Muscle Biological Characteristics of Differentially Expressed Genes in Wujin and Landrace Pigs

Journal of Integrative Agriculture 2014, 13(10): 2236-2242

October 2014

RESEARCH ARTICLE

Muscle Biological Characteristics of Differentially Expressed Genes in Wujin and Landrace Pigs XU Hong1*, HUANG Ying2*, LI Wei-zhen3, YANG Ming-hua1, GE Chang-rong1, ZHANG Xi1, LI Liu-an4, 5, GAO Shi-zheng1 and ZHAO Su-mei1 1

School of Public Finance and Economics, Yunnan University of Finance and Economics, Kunming 650221, P.R.China Yunnan Key Laboratory of Animal Nutrition and Feed Science, Yunnan Agricultural University, Kunming 650201, P.R.China 3 Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, P.R.China 4 Animal Husbandry Post-Doctoral Station, Nanjing Agricultural University, Nanjing 210095, P.R.China 5 Department of Animal Science, Tianjin Agricultural University, Tianjin 300384, P.R.China 2

Abstract The biological chemistry would be responsible for the meat quality. This study tried to investigate the transcript expression profile and explain the characteristics of differentially expressed genes between the Wujin and Landrace pigs. The results showed that 526 differentially expressed genes were found by comparing the transcript expression profile of muscle tissue between Wujin and Landrace pigs. Among them, 335 genes showed up-regulations and 191 genes showed down-regulations in Wujin pigs compared with the Landrace pigs. Gene ontology (GO) analysis indicated that the differentially expressed genes were clustered into three groups involving in protein synthesis, energy metabolism and immune response. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis found that these differentially expressed genes participated in protein synthesis metabolism, energy metabolism and immune response pathway. The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of protein function and protein domains function also confirmed that differentially expressed genes belonged to protein synthesis, energy metabolism and immune response. Genes related protein synthesis metabolism pathway in Landrace was higher than in Wujin pigs. However, differentially expressed genes related energy metabolism and immune response was up-regulated in Wujin pigs compared with Landrace pigs. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. These suggested that the genes related to protein synthesis, energy metabolism and immune response would contribute to the growth performance, meat quality as well as anti-disease capacity. Key words: pigs, muscle tissue, microarray, bioinformatics analysis, differentially expressed genes

INTRODUCTION It is well known that western pig breeds owed the characteristics of high growth rate and high lean meat percentage. However, Chinese pigs own the high intra-

muscular fat and excellent meat quality. The Landrace pig is considered a lean breed. The Wujin pig is one of the Chinese local pigs, which is regarded as fatty genotype (Zhao et al. 2009). Therefore, the underlying mechanism of intramuscular fat deposition in fatty and lean pigs could be elucidated using these two pig models. The identification of genes underlying body composi-

Received 15 April, 2013 Accepted 16 September, 2013 XU Hong, E-mail: [email protected]; Correspondence ZHAO Su-mei, E-mail: [email protected] * These authors contributed equally to this study.

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60605-X

Muscle Biological Characteristics of Differentially Expressed Genes in Wujin and Landrace Pigs

tion is of major interest for the improvement of livestock animal production. Previous reports had described the histochemical, physiological and genetic properties of muscle tissue and examined the meat quality and carcass traits in different pig breeds (Lefaucheur et al. 2004). Our previous reports indicated that the mechanism of higher intramuscular fat (IMF) content in fatty pigs may be due to the higher capacity of lipogenesis and fatty acid transportation and the lower capacity of lipolysis (Zhao et al. 2009). However, these experiments, based on monitoring the expression of a limited number of genes, did not completely take into account the complexity and multiplicity of interwoven molecular mechanisms. Recently, the focus of research has moved to the analysis of the skeletal muscle transcriptome and compare the gene expression profile in the muscles of different quality using expression microarrays (Bai et al. 2003; Lobjois et al. 2008; Ponsuksili et al. 2008). Microarray technology can simultaneously measure the differential expression of a large number of genes in a given tissue and may identify the genes involved in different phenotypes. Typically, microarray experiments produced long lists of genes that were differentially expressed between two different physiological situations. Combined with bioinformatics analysis, these tools would provide information on networks of expressed genes in muscle tissue and increase the knowledge of the biological pathways controlling the phenotypes. Therefore, we performed microarray analysis to characterize and compare transcript expression profiles in the muscle tissue between Wujin and Landrace pigs in the present study. Our aim was to reveal the differences of breed-related gene expression between the indigenous Wujin pig and the Landrace pigs, because it might be useful to understand the molecular mechanisms which would be responsible for breed-specific differences in growth performance and meat quality as well as anti-disease capacity.

RESULTS Carcass traits Table 1 showed carcass traits of Wujin and Landrace pigs. Lean meat percentage and eye loin area for Wujin pigs was significantly lower than that for Landrace pigs

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Table 1 Carcass traits of Wujin and Landrace pigs Indexes Body weight (kg) Lean meat percentage (%) Fat meat percentage (%) Backfat thickness (cm) Eye loin area (cm2)

Wujin pigs 104.33±9.89 a 39.18±1.02 a 37.93±1.45 a 4.61±0.14 a 22.79±1.22 a

Landrace pigs 102.72±10.01 a 63.89±1.23 b 18.67±1.07 b 2.30±0.11 b 35.14±1.33 b

The means with different lower letters are significantly different (P<0.05). The same as in Table 2.

(P<0.05). On the contrary, fat meat percentage and backfat thickness for Wujin pigs was significantly higher than that for Landrace pigs (P<0.05).

Meat quality The meat quality of Wujin and Landrace pigs was shown in Table 2. The results showed that marbling, a*, b* and water holding capacity in Wujin pigs was significantly higher than in Landrace pigs (P<0.05). However, shear force, cooking loss and drip loss in Wujin pigs was significantly lower than in Landrace pigs (P<0.05). Table 2 Meat quality of Wujin and Landrace pigs Indexes pH 45 min pH 24 h Marbling L* a* b* Water holding capacity (%) Shear force (kg) Cooking loss (%) Drip loss (%)

Wujin pigs 6.36±0.13 a 6.05±0.14 a 3.83±0.20 a 45.42±1.46 a 9.12±0.40 a 5.90±0.33 a 79.72±1.20 a 2.95±0.17 a 28.90±1.36 a 1.54±0.05 a

Landrace pigs 6.30±0.06 a 6.15±0.13 a 2.26±0.03 b 46.14±1.11 a 7.20±0.28 b 3.15±0.42 b 71.63±2.40 b 3.35±0.15 b 35.43±1.15 b 1.59±0.07 a

Differential expression profile analysis Comparative expression profiling between the Wujin and Landrace pigs revealed 526 differentially expressed transcripts. Among them, 335 showed mainly up-regulation and 191 showed mainly up-regulation in the Wujin pigs compared to the Landrace pigs.

Ontological analysis of differentially expressed genes Gene ontology (GO) term annotations were used to perform a functional analysis of the resulting list of differen© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

XU Hong et al.

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tially expressed genes. The three GO categories (biological process, metabolic function and cell component) were explored with the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics tools. Significant results are summarized in Table 3. The differentially expressed genes belong to groups of genes functionally categorized as genes involved in the biological processes including: 1) protein synthesis (translational elongation and translation); 2) energy metabolism (oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport, ATP synthesis coupled electron transport); and 3) immune response (response to wounding, inflammatory response and defence response). These biological processes were localized to ribosomal subunit, ribosome, small ribosomal subunit, cytoplasmic part and cytoplasm, as well as respiratory chain and mitochondrial inner membrane. The differentially expressed genes participated in the metabolic function relating to: 1) protein synthesis (structural constituent of ribosome); 2) energy metabolism, especially consuming oxygen to supply energy (hydrogen ion transmembrane transporter activity, monovalent inorganic cation); and 3) immune response.

KEGG pathway analysis Mining the KEGG database using the differential ex-

pressed genes list retrieved three pathways according to Benjamini<0.05. The number of genes with microarray information varied per pathway. Microarray data from a single gene of a pathway is not sufficient to describe a common regulatory mechanism of a pathway. Pathways with at least two genes with information on the microarray and localized on one biochemical path were further analysed (te Pas et al. 2007). In this study, all three pathways including protein synthesis pathway, energy metabolism and immune response pathways were further analyzed and three biological functional categories could be categorized (Table 4). 1) Protein synthesis metabolism pathway. A number of ribosome protein (RP) genes were down-regulated in the Wujin pigs compared with Landrace pigs. These differentially expressed RP genes composed of ribosome pathway. The ribosome pathway may be related to genetic information processing and translation processing, which could supply the place for protein synthesis. The pathways may underlie the muscle tissue protein metabolism features. 2) Energy metabolism pathway. There were differentially expressed genes related to mitochondrion oxidative phosphorylation between Wujin and Landrace pigs. The oxidative phosphorylation pathway was consisted one of the energy metabolism pathways. The differentially expressed genes in energy metabolism pathways may relate to the muscle fibre types with the oxidative energy.

Table 3 Analysis of gene ontology (GO) terms for biological process (BP), cellular components (CC) and metabolic function (MF) Category

Fold-enrichment

Benjamini

GOTERM_BP_ALL

GO:0006952~defense response

Term

0.99

0.02

FDR1) 0.12

GOTERM_BP_ALL

GO:0006119~oxidative phosphorylation

2.76

0.01

0.20

GOTERM_BP_ALL

GO:0006954~inflammatory response

2.90

0.02

0.45

GOTERM_BP_ALL

GO:0042775~mitochondrial ATP synthesis coupled electron transport

3.44

0.03

0.21

GOTERM_BP_ALL

GO:0042773~ATP synthesis coupled electron transport

3.65

0.03

0.21

GOTERM_BP_ALL

GO:0009611~response to wounding

4.12

0.03

0.12

GOTERM_BP_ALL

GO:0006412~translation

4.23

5.09×10-7

6.99×10-5

GOTERM_CC_ALL

GO:0005737~cytoplasm

1.30

2.33×10-8

4.99×10-7

GOTERM_CC_ALL

GO:0043226~organelle

1.55

6.77×10-6

6.11×10-4

GOTERM_CC_ALL

GO:0005829~cytosol

2.22

-11

1.99×10

4.08×10-10

GOTERM_CC_ALL

GO:0005840~ribosome

3.88

6.09×10-11

8.67×10-10

GOTERM_CC_ALL

GO:0015935~small ribosomal subunit

5.33

7.22×10-6

7.90×10-4

GOTERM_CC_ALL

GO:0005743~mitochondrial inner membrane

5.34

3.45×10

8.78×10-2

GOTERM_CC_ALL

GO:0070469~respiratory chain

6.43

2.31×10-5

4.55×10-4

GOTERM_CC_ALL

GO:0033279~ribosomal subunit

7.67

2.00×10-14

1.56×10-12

GOTERM_MF_ALL

GO:0005198~structural molecule activity

3.09

2.01×10-10

5.32×10-10

GOTERM_MF_ALL

GO:0015077~monovalent inorganic cation transmembrane transporter activity

4.78

0.03

0.42

GOTERM_MF_ALL

GO:0015078~hydrogen ion transmembrane transporter activity

5.98

0.01

0.12

GOTERM_MF_ALL

GO:0003735~structural constituent of ribosome

6.78

3.05×10-10

6.12×10-9

1)

-3

FDR, false discovery ratio. The same as in Table 5.

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Muscle Biological Characteristics of Differentially Expressed Genes in Wujin and Landrace Pigs

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Table 4 KEGG pathways analysis

Quantitative real-time PCR validation Down-regulated genes (mostatin, MSTN) and up-regulated genes (uncoupling protein 3, UCP3; and interleukin-1, IL1β) in Wujin pigs compared with Landrace pigs were selected from all differential expressed genes. The result revealed that the gene expression is in consonant with the microarray result. Expression level of MSTN gene was lower in Wujin pigs than in Landrace pigs, while UCP3 and IL1β exhibited higher expression in the Wujin pigs compared with Landrace pigs (Fig.).

DISCUSSION Our previous study showed that IMF content in Wujin pigs was higher than in Landrace pigs (Zhao et al. 2009). In the present study, we indicated that lean meat percentage and eye loin area in Wujin pigs was reduced compared with the Landrace pigs. On the contrary, fat meat percentage and backfat thickness was increased in the former pig breed contrast with the latter pig breed. Meanwhile, our data also reported that marbling and shear force in Wujin pigs was higher than in Landrace pigs. All these results would suggest that Wujin and Landrace pigs exhibited the phenotypes difference, which would supply the ideal model to investigate the molecular mechanism govern the phenotypic characteristics of muscles. Meat is postmortem aspect of muscles, differing from them due to a series of biochemical modification. Therefore, the biological metabolism pathway in the muscle would be responsible for the meat quality. In the present study, the differentially expressed genes were classified into three biological processes including protein synthesis, energy metabolism and immune response. The differentially expressed genes related to protein synthesis in

Benjamini 3.15×10-12 3.08×10-5 5.41×10-3

FDR 2.09×10-10 5.99×10-4 0.01

A Relative expression level of MSTN gene

3) Immune response pathway. The genes related to complement and coagulation cascades pathway in Wujin pigs were higher than in Landrace pigs.

Fold-enrichment 6.52 4.55 1.69

3 b 2

a 1

0 Wujin

Landrace Pigs

B Relative expression level of IL1β gene

Term ssc03010: ribosome ssc00190: oxidative phosphorylation ssc04610: complement and coagulation cascades

1.2

a

0.8 b 0.4

0 Wujin

Landrace Pigs

C Relative expression level of UCP3 gene

Category KEGG_PATHWAY KEGG_PATHWAY KEGG_PATHWAY

1.2

a

1 0.8 b

0.6 0.4 0.2 0 Wujin

Landrace Pigs

Fig. The expression of differentially expressed genes. A, B and C represented MSTN, IL1β and UCP3 genes, respectively. The means with different lower letters are significantly different (P<0.05).

Wujin pigs were mainly down-regulated compared with Landrace pigs. However, the genes related to energy metabolism and immune responses were up-regulated in Wujin pigs. It is well accepted that total protein synthesis in muscle is related to both the ribosome content of the tissue © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

XU Hong et al.

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and the activity per ribosome (Liu and Hsu 2005). The increase in protein synthesis may not be attributable solely to an increase in ribosomal and mRNA in muscle. However, the high ribosome concentration may be associated with the rapid rate of protein synthesis (Zhao et al. 2003; te Pas et al. 2007, 2010). Our result showed that protein synthesis metabolism-related genes in Wujin pigs were down-regulated compared with Landrace pigs, especially the ribosome pathway and a number of ribosome protein genes. This reveals that the protein synthesis would be the basic process for lean meat deposition. The down-regulated genes in Wujin pigs would contribute to lower lean meat percentage. Muscle fiber is considered an important factor influencing many biochemical processes and thereby meat quality (Klont et al. 1998). The intensive selection for lean muscle growth in modern pigs has, over time, induced a shift in muscle metabolism toward a more glycolytic and less oxidative fiber type (Lefaucheur et al. 2004). Less mitochondrial function is evident in muscles with higher type II muscle fiber content (Conley et al. 2007). Mitochondria are central to the conversion of energy by oxidizing substrates and generating ATP via oxidizing phosphorylation. High proportions of type II fibers are associated with unfavorably meat quality (Fieldler et al. 2004). Oxidative-type fibers generally contain more intramuscular fat. Our results also indicated that the differentially expressed genes take part in the oxidative phosphorylation pathway in the mitochondria. Moreover, the genes of oxidative phosphorylation pathway were upregulated in Wujin pigs compared with the Landrace pigs. Meanwhile, Wujin pigs contained more IMF and oxidative fiber types. Another biological process of the differentially expressed genes being clustered into was the immune system response activation and regulation against different stimulus like wounding and inflammation. One of KEGG pathways that differently expressed genes involved in regulation of homeostasis through the complement and coagulation cascades, which are also interacting with the immune system (Li and Huang 2009). Chinese pigs have better anti-disease abilities (Johns 1998). Wujin pigs belong to Chinese local breed, which own the good adaptability to crude diet, good anti-disease competence, excellent meat quality and so on (Ge et al. 2008; Zhang et al. 2008). In this study, the genes related to the immune systems were up-regulated in Wujin pigs

compared with Landrace pigs. This result would help to explain the difference of anti-disease capacity between these two pig breeds.

CONCLUSION The present study investigated the differentially expressed genes between Wujin and Landrace pigs. These differentially expressed genes were clustered into three biological processes involving in protein synthesis, energy metabolism and immune response. The result may contribute to a better understanding of the mechanisms involved in porcine growth performance, meat quality and anti-disease capacity.

MATERIAL AND METHODS Animals and muscle tissue samples, carcass traits and meat quality measurement All experiment procedures were performed according to the Guide for Animal Care and Use of Laboratory Animals in the Institutional Animal Care and Use Committee of Yunnan Agricultural University, China. The experimental protocol was approved by the Department Animal Ethics Committee of Yunnan Agricultural University. Six Wujin and six Landrace pigs were used. All pigs had free access to water from nipple drinkers. The diets were not isonitrogenous or isoenergetic because the growth rate of the two breeds was so divergent and were provided ad libitum. The growth performance was indicated by Zhao et al. (2009). When the pigs reached 100 kg body weight, they were transported to Yunnan Agricultural Center Meats Laboratory and slaughtered after electrical stunning. Longissimus muscle samples from the last ribs were collected from every animal. The determined technical meat quality parameters included: drip loss, marbling, color, pH at 24 h and IMF in the longissimus muscles. Technical meat quality traits were measured using standard methodology (Honikel 1998). Furthermore, carcass parameters were measured including carcass weight, lean meat percentage, fat meat percentage, and eye loin area and backfat thickness. Part of longissimus muscle tissue samples were taken after slaughter and immediately snap frozen. Samples were stored at -80°C until use.

RNA extraction Samples were homogenized in the Fastprep ® Instrument © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Muscle Biological Characteristics of Differentially Expressed Genes in Wujin and Landrace Pigs

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(FP120, Bio101 Thermo Savant) and total RNA was extracted from 100 mg tissue using the RNeasy® Mini Kit (Qiagen, UK) according to manufacturer’s instructions. RNA quality and quantity was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, Germany) and the ND-1000 Nanodrop spectrophotometer (ND-1000 V3.1.2, USA) respectively and quality checked on a 1% agarose gel.

Dennis et al. 2003; Huang et al. 2009). Analysis was done using the total porcine genes as suggested by DAVID (te Pas et al. 2010). The up-/down-regulated expression genes were individually analyzed (Zhao et al. 2013).

Microarray analysis For microarray analysis, double stranded cDNA was synthesized from RNA samples using Affymetrix One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA). The cDNA product was purified using GeneChip sample cleanup module (Affymetrix, Santa Clara, CA). cRNA was synthesized and biotin was labeled using GeneChip IVT Labeling Kit (Affymetrix, Santa Clara, CA). The product was purified using GeneChip sample cleanup module and subsequently chemically fragmented. The labeled cRNA fragment then was applied to a GeneChip porcine genome array (Affymetrix, SantaClara, CA) containing 23 256 transcripts with 23 937 probe sets. The fragmented, biotinylated cRNA was hybridized in a GeneChip Hybridization Oven 640, Affymetrix, USA. Arrays were placed in a GeneChip fluidic station 400 for a series of washes, and arrays were scanned with GeneArrayTM scanner 3000 (Affymetrix, USA), finally, and arrays were analyzed by GeneChip operating software (GCOS) (Affymetrix, Santa Clara, CA). MAS5.0 algorithm was employed to analyze the obese pigs differentially expressed genes. The lean pigs were taken as the control.

2 µg of total RNA was used for reverse transcript (RT) in a final volume of 25 µL according to the manufacturer’s instructions (Promega, USA). Quantitative real-time PCR was performed to quantitate mRNA expression of genes. Each 25-µL PCR mixture contained 12.5 µL of 29 iQTM SYBR green supermix, 0.5 µL (10 mmol L-1) of each primer and 1 µL of cDNA. As an internal control, the same reverse transcription products were also subjected to PCR in the presence of a second pair of primers specific to pig 18S rRNA. Mixtures were incubated in an iCyler iQ Real-time Detection System (Bio-Rad, USA). Quantitation of the transcripts was performed using a standard curve with 10-fold serial dilutions of cDNA. A melting curve was constructed to ensure that only a single PCR product was amplified. Each experiment was repeated at least twice. Each primer (Table 5) was designed according to porcine nucleotide sequences in GenBank using Primes Premier 5, and synthesized by Shanghai Sangon Biological Company (Shanghai, China). Because PCR efficiencies for target genes and 18S rRNA were close to 1, the amount of the specific target normalized to 18S rRNA, and relative to a calibrator (i.e., one sample of the lean BMI group), was calculated according to the following formula: Ratio=2-ΔCTtarget(sample-calibrator)/2-ΔCT18S(sample-calibrator).

Bioinformatics analysis

Statistical analysis

Gene function analysis of genes with differential expression as compared with the reference was done with the sixth version of DAVID (the Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/home.jsp;

The results were expressed as means±SE and differences were considered significant when P<0.05 tested by a t-test with Statistical Packages for Social Science 12.0 and Excel 2003 in Microsoft.

Quantitative real-time PCR confirmation

Table 5 Selected genes primer information of quantitative real-time PCR for validation Gene 18S rRNA MSTN UCP3 IL1β

Primer F: 5′-GCGGCTTTGGTGACTCTA-3′ R: 5′-CTGCCTCCTTGGATGTG-3′ F: 5′-GTTTCCGTCGTAGCGTGA-3′ R: 5′-TGAATGAGAACAGCGAGCAA-3′ F: 5′-TGCTGGGCACCATTCTC-3′ R: 5′-TTGACGGAGTCGTAGAGGC-3′ F: 5′-TCGATGCCCAGCTGTCTTCCCT-3′ R: 5′-TGTGCCATGGTTTCCACCAGGC-3′

Acknowledgements

This work was sponsored by the Yunnan Natural Science Foundation, China (2009CD056), the National Natural Science foundation of China (30660132, 31060331 and 31260592),

NCBI accession NR_002170.3

Size of product(bp) 194

Tm (°C) 55

AF188635.1

283

55

AF128837.1

127

57

NM_214055

168

60

the Special Program for Key Basic Research of the Ministry of Science and Technology, China (2007CB116201) and the National Key Program of Transgenic Project of China (2009ZX08009-140B).

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

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