor alpha (PPARA). A study by Rakhshandehroo [88] has demonstrated higher expression levels of ALAS1 specifically induced by PPARA in human hepatocytes. 3.4. Functional Analysis A total of 475 biological functions (p <0.05) were retrieved by the IPA software for our dataset (Table 2). Four functions achieved a z-score enabling prediction of the ac-tivation direction， namely quantity of cells (z-score =2.185) and quantity of leucocytes (z-score=2.152)， which were both predicted to be increased in AL， while neuronal cell death (z-score=-2.164) and apoptosis of tumour cell lines (z-score=-2.043) were pre-dicted to be increased in BI. As expected， MAP3k14's coding product NIK is signalling the noncanonical NF-kB pathway， an alternative signalling cascade involved in the recruitment of leukocytes，macrophages， and lymphocytes. Though the respective retrieved z-score was below the threshold， lipid synthesis is suggested to be enhanced in AL which agrees with the previous phenotypical data. A total of 64 upstream regulators were found related to the dataset (p<0.05， Table S5)though none surpassed the required activation z-score threshold. Ten networks associated to our gene dataset were identified with IPA. The top network found is represented in Figure 2， resuming 15 focus molecules and a total score of 3. This network illustrates the major involvement of the NFxB complex (mediated by MAP3K14)，Histone h3 complex (mediated by KDM2B)， extracellular-signal-regulated protein kinase 1/2 (ERK 1/2， mediated by KLB) as main contributors for numerous gene interactions related to cell proliferation， differentiation and biochemistry. Table 2. Top f unctions found with IPA associated with the target molecules in t he Deseq2 dataset and t heir respective predicted activation state i n Alentejano pigs (AL). Functions p-Value Activation z-Score Predicted Activation in AL Target Molecules IL12RB1，ITPR1， Quantity of cells 3.64×10一么 2.185 Increased KDM2B，LSP1， MAP3K14，PSMB9， SPHK2， THRA，TRIB1 Neuronal cell death 1.69×10一么 -2.164 Decreased ITPR1，KDM2B， P4HB， SLC9A1，UCN IL12RB1，ITPR1，LSP1， Quantity of leukocytes 1.75×10一3 2.152 Increased MAP3K14，PSMB9， SPHK2，THRA， TRIB1 Decreased ITPR1， KDM2B，LSP1， Apoptosis of tumour cell lines 1.32×10一- -2.043 MAP3K14，P4HB， SLC9A1， SPHK2， THRA Binding of DNA 1.77×10-2 1.993 CBX1，MAP3K14， THRA，UCN Cell cycle progression 3.52×10-2 1.964 KDM2B， SLFN11， 一 ANGEL2， CSNK1D， SPHK2， THRA Quantity of macrophages 1.08×10一3 1.961 IL12RB1，LSP1，THRA， TRIB1 Quantity of B 9.65×10-3 1.190 ITPR1，MAP3K14， SPHK2， THRA lymphocytes Synthesis of lipid 3.57×10-2 1.186 一 MAP3K14， PTGES2， RUFY1，SPHK2，TRIB1 3.5. Candidate Gene Expression Analysis with Real Time Quantitative PCR A panel i ncluding the most relevant known genes involved in lipid metabolism was selected for quantification by RT-qPCR， as those were excluded in the RNAseq analysis due to their low reads counts. Results on these tested genes suggest a much more important role of lipid metabolism in defining the biochemical properties of each breed’s muscle tissue (Figure 3) when compared to our RNA-seq results. Overall， lipogenic related genes were found signif i cantly overexpressed in AL when compared to BI， such as in the cases of ACLY (p<0.01)，ELOVL6(p<0.01)， and ME1(p=0.01)， which agree with the previously mentioned higher IMF content of AL pigs' muscle t issue. A key gene in the de novo fatty acid synthesis，FASN， was only found with a numerical difference towards AL (p=0.115)， and the main fatty acid desaturation inducing gene， SCD， followed t he same t rend (p=0.131). Similarly，expression levels of the complex multifunctional enzyme system coded by ACACA and responsible for catalysing the l imiting step in fatty acid synthesis， was not stat i stically different between breeds (p=0.338). Figure 2. Cellular movement， lipid metabolism and small molecule biochemistry Ingenuity Pathway Analysis (IPA) Network. No rmalised a r bitrary un i t s O A L 口B I Figure 3. Est i mated rela t ive expression of candidate genes involved i n l ipid metabolism in the Longissimus lumborum of Alentejano (AL) and Bisaro (BI) pigs (n =5 for each genotype) through Rea l T ime qPCR. ACACA (Acetyl-CoA carboxylase alpha)， ACLY (ATP citrate lyase)， ADIPOQ (Adiponectin)， ELOVL6 (Elongation of long-chain fatty acids family member 6)，FASN (Fatty acid synthase)， LEP (Leptin)， ME1 (Malic enzyme 1)， SCD (Stearoyl-CoA desaturase). Represented values are means of relative gene expression with their respective standard errors represented by vertical bars. Significance： ** p<0.01，*p<0.05. Adiponec t in， coded by ADIPOQ， is widely known to modulate the lipid and glucose metabolisms by activating fatty acid oxidation pathways and increasing blood glucose utilisation， which culminate in the activation of the AMPK pathway and an increase in energy supply [89]. A study by Choudhary [67] demonstrated that NIK overexpression can induce skeletal muscle insulin resistance， while ADIPOQ is responsible for suppressing NIK expression and restoring insulin sensitivity. In our dataset ， the gene coding for NIK expression was found overexpressed in AL when compared to BI samples， suggesting an overcompensation in adiponectin levels. This was confirmed by the qPCR results (Log2 FC =0.783， p <0.01). These resul t s suggest that adiponectin resistance is occurring，particularly in AL pigs， since higher ADIPOQ levels are associated with leaner i ndividuals while lower l evels of this cytokine are detected in obese individuals [90]. According to our RNAseq data， ADIPOQ receptors ADIPOR1 and ADIPOR2 were numerically higher in BI ，without statistical significance. This may suggest that a potential lower signalling could be occurring in AL . Future investigation on the expression of these receptors， with a higher biological replication， as well as the circulating levels of adiponectin are needed to confirm this hypothesis. Leptin， coded by LEP， is a cell-signalling hormone responsible for appetite regulation by informing the central nervous system when the total fat stored in the body rises. Individ-uals with high body fat composition exhibi t higher levels of leptin， which signals the brain to decrease feeding and increase the use of stocked energy. Leptin is primarily secreted by the main energy store tissue of the body， white adipocytes， but i t can also be found expressed in other t issues including skeletal muscle [91，92]. In our trial， LEP presented a tendency to be upregulated in AL pigs (p=0.056) which agrees with the typically fatter composition of this breed when compared to BI. A suggested state of leptin resistance may be occurring in the AL， comparable to what happens with the genetically similar Iberian pig[93]. Our results from AL and BI breeds， particularly the differences in expression levels of lipogenic genes such as ACLY and ME1 suggest that these may play an important role defining the synthesis of new fatty acids， overruling the importance of more central-rolled genes such as ACACA and FASN. ACLY and ME1 are responsible for catalysing reactions that produce specific non-lipid precursors， including cytosolic acetyl-CoA and NADPH，that can later be used by ACACA and FASN to assemble palmitic acid (C16：0) [94，95].This fatty acid is the main precursor for the synthesis of stearic acid (C18：0) through ELOVL6， and oleic acid (C18：1 n-9) through SCD. ELOVL6 is responsible for catalysing the reaction that introduces two carbon groups to several SFAs and MUFAs [96] while SCD is accountable for introducing a cis double bond at the delta-9 position i n fatty acyl-CoA substrates， including stearic and palmitic acids [97]. Higher expression levels of ELOVL6and SCD i n the loin muscle agree with the previously mentioned higher oleic acid content of AL and may also justify the lower proportion of stearic acid (C18：0) when compared to BI . Nevertheless， we cannot exclude t he possibility of more gene regulators being involved in influencing these traits， particularly ones related to fatty acid desaturation since SCD expression was only numerically higher in AL pigs. The generic overexpression of lipogenic related genes i n t he LL muscle of the AL breed agrees with the higher IMF content when compared to BI pigs. The contribution of lipoly t ic genes in the regulation of this metabolic balance remains unclear while the higher leptin and adiponectin signalling in the obese AL suggest that these hormones fail to stimulate l ipolytic pathways， possibly through post-transcriptional regulation. 3.6. Linking Adipose and Skeletal Muscle Tissue Transcriptomes In a previous study， we explored the genome function of these two local breeds at t he adipose tissue level [34]. Several DE genes involved in l ipid metabolism were previously detected in adipose tissue through RNAseq but were not detected in muscle tissue， including ACLY，ELOVL6，FASN，LEP，ME1 and SCD. This agrees with the suggestion that lipid metabolism in muscle and adipose t issues are differently regulated [98，99]. Furthermore， the total DEG output (p <0.05) was also much larger in the adipose tissue when compared to the muscle one (458 vs. 49， respectively)， with a greater proportion found overexpressed in the AL breed (57 vs. 69%， respectively). This pattern agrees with the lower relevance of muscle tissue in influencing lipid composition through t ranscriptional and signalling regulation. Perception of adipose tissue as a mere energy storage element i s outdated and its functions have been extended to a pivotal endocrine organ that secretes numerous substances that influence homeostasis and metabolism. On the other hand， the combining interactions between skeletal muscle cells and adipocytes have the most impact in defining fat and lean tissue depositions and their respective efficiency rates [100]. Both studies share a tota l of four DEGs in common， namely chromobox 1 (CBX1)，integrator complex subunit 11 (INTS11)， STMN3 and RUN and FYVE domain containing 1(RUFY1). CBX1 encodes for a highly conserved protein that binds to methylated histone 3 tails at the lysine 9 residue， acting as an epigenetic agent that modulates chromatin structure and gene expression [101]. This gene was found to be consistently overexpressed in AL pigs in either adipose or LL (log2 FC=0.954， q<0.05 vs. log2 FC=1.300，q<0.05，respectively) which indicates the relevance of epigenetic mechanisms in regulating gene expression through histone modifications across different tissues， particularly in AL pigs.INTS11 encodes for the integrator complex subunit 11， an element of the 12-subunit com-plex that participates in t he transcription and processing of small nuclear RNAs [102]and was found consistently overexpressed in BI in both tissues (log2 FC=-1.297，q<0.01vs. log2 FC=-1.675， q<0.01， respectively). On the other hand， STMN3 encodes for a highly conserved phosphoprotein in vertebrates， generally associated with the dereg-ulation of microtubule dynamics and tubulin sequestering [103]. STMN3 is commonly associated with various functions in the nervous system and， in our results， presented similar overexpression patterns in BI pigs in both adipose tissue and LL (log2 FC=-2.058，q<0.01 vs. log2 FC= -4.033，q<0.01). RUFY1 is a gene responsible for encoding a protein that binds to several signalling molecules and is suggested to participate in cell polarity and membrane trafficking mediated by small GTPases [104]. Curiously， RUFY1was found overexpressed in BI's adipose tissue and in AL’s longissimus lumborum tissue (log2 FC=-1.809， q<0.01 vs. log2 FC = 1.060，q<0.05). This suggests that different Intramuscular fat participates in important regulatory roles in muscle， particularly through insulin-mediated glucose uptake and l ipid peroxidation. Oversupply of fat stores is then strongly associated with decreased i nsulin sensitivity i n skeletal muscle [106]. The proposed obesity-induced chronic inflammatory state in AL pigs， caused by exacerbated lipid accumulation， induces expression of pro-inflammatory cytokines and activation of numerous signalling pathways， including t he nuclear factor-kappa B (NFxB) pathway，which ultimately a 1071.1re suggested to inhibi t insulin signalling and action [107]. Interestingly，while we associated MAP3K14 with the activation of the NFxB pathway in the LL of AL pigs (log2 FC=1.829， q<0.01)， in the adipose tissue this role seems to be played by MAP3K15 (log2 FC=1.036，q<0.05). However， development of insulin resistance involves several complex biological mechanisms that are not fully understood yet. BI’s lower tendency to store fat when compared to AL pigs does not exclude the possibility of decreased insulin sensitivity to be occurring. BI pigs presented significantly higher total collagen content than AL pigs (13.7 vs. 16.3 mg/g，p <0.05) which has previously been positively associated with insulin resistant skeletal muscle t issue in human patients [108]. Overall expression of the selected candidate genes involved in lipid metabolism was similar between adipose and muscle tissues. In short ， l ipogenic related genes were consistently more expressed in the AL breed across both studies which agrees with the fatter profile of this breed. Higher absolute fold change values were higher in adipose tissue though more significant differences between breeds were observed in the muscle tissue (Table 3). Table 3. Candidate gene expression for l ipid metabol i sm comparison between adipose tissue and longissimus lumborum in Alentejano (AL) and Bisaro (BI) pigs assessed with qPCR. Positive values indicate overexpression in AL and negative values overexpression in BI. Genes Adipose Tissue Longissimus lumborum log2 FC p-Value log2 FC p-Value ACACA 1.055 0.077 0.150 0.338 ACLY 1.601 0.068 0.362 0.017 ADIPOQ -0.685 0.110 0.783 0.007 ELOVL6 0.671 0.136 0.540 0.001 FASN 1.359 0.100 0.367 0.115 LEP 0.929 0.046 1.524 0.056 ME1 1.008 0.106 0.627 0.010 SCD 1.351 0.087 1.325 0.131 Next-generation sequencing techniques such as RNA-seq provide a broad and in-sightful perspective over a tissue transcriptomics but still struggle with the analysis of low expression data， while differences within high read counts are more easily detected [109].In our dataset， several key genes involved in li pid synthesis and regulation were found below the cut-off point regarding total read count in the filtering step， which determined their premature withdrawal of the differentia l expression analysis. FASN， for example，in the adipose tissue scored a great correlation coefficient (0.92， p < 0.01) while in the muscle， where i t was excluded in the i nitial f iltering (<50 reads per group)， presented a much lower， barely significant score (0.58， p<0.05). This is why we suggest that genes with fewer reads detected through RNAseq should be accounted for with care since they arouse more associated noise and differential expression estimations may be biased against low read count values [110，111]. Besides the utility of RNA-seq at a standard read depth (30-60 million reads) for a general overview of the transcriptomic universe， Real Time qPCR provides a more reliable source of information to assess low expressed genes， as our results suggest. In conclusion， this study intended to determine the nature of the biological events occurring in the muscle of the two main Portuguese autochthonous pig breeds and their interpretation regarding the phenotypical diversity shown by the two breeds. Our data allowed the identification of 49 differentially expressed genes through RNA-seq. Specific myosin heavy chain components have been associated with AL (MYH7) and BI (MYH3)pigs， while an overexpression of MAP3K14 in AL may be associated with their lower loin proportion， induced insulin resistance， and increased inflammatory response. Our Real time qPCR data acknowledged the importance of several lipogenic genes and regulators，including ACLY， ADIPOQ，ELOVL6，LEP and ME1， disregarded in the RNA-seq by their lower total read count. These l atter results agree with the fatter profile of the AL pig breed and adiponectin resistance may be responsible for the overexpression of MAP3K14's coding product NIK， failing to restore insulin sensitivity. Supplementary Materials： The following are available online at ht t ps：//www.mdpi .com/article/10.3390/ani11051423/s1， Table S1： Primer design for qPCR， Table S2： Plasma parameters from Alentejano (AL) and Bisaro (BI) pigs at ~150 kg LW， Table S3： Plasma parameters f rom Alentejano (AL ) and Bisaro (BI) pigs slaughtered at 150 kg LW， Table S4： The full detailed l ist of DEGs， Table S5：Predicted upstream regulators for the set of differentially expressed genes. Author Contributions： Conceptualisation， J.M.M. and A.A.； methodology and investigation， A.A.，C.O.， Y.N.， R.B.， F.G .， A.L.-G. and J.M.M.； writing-original draft preparation， A.A.； writing-review and editing， J.M.M.， C.O.， M.L .， R.B.， Y.N.， R.C. and A.A.； supervision， J.M.M.， C.O.，M.d.R.F. and M.L.； funding acquisition， J .M.M.， C.O.， R.C.， M.d.R.F. and M.L. Al l authors have read and agreed to the published version of the manuscript. Funding： This work was funded by European Union’s H2020 RIA program (grant agreement no.634476)， by Portuguese national funds through FCT/MCTES under project UIDB/05183/2020，and a research grant SFRH/BD/132215/2017 for A. Albuquerque. Institutional Review Board Statement： The study was conducted according to the regulations and ethical guidelines set by the Portuguese Animal Nutrition and Welfare Commission (DGAV一 Directorate-General for Food and Veterinary， Lisbon， Portugal) following the 2010/63/EU Directive. Data Availability Statement： The results from data analyses performed in this study are included in this artic l e and i ts tables. Raw sequencing data are avai l able through the GEO Series accession number GSE159817 or from the corresponding author on reasonable request. Conflicts of Interest： The authors declare no conflict of interest. References 1 Porter， V. Spain and Portugal. 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