RNA sequencing for global gene expression associated with muscle growth in a single male modern broiler line compared to a foundational Barred Plymouth Rock chicken line.

Modern broiler chickens exhibit very rapid growth and high feed efficiency compared to unselected chicken breeds. The improved production efficiency in modern broiler chickens was achieved by the intensive genetic selection for meat production. This study was designed to investigate the genetic alterations accumulated in modern broiler breeder lines during selective breeding conducted over several decades. To identify genes important in determining muscle growth and feed efficiency in broilers, RNA sequencing (RNAseq) was conducted with breast muscle in modern pedigree male (PeM) broilers (n = 6 per group), and with an unselected foundation broiler line (Barred Plymouth Rock; BPR). The RNAseq analysis was carried out using Ilumina Hiseq (2 x 100 bp paired end read) and raw reads were assembled with the galgal4 reference chicken genome. With normalized RPM values, genes showing >10 average read counts were chosen and genes showing <0.05 p-value and >1.3 fold change were considered as differentially expressed (DE) between PeM and BPR. DE genes were subjected to Ingenuity Pathway Analysis (IPA) for bioinformatic functional interpretation. The results indicate that 2,464 DE genes were identified in the comparison between PeM and BPR. Interestingly, the expression of genes encoding mitochondrial proteins in chicken are significantly biased towards the BPR group, suggesting a lowered mitochondrial content in PeM chicken muscles compared to BPR chicken. This result is inconsistent with more slow muscle fibers bearing a lower mitochondrial content in the PeM. The molecular, cellular and physiological functions of DE genes in the comparison between PeM and BPR include organismal injury, carbohydrate metabolism, cell growth/proliferation, and skeletal muscle system development, indicating that cellular mechanisms in modern broiler lines are tightly associated with rapid growth and differential muscle fiber contents compared to the unselected BPR line. Particularly, PDGF (platelet derived growth factor) signaling and NFE2L2 (nuclear factor, erythroid 2-like 2; also known as NRF2) mediated oxidative stress response pathways appear to be activated in modern broiler compared to the foundational BPR line. Upstream and network analyses revealed that the MSTN (myostatin) -FST (follistatin) interactions and inhibition of AR (androgen receptor) were predicted to be effective regulatory factors for DE genes in modern broiler line. PRKAG3 (protein kinase, AMP-activated, gamma 3 non-catalytic subunit) and LIPE (lipase E) are predicted as core regulatory factors for myogenic development, nutrient and lipid metabolism. The highly upregulated genes in PeM may represent phenotypes of subclinical myopathy commonly observed in the commercial broiler breast tissue, that can lead to muscle hardening, named as woody breast. By investigating global gene expression in a highly selected pedigree broiler line and a foundational breed (Barred Plymouth Rock), the results provide insight into cellular mechanisms that regulate muscle growth, fiber composition and feed efficiency.

Kong BW ，Hudson N ，Seo D ，Lee S ，Khatri B ，Lassiter K ，Cook D ，Piekarski A ，Dridi S ，Anthony N ，Bottje W ... -  《BMC GENOMICS》

MicroRNA profiling associated with muscle growth in modern broilers compared to an unselected chicken breed.

Genetically selected modern broiler chickens have acquired outstanding production efficiency through rapid growth and improved feed efficiency compared to unselected chicken breeds. Recently, we analyzed the transcriptome of breast muscle tissues obtained from modern pedigree male (PeM) broilers (rapid growth and higher efficiency) and foundational Barred Plymouth Rock (BPR) chickens (slow growth and poorer efficiency). This study was designed to investigate microRNAs that play role in rapid growth of the breast muscles in modern broiler chickens. In this study, differential abundance of microRNA (miRNA) was analyzed in breast muscle of PeM and BPR chickens and the results were integrated with differentially expressed (DE) mRNA in the same tissues. A total of 994 miRNA were identified in PeM and BPR chicken lines from the initial analysis of small RNA sequencing data. After filtering and statistical analyses, the results showed miR-2131-5p, miR-221-5p, miR-126-3p, miR-146b-5p, miR-10a-5p, let-7b, miR-125b-5p, and miR-146c-5p up-regulated whereas miR-206 down-regulated in PeM compared to BPR breast muscle. Based on inhibitory regulations of miRNAs on the mRNA abundance, our computational analysis using miRDB, an online software, predicated that 118 down-regulated mRNAs may be targeted by the up-regulated miRNAs, while 35 up-regulated mRNAs appear to be due to a down-regulated miRNA (i.e., miR-206). Functional network analyses of target genes of DE miRNAs showed their involvement in calcium signaling, axonal guidance signaling, and NRF2-mediated oxidative stress response pathways suggesting their involvement in breast muscle growth in chickens. From the integrated analyses of differentially expressed miRNA-mRNA data, we were able to identify breast muscle specific miRNAs and their target genes whose concerted actions can contribute to rapid growth and higher feed efficiency in modern broiler chickens. This study provides foundation data for elucidating molecular mechanisms that govern muscle growth in chickens.

Khatri B ，Seo D ，Shouse S ，Pan JH ，Hudson NJ ，Kim JK ，Bottje W ，Kong BC ... -  《BMC GENOMICS》

Identification and Differential Abundance of Mitochondrial Genome Encoding Small RNAs (mitosRNA) in Breast Muscles of Modern Broilers and Unselected Chicken Breed.

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Bottje WG ，Khatri B ，Shouse SA ，Seo D ，Mallmann B ，Orlowski SK ，Pan J ，Kong S ，Owens CM ，Anthony NB ，Kim JK ，Kong BC ... -  《Frontiers in Physiology》

Messenger RNA sequencing and pathway analysis provide novel insights into the biological basis of chickens' feed efficiency.

Advanced selection technologies have been developed and continually optimized to improve traits of agricultural importance; however, these methods have been primarily applied without knowledge of underlying biological changes that may be induced by selection. This study aims to characterize the biological basis of differences between chickens with low and high feed efficiency (FE) with a long-term goal of improving the ability to select for FE. High-throughput RNA sequencing was performed on 23 breast muscle samples from commercial broiler chickens with extremely high (n = 10) and low (n = 13) FE. An average of 34 million paired-end reads (75 bp) were produced for each sample, 80% of which were properly mapped to the chicken reference genome (Ensembl Galgal4). Differential expression analysis identified 1,059 genes (FDR < 0.05) that significantly divergently expressed in breast muscle between the high- and low-FE chickens. Gene function analysis revealed that genes involved in muscle remodeling, inflammatory response and free radical scavenging were mostly up-regulated in the high-FE birds. Additionally, growth hormone and IGFs/PI3K/Akt signaling pathways were enriched in differentially expressed genes, which might contribute to the high breast muscle yield in high-FE birds and partly explain the FE advantage of high-FE chickens. This study provides novel insights into transcriptional differences in breast muscle between high- and low-FE broiler chickens. Our results show that feed efficiency is associated with breast muscle growth in these birds; furthermore, some physiological changes, e.g., inflammatory response and oxidative stress, may occur in the breast muscle of the high-FE chickens, which may be of concern for continued selection for both of these traits together in modern broiler chickens.

Zhou N ，Lee WR ，Abasht B 《BMC GENOMICS》

Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria.

As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P < 0.001). Over 1800 proteins were identified, of which 152 were different (P < 0.05) by at least 1.3 fold and ≤ 15 fold between the high and low FE phenotypes. Data were analyzed for a modified differential expression (DE) metric (Phenotypic Impact Factors or PIF) and interpretation of protein expression data facilitated using the Ingenuity Pathway Analysis (IPA) program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P < 0.00001). Within the top up and down 5% PIF molecules in the dataset, there were 15 mitoproteome proteins up-regulated and only 5 down-regulated in the high FE phenotype. Pathway enrichment analysis also identified mitochondrial dysfunction and oxidative phosphorylation as the number 1 and 5 differentially expressed canonical pathways (up-regulated in high FE) in the proteomic dataset. Upstream analysis (based on DE of downstream molecules) predicted that insulin receptor, insulin like growth receptor 1, nuclear factor, erythroid 2-like 2, AMP activated protein kinase (α subunit), progesterone and triiodothyronine would be activated in the high FE phenotype whereas rapamycin independent companion of target of rapamycin, mitogen activated protein kinase 4, and serum response factor would be inhibited in the high FE phenotype. The results provide additional insight into the fundamental molecular landscape of feed efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE. Funding provided by USDA-NIFA (#2013-01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville).

Kong BW ，Lassiter K ，Piekarski-Welsher A ，Dridi S ，Reverter-Gomez A ，Hudson NJ ，Bottje WG ... -  《PLoS One》