LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs

gb-2007-8-6-r115 GBJ Research LongSAGE analysis of skeletal muscle at three prenatal stages in Tongcheng and Landrace pigs Tang Zhonglin zhonglinqy_99@sina.com Li Yong leombab@yahoo.com.cn Wan Ping wanping@hgbiochip.com Li Xiaoping xiaoping@webmail.hzau.edu.cn Zhao Shuhong shzhao@mail.hzau.edu.cn Liu Bang liubang@mail.hzau.edu.cn Fan Bin fanbin@mail.hzau.edu.cn Zhu Mengjin zhumengjin@mail.hzau.edu.cn Yu Mei ymcgxy@public.wh.hb.cn Li Kui likuihau@yahoo.com

Department of Gene and Cell Engineering, State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, PR China

Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education of China, Huazhong Agricultural University, Wuhan 430070, PR China

Shanghai Huaguan Biochip Co. Ltd, Shanghai, 201203, PR China

Life and Environment Science College, Shanghai Normal University, Shanghai, 200234, PR China

Genome Biology 1465-6906 2007 8 6 R115 http://genomebiology.com/2007/8/6/R115 17573972 10.1186/gb-2007-8-6-r115 6 9 2006 30 1 2007 16 6 2007 16 06 2007 2007 Tang et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Profiling obesity in pigs

Transcriptional profiling of Tongcheng and Landrace pigs using long serial analysis of gene expression provides insight into the molecular mechanism underlying differences in muscle growth.

Abstract

Background

Obese and lean pig breeds show obvious differences in muscle growth; however, the molecular mechanism underlying phenotype variation remains unknown. Prenatal muscle development programs postnatal performance. Here, we describe a genome-wide analysis of differences in prenatal skeletal muscle between Tongcheng (a typical indigenous Chinese breed) and Landrace (a leaner Western breed) pigs.

Results

We generated transcriptome profiles of skeletal muscle from Tongcheng and Landrace pigs at 33, 65 and 90 days post coitus (dpc), using long serial analysis of gene expression (LongSAGE). We sequenced 317,115 LongSAGE tags and identified 1,400 and 1,201 differentially expressed transcripts during myogenesis in Tongcheng and Landrace pigs, respectively. From these, the Gene Ontology processes and expression patterns of these differentially expressed genes were constructed. Most of the genes showed different expression patterns in the two breeds. We also identified 532, 653 and 459 transcripts at 33, 65 and 90 dpc, respectively, that were differentially expressed between the two breeds. Growth factors, anti-apoptotic factors and genes involved in the regulation of protein synthesis were up-regulated in Landrace pigs. Finally, 12 differentially expressed genes were validated by quantitative PCR.

Conclusion

Our data show that gene expression phenotypes differ significantly between the two breeds. In particular, a slower muscle growth rate and more complicated molecular changes were found in Tongcheng pigs, while genes responsible for increased cellular growth and myoblast survival were up-regulated in Landrace pigs. Our analyses will assist in the identification of candidate genes for meat production traits and elucidation of the development of prenatal skeletal muscle in mammals.

Genome studies Physiology

Background

The pig (Sus scrofa) was domesticated over 7,000 years ago and has become one of the most important farm animals 1. Anatomical, physiological, pathological and genomic similarities between pig and human have suggested that the pig could be considered a model species for human health issues 123. Moreover, pigs have distinct advantages over other animals for studying the underlying mechanisms of phenotype variation within species: highly differentiated phenotypes resulting from intensive selection, and excellent phenotype records 4. Therefore, use of pigs as research animals will benefit both animal agriculture and biomedical research.

Western pig breeds have been intensively selected over the past two decades for rapid, large and efficient accretion of muscle, which is believed to have led to deterioration in meat quality 5. Landrace, a typical lean-type western breed, is now widely used for commercial production throughout the world. While indigenous Chinese pig breeds have lower growth rates and a lower lean meat content than conventional western pig breeds 67, they have proved superior in terms of perceived meat quality 89. The Tongcheng variety is a typical indigenous Chinese breed of pig, and is one of the main groups derived from breeds in central China that have a coat color featuring two black ends. Tongcheng was also listed as an important breed for resource conservation by the Chinese Ministry of Agriculture in 2000.

In the pig, genotype has a major effect on embryonic growth rate 10. Preimplantation embryos from Meishan (an indigenous Chinese breed) females have markedly slower growth rates through day 12 than embryos from Yorkshire (a western breed) females 101112. However, there are no current reports of the differences between indigenous Chinese and western pigs in prenatal skeletal muscle development. The lower potential for postnatal muscle growth in indigenous Chinese breeds compared with exotic breeds is already evident at birth in the lower total number of fibers (TNF), which is fixed before birth 1314. Hence, prenatal skeletal muscle development is an important determinant of both muscle growth and meat quality 15. Myogenesis is a highly ordered process that can be subdivided into a sequence of temporally separable events: myogenic progenitor cell determination and proliferation, myoblast differentiation, and subsequent myotube modulation. Establishment of the TNF involves two major waves of fiber generation: a primary generation from 35 to about 60 days post coitus (dpc), and a secondary generation from about 54 to 90 dpc 13. Hence, around 35 dpc, 60 dpc and 90 dpc are key time points in prenatal skeletal muscle development. More systematic analyses of these particular stages are required to elucidate these phenomena further.

Comparative analyses of expression profiles are useful for identifying the molecular differences between variant muscle phenotypes 16. Full-transcriptome analysis of skeletal muscle may be particularly valuable for such studies. In recent years, several techniques have been used to elucidate the molecular basis of prenatal skeletal muscle development 171819. However, the genetic complexity underlying the development of skeletal muscle remains only partially understood. In particular, there have been no reports on the differences in the global transcription profiles of prenatal skeletal muscle between indigenous Chinese and western breeds of pig. Consequently, a genome-wide profiling of transcription is needed as a basis for further understanding of the molecular basis of prenatal skeletal muscle development by analyzing gene expression patterns of prenatal skeletal muscle development at key stages and assembling molecular mechanisms. This would also help to identify putative candidate genes for meat production traits. The analysis of gene expression will also facilitate the study of gene function.

Serial analysis of gene expression (SAGE) is a powerful tool for the comprehensive and quantitative measurement of gene expression and for identifying novel genes 2021. In addition, the results from experiments undertaken in different laboratories can be compared 22. Long serial analysis of gene expression (LongSAGE) has a higher specificity for gene identification than conventional SAGE 23. In this study, LongSAGE was used to investigate the molecular basis of the differences in postnatal development between indigenous Chinese and western breeds by analyzing and comparing prenatal muscle gene expression in Tongcheng and Landrace pigs. We describe the construction and screening of six LongSAGE libraries constructed from Tongcheng (T) and Landrace (L) pigs at 33, 65, 90 dpc, designated T33, T65, T90, L33, L65 and L90. To delineate the genes that were differentially expressed at these three developmental stages and also between breeds, the LongSAGE libraries were further subjected to pairwise comparisons. Through Gene Ontology (GO) annotation and cluster analyses for these differentially expressed transcripts, we have obtained the first results showing the gene regulation patterns during prenatal skeletal muscle development in these two breeds of pig.

Results

LongSAGE libraries

A combined total of 317,115 LongSAGE tags were sequenced from the six LongSAGE libraries. This translated into 98,437 distinct transcripts. Approximately 75% to 80% (83,754) of these unique tags were observed only once in each library (Figure 1a). All the libraries were very similar in the total number of tags identified (approximately 50,000 per library), as well as average GC content (44.56% to 50.02%) (Table 1; also deposited in the NCBI database (GSM125246, GSM125247, GSM125248, GSM125249, GSM125250, and GSM125251)). Moreover, the ratio of unique tags to total tags was reduced in parallel with the development of skeletal muscle for Tongcheng pigs (Table 1). This suggested that more genes were detected at early stages than at later stages in Tongcheng pigs. Also, more transcripts were expressed at lower levels during early stages of skeletal muscle development in this breed. However, we observed the opposite change in Landrace pigs (Figure 1b). These results suggest that more intricate molecular events occur during early stages of skeletal muscle development in Tongcheng pigs, but during later stages in Landrace pigs.

Table 1

Summary of data obtained from the LongSAGE libraries

LongSAGE library

T33

T65

T90

L33

L65

L90

Total tags¹

50,450

53,927

53,761

53,104

54,483

51,188

Unique tags²

25,738

24,655

22,035

24,408

24,829

22,464

Unique tags/total tags (%)³

51.0

45.7

41.0

46.0

45.6

43.9

Tags/clone

23.4

26.5

31.7

25.97

24.8

25.9

Average GC content⁴

45.16

50.02

44.56

47.24

47.15

46.20

Remaining total tags⁵

32,722 (64.8)

37,058 (68.6)

40,074 (74.5)

37,135 (69.8)

37,948 (69.6)

36,596 (71.4)

Remaining unique tags⁶

8,081

7,786

8,348

8,439

8,294

7,872

Unmatched tags⁷

2,458 (30.4%)

2,377 (30.5%)

2,621 (31.4%)

2,362 (28.0%)

2,740 (33.0%)

2,513 (31.9%)

Matched tags⁸

5,623 (69.6%)

5,409 (69.5%)

5,727 (68.6%)

6,077 (72.0%)

5,554 (67%)

5,359 (68.1%)

Single match⁹

5,437 (96.7%)

5,249 (96.9%)

5,550 (97%)

5,887 (97%)

5,358 (96.8%)

5,163 (96.7%)

Multiple matches¹⁰

188 (3.3%)

169 (3.1%)

174 (3.0%)

185 (3.0%)

175 (3.2%)

176 (3.3%)

¹Total tags were obtained in each library. LongSAGE tags containing wildcard characters not in {A, C, G, T} were discarded. ²Unique tags were obtained in each library. ³The differences in accrual rates (the ratio of unique tags to total tags) indicate that the number of genes expressed regularly changed during myogenesis. ⁴For an explanation of this value, in the context of the quality of a SAGE library, see Margulies et al. [31]. ⁵The total tags remaining in each library after eliminating the unique tags with a frequency <2 in all six libraries. The percentage of non-singleton tags is shown in parentheses. ⁶The unique tags remaining in each library after eliminating the unique tags with a frequency <2 in all six libraries. ⁷Unique tags unmatched with any known sequence. The values in parentheses indicate the percentage of unique tags in the total. ⁸Unique tags that correspond to UniGene entries. The values in parentheses indicate the percentage of unique tags in the total. ⁹Unique tags matched with a single UniGene sequence. ¹⁰Unique tags matched with more than one UniGene sequence. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus.

Figure 1

Genetic complexity of prenatal skeletal muscle of pigs

Genetic complexity of prenatal skeletal muscle of pigs. (a) Distribution of LongSAGE tags in abundance categories. The number of unique transcripts (tags) for each abundance category is shown. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus; 1, 2-4, 5-9, 10-100 and >100 indicate tag abundance categories in our LongSAGE libraries. (b) Genetic complexity of the Tongcheng pigs in comparison with the Landrace variety during skeletal muscle development.

A total of 83,754 unique tags, which were not observed more than twice in any of the six libraries, were eliminated from the analysis to compensate for possible sequencing errors 24. The remaining 14,683 valid unique tags were then selected for further comparative analysis. As shown in Table 1, the percentage of unique tags assigned to UniGene entries ranged from 67% to 72%. Of these, about 97% corresponded to single UniGene entries, whereas approximately 3% matched more than one UniGene cluster because they contained a 3' region conserved between different genes. In addition, these unique tags matched at the punctuation mark (CATG) in all the UniGene clusters. A total of 5,953 unique tags were unmatched by any known sequence in the combined LongSAGE libraries, while the occurrence of unknown tags was probably due to the incompleteness of pig genome sequencing 225.

Validation of LongSAGE data by quantitative PCR

To confirm that the genes identified were differentially expressed, we selected 12 genes for validation by quantitative PCR (QPCR) on the basis of their functional roles in skeletal muscle development and expression patterns in these libraries. Among these genes, five encoding myofibrillar proteins (fast skeletal myosin light chain 2 (MYLPF); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, light chain 1, alkali, skeletal, fast (MYL1); sarcolipin (SLN); and troponin C type 2, fast, (TNNC2)), and two encoding proteins involved in regulation of myoblast proliferation and differentiation (lectin, galactoside-binding, soluble, 1 (galectin 1; LGALS1); and transducer of ERBB2, 1 (TOB1)) were selected for validation. Three genes, RPS28 (ribosomal protein S28), GNB2L1 (guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1), and TPT1 (tumor protein, translationally controlled 1), which are associated with protein synthesis, were selected because their expression levels differed significantly between the two breeds at 65 dpc. Validation was also performed for the cellular retinoic acid binding protein 1 (CRABP1) gene, which was expressed specifically at 33 dpc in both breeds. Finally, a noncoding RNA, named trophoblast-derived noncoding RNA (TncRNA), which was up-regulated during myogenesis in both breeds, was identified and selected for validation by QPCR. Housekeeping genes such as those encoding β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), commonly used as internal controls for such analysis, were not suitable for normalization in these experiments because their transcription was altered during myogenesis 1826. Histone 3 mRNA (H3 histone, family 3A (H3F3A)), which was consistently expressed in our study, was therefore used as an internal control. The results for a panel of the 12 genes were in good agreement with the LongSAGE data (Table 2) and there was a highly significant correlation (r = 0.79, p = 8.52E-17) between the two techniques. For example, genes encoding myofibrillar proteins, such as MYL1, SLN, MYLPF and TNNC2, were shown to be up-regulated during myogenesis in both the LongSAGE and QPCR experiments, while QPCR also showed a significant difference between the two breeds in the expression of GNB2L1 and TPT1 at 65 dpc. For CRABP1, although LongSAGE tags were not detected in skeletal muscle from either breed at 65 or 90 dpc, QPCR indicated that it was expressed at low levels. This correlation indicated that our LongSAGE results reliably reveal the differences in gene expression profiles in skeletal muscle.

Table 2

Genes differentially expressed in LongSAGE data and validated by QPCR

Gene

Method*

Fold changes of gene expression in different skeletal muscle samples

T33

T65

T90

L33

L65

L90

MYLPF

QPCR

1.00^a

8.00^b

16.00^c

0.71^a

10.56^b

39.40^d

SAGE

1.00

4.10

4.63

0.67

2.31

2.34

MYL2

QPCR

1.00^a

4.92^b

2.46^ab

0.31^c

4.59^b

2.64^b

SAGE

1.00

1.41

2.56

0.23

2.70

2.23

MYL1

QPCR

1.00^a

9.85^b

13.00^c

0.41^a

12.13^bc

34.30^d

SAGE

1.00

6.62

11.85

0.38

6.31

5.77

SLN

QPCR

1.00^a

40.50^b

78.79^c

0.71^a

64.00^b

207.94^d

SAGE

1.00

16.70

29.30

0.20

14.70

24.00

TNNC2

QPCR

1.00^a

22.63^b

6.50^c

0.27^d

6.96^c

8.57^c

SAGE

1.00

6.74

5.52

0.26

4.03

3.58

TOB1

QPCR

1.00^a

2.30^a

7.46^b

1.53^a

3.73^c

1.87^a

SAGE

1.00

3.00

11.00

1.00

3.00

2.00

CRABP1

QPCR

1.00^a

0.07^b

0.04^b

2.64^c

0.05^b

SAGE

1.00

0.00

3.36

0.00

LGALS1

QPCR

1.00^a

0.57^b

0.62^bc

0.41^b

0.74^b

0.38^b

SAGE

1.00

0.86

0.88

1.26

0.80

GNB2L1

QPCR

1.00^a

0.01^b

0.76^a

1.00^a

0.54^a

SAGE

1.00

0.30

1.02

0.93

1.02

0.80

TPT1

QPCR

1.00^a

0.00^b

2.00^c

1.07^a

2.83^c

1.74^ac

SAGE

1.00

0.74

2.69

0.93

1.61

1.41

RPS28

QPCR

1.00^a

0.81^ab

1.00^a

0.62^b

0.81^ab

SAGE

1.00

0.39

1.02

0.81

1.06

TncRNA

QPCR

1.00^a

0.81^a

5.66^b

0.27^c

1.00^a

1.62^d

SAGE

1.00

1.50

29.50

0.50

1.00

5.00

*The QPCR row provides the ratio of the 2^-ΔΔCtvalue for each breed/stage sample to the 2^-ΔΔCtof T33. For the T33 sample, the fold change in gene expression relative to the T33 equals one, by definition. For each row, different superscript letters (a, b or c) indicate a statistically significant difference in gene expression between skeletal muscles at p < 0.05 in the QPCR experiment; the same superscript letters indicate no statistically significant difference (p > 0.05). For values with two superscript letters, gene expression in that sample was not significantly different from the expression in the other two samples compared, but the other two samples were significantly different in gene expression; for example, for the MYL2 gene, the value 2.46^abindicates that the expression in T90 was not statistically significantly different from that of both T33 and T65, but there was a significant difference between the expression of T33 (1.00^a) and T65 (4.92^b). The SAGE row provides the frequency of each breeds/stage sample relative to T33 frequency in the LongSAGE data. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus.

Cluster analysis

To gain insight into transcriptome-scale similarities among all six skeletal muscle libraries, we performed systematic cluster analysis using two different methods (Cluster 3.0 and TreeBuild 3D software) independently. Both sets of results indicated that the six different transcription profiles could be divided into three distinct classes (Figure 2). L65 and L90 were initially clustered together because their expression profiles were most similar, and T90 was then grouped into this class by similarity to both of them. T33 and L33 were clustered to form another class. Interestingly, T65 differed from the other five samples in transcriptional profiling and was clustered into a single class. Also, the gene expression patterns in Landrace pigs at 65 and 90 dpc were more similar than those in Tongcheng pigs.

Figure 2

Similarity of transcriptome profiles between six muscle tissues using cluster analysis

Similarity of transcriptome profiles between six muscle tissues using cluster analysis. (a) Clustering dendrogram of LongSAGE libraries generated by Cluster 3.0 and TreeView software. (b) Hypothetical tree-like diagram generated by TreeBuild 3.0 software, indicating the relatedness of these six libraries.

Comparisons of the gene expression profiles between Landrace and Tongcheng pigs during skeletal muscle development

Table 3 shows the comparison of differentially expressed tags between the libraries. A total of 1,400 and 1,201 unique tags were differentially expressed during skeletal muscle development in Tongcheng and Landrace pigs, respectively. Among these tags, 234 (corresponding to 182 annotated transcripts) and 203 (corresponding to 153 annotated transcripts) matched annotated genes in the Tongcheng and Landrace breeds, respectively. Figure 3 shows the distribution of differentially expressed tags at each stage. It reveals that most of these transcripts were expressed in all the skeletal muscle samples at each of the three selected stages. Only a few were restricted in regulation of expression to a single stage.

Table 3

Number of differentially expressed genes and node distance between six skeletal muscle samples

T33

T65

T90

L33

L65

T65

701 (2.24)

T90

751 (1.96)

781 (1.99)

L33

532 (1.38)

988 (2.51)

1,008 (2.42)

L65

645 (1.51)

653 (1.52)

577 (1.43)

741 (1.76)

L90

684 (1.83)

697 (1.76)

459 (1.3)

812 (2.11)

341 (1.26)

The values in parentheses indicate the node distance between skeletal muscles in cluster analysis using TreeBuild 3.0. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus.

Figure 3

Venn diagrams of genes differentially expressed at different stages

Venn diagrams of genes differentially expressed at different stages. There were 1,400 and 1,201 differentially expressed tags for (a) Tongcheng and (b) Landrace pigs, respectively. This figure is not drawn to scale. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus.

Gene Ontology analysis

To gain further insight into the biological importance of the differentially expressed transcripts identified, we further analyzed the functional categories of the annotated genes by querying their associated Gene Ontologies. In general, the categories of biological processes involved in myogenesis were similar in Tongcheng and Landrace pigs. Mainly, they included cellular physiological pathways, metabolism, localization processes, cell communication, responses to stimuli and development (Figure 4) (at level 3). However, the numbers of differentially expressed genes involved in certain biological processes (at level 5) were quite different in Tongcheng and Landrace pigs. For instance, more genes involved in cellular biosynthesis (T versus L = 21.32% versus 9.77%, p = 0.00646), regulation of cell proliferation (T versus L = 3.55% versus 0%, p = 0.04446), organic acid metabolism (T versus L = 6.70% versus 0.79%, p = 0.07322), macromolecule biosynthesis (T versus L = 14.21% versus 7.52%, p = 0.07818), and regulation of cell size (T versus L = 3.05% versus 0%, p = 0.08482) were differentially expressed in Tongcheng pigs. In contrast, there was a tendency for more differentially expressed genes involved in biopolymer metabolism (L versus T = 30.83% versus 22.34%, p = 0.09562) to be identified in Landrace pigs.

Figure 4

GO classifications of biological processes of genes differentially expressed during skeletal muscle development

GO classifications of biological processes of genes differentially expressed during skeletal muscle development. On the basis of the annotated genes that matched our unique tags, GO analysis was carried out using the Blast2GO program [66]. The numbers shown indicate the exact number of genes for each GO classification. (a) GO categories for Tongcheng pigs. (b) GO categories for Landrace pigs.

Expression patterns

In order to determine whether the temporal pattern of expression of a gene during prenatal skeletal muscle development might predict its molecular function, clusters of differential expression tags were assembled. The differentially expressed genes identified in our screening were found to exhibit eight types of pattern in both Tongcheng and Landrace pigs (Additional data files 1 and 2 list all the LongSAGE tags used in this analysis and their corresponding cluster assignments for Tongcheng and Landrace pigs, respectively). These patterns are shown graphically for each breed in Additional data file 6. Table 4 lists the genes that had previously been confirmed (Additional data file 7) to be either highly or specifically expressed in developing skeletal muscle and for which the specific GO category assignments were enriched in each expression pattern cluster for both pig breeds.

Table 4

Summary of LongSAGE tag cluster data according to breed type

Cluster*

Tags per cluster^†

Previously characterized genes^‡

GO categories enriched in the cluster^§

Tongcheng

191

APOBEC2, BIN1, BTBD1, CACNB1, MYBPC1, MYL1, MYOZ1, RRAS, RTN4, SH3BGR, SLN, TCAP, TMOD1, TNNC2, TNNT3, TPM1

Muscle development (0.0007806)

245

ACTN2, AMPD1, ATP2A1, CKM, DES, ENO3, MYH2, RYR1, TMOD4, TPM2, GAPDH, IGF2

Carbohydrate metabolism (0.001492) Mitochondrion (0.016297)

204

LASS2

Signal transduction (0.008368)

160

SPARC, HSPB2, ATF4, MYL2

Tricarboxylic acid cycle (0.021523489)

152

MYH3, S100A11

Ribosomal (1.25E-06)

218

PSMD2, RTN2, SERF1A, THBS4, TIMM13, TNNC1, TNNI1, TNNT1, FAU, HSPA8, FHL1C, HDAC5, MYOT

Obsolete molecular function (0.048234)

153

DGKZ, GNB2L1

Binding (0.023903)

Total

1,400

Landrace

158

APOBEC2, CFL2, HSPB2, ITGB1, MYH3, PGM5, RTN4, TNNT1, TPM2, FHL1C

Muscle development (0.003181)

SPARC, TIMM13

Mitochondrion (0.002539)

126

AMPD1, CKM, DES, MYBPC1, MYL1, MYOZ1, PFDN5, RYR1, SLN, TNNT3, MYL2

Muscle contraction (0.035242)

160

LGALS1, SERF1A1, FAU, HSPA8

Ribosomal (4.83E-07)

Cytoskeleton organization and biogenesis (0.012664)

301

GSN, TPM3, TPM4, TRIO, S100A11, NACA, SUMO2

Cell cycle (0.002587)

CACNB1, MYLPF, TMOD1, TNNI1, IGF2

Development (0.014224)

183

ACTN2, ATP2A1, BIN1, CCNG1, ENO3, MYH2, RTN2, SH3BGR, TCAP, TPM1, GAPDH, SDHD, HDAC5

Protein complex assembly (0.035886)

Total

1,201

*The expression patterns in each cluster for Tongcheng and Landrace pigs are shown as (A) and (B) in Additional data file 6, respectively. ^†Tags in cluster value corresponds to the number of LongSAGE tags in each cluster. ^‡Selected genes previously detected during myogenesis are indicated (references for these are given in Additional data file 7). ^§All significant (p < 0.05) GO categories over-represented in individual clusters were determined by EASE scores [68]; raw EASE scores (Jackknife one-sided Fisher exact p values) for the categories in question are given in parentheses.

Most of the genes previously reported to be regulated in porcine prenatal skeletal muscle were detected in our analysis and shared similar expression patterns 1718. For instance, expression of desmin (DES) and GAPDH was increased during myogenesis in both breeds, but both vimentin (VIM) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) showed lower expression levels. These data are consistent with previous reports 1718. Some genes that have been shown to play important roles in the development of skeletal muscle in humans and model animals 2728, but had not been identified in pig, were also detected in our analysis. These included SUMO2 (SMT3 suppressor of mif two 3 homolog 2 (Saccharomyces cerevisiae) and LGALS1, which have essential functions during myotube formation 2728. SUMO2, a member of the SUMO gene family, and LGALS1 were the only differentially expressed genes of this type found in Landrace pigs.

Certain functional categories of genes were over-represented in a number of LongSAGE tag clusters (Table 4). In Tongcheng pigs, muscle development genes, which are typically up-regulated in development, were enriched in cluster 1. Cluster 2 was enriched in mitochondrial proteins and carbohydrate metabolism. Tricarboxylic acid cycle genes were concentrated in cluster 4. Ribosomal proteins, which showed lower expression in the later stages of development, were highly enriched in cluster 5. Genes representing a number of other functional categories were also enriched in specific clusters; for example, genes involved in signal transduction, obsolete molecular function and protein binding in clusters 3, 7 and 8, respectively. In Landrace pigs, by contrast, muscle development and muscle contraction genes were enriched in clusters 1 and 3, respectively. Mitochondrial proteins were concentrated in cluster 2. Ribosomal proteins were obviously enriched in cluster 4. In addition, genes involved in cytoskeleton organization and biogenesis, cell cycle and protein complex assembly, which were concentrated in clusters 5, 6 and 8, respectively, were not enriched in the Tongcheng clusters. On the other hand, genes for signal transduction, the tricarboxylic acid cycle and obsolete molecular function were not over-represented in Landrace pigs.

Differential expression of genes between Tongcheng and Landrace pigs at the same stage of skeletal muscle development

T33 versus L33

We identified 532 tags that were differentially expressed between the T33 and L33 samples, including 327 known genes or expressed sequence tags (ESTs) and 105 novel tags. Among these genes, 221 were expressed more abundantly in T33, while 311 were expressed at higher levels in L33. Analysis of the GO annotations indicates that more genes encoding proteins associated with muscle development (18.03% versus 1.69% for T33 versus L33, p = 0.00423) were up-regulated in Tongcheng pigs, whereas more genes related to cellular biosynthesis (16.39% versus 32.20% for T33 versus L33, p = 0.05927) and cofactor metabolism (1.64% versus 10.17% for T33 versus L33, p = 0.05532) were up-regulated in Landrace pigs (Figure 5a). We further focused on 67 transcripts that showed significant fold differences ≥2.0 (p < 0.01) and tag counts ≥10 in any of our SAGE libraries (Additional data file 3). Among these genes, the following were more highly expressed in T33: PDLIM7 (PDZ and LIM domain 7 (enigma)), CAPNS1 (calpain, small subunit 1), ACTC (actin, alpha, cardiac muscle), TNNC2, FSCN1 (fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus)), COL1A1 (collagen, type I, alpha 1), MYL2, ACTG1 (actin, gamma 1), and MYH3 (myosin, heavy polypeptide 3, skeletal muscle, embryonic). It is obvious that most of these genes are related to muscle fiber formation. In contrast, the following were more highly expressed in L33: MARCKS (myristoylated alanine-rich protein kinase C substrate), TSC22D1 (TSC22 domain family, member 1), CRABP1, PTMA (prothymosin, alpha (gene sequence 28)), GSTP1 (glutathione S-transferase pi), FAU (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (fox derived)), UCHL1 (ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)), MDK (midkine (neurite growth-promoting factor 2)), and GNAS (GNAS complex locus). Interestingly, we also detected several genes in one breed only. For example, DNAJC5 (DnaJ (Hsp40) homolog, subfamily C, member 5) and RPL9 (ribosomal protein L9) were not detectable in L33, whereas RPL29 (ribosomal protein L29), PSMB2 (proteasome (prosome, macropain) subunit, beta type, 2), RPS4X (ribosomal protein S4), and SLC25A6 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6) were absent from T33.

Figure 5

GO annotations for 'biological process' for differentially expressed genes between breeds at specific stages

GO annotations for 'biological process' for differentially expressed genes between breeds at specific stages. These categories include only Gene Ontologies with significant difference in gene numbers between breeds (p < 0.10). Numbers of up-regulated genes in Tongcheng pigs were compared with those in Landrace pigs by the FatiGO tool and p values <0.10 were considered significant. Gene Ontologies are listed on the vertical axis. The score on the horizontal axis is the percentage of up-regulated genes. T, Tongcheng; L, Landrace; 33, 65 and 90 refer to days post coitus.

T65 versus L65

A total of 653 transcripts were differentially expressed between T65 and L65, including 497 annotated genes or EST sequences and 156 novel tags. Of these, 342 were up-regulated in T65 and 311 were more highly expressed in L65. Analysis of the biological processes associated with these factors suggests that more genes related to programmed cell death (0% versus 5.88% for T65 versus L65, p = 0.03521), lipid biosynthesis (0% versus 4.41% for T65 versus L65, p = 0.08233), response to heat (0% versus 4.41% for T65 versus L65, p = 0.08233) and responses to abiotic stimuli (0% versus 4.41% for T65 versus L65, p = 0.08233) were up-regulated in Landrace pigs (Figure 5b). One hundred and nineteen unique tags were differentially expressed with ≥2.0-fold difference (p < 0.01) between the two breeds at 65 dpc (Additional data file 4). Among these transcripts, ribosome families were the most variable. Most of these genes were more highly expressed in Landrace pigs, for example, those encoding ribosomal proteins L36 (RPL36), L38(RPL38), S26 (RPS26) and S28 (RPS28). The following were also more highly expressed in L65: IGF2 (insulin-like growth factor 2 (somatomedin A)), GNB2L1, DES (desmin), ALDOA (aldolase A, fructose-bisphosphatase),CD63 (CD63 molecule), TTN (titin), TPT1, and RYR1 (ryanodine receptor 1). On the other hand, the following were more highly expressed in T65: Cox6c (cytochrome c oxidase subunit Vic), FAU, PCBP4 (poly(rC) binding protein 4), PPP1R14B (protein phosphatase 1, regulatory (inhibitor) subunit 14B), FHL1C (four and a half LIM domains 1 protein, isoform C), THBS4 (thrombospondin 4), TMOD1 (tropomodulin 1), and YWHAQ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide). Interestingly, four genes were found to be absent from T65: VCP (valosin-containing protein), RPL29, SULT1E1 (sulfotransferase family 1E, estrogen-preferring, member 1), and RPLP0 (ribosomal protein, large, P0). Moreover, six transcripts were detectable in T65 only, including PRDX3 (peroxiredoxin 3), BCAP31 (B-cell receptor-associated protein 31) and TH1L (TH1-like).

T90 versus L90

We found that 459 transcripts, including 330 annotated genes and ESTs and 129 novel tags, were differentially expressed between T90 and L90. Of these transcripts, 273 were up-regulated in T90. More genes related to the regulation of cellular metabolism (2.08% versus 18.52% for T90 versus L90, p = 0.02071), macromolecule biosynthesis (12.50% versus 33.33% for T90 versus L90, p = 0.03901), and cellular biosynthesis (16.67% versus 37.04% for T90 versus L90, p = 0.05552) were up-regulated in Landrace pigs, and more genes encoding proteins associated with cellular catabolism (12.50% versus 0% for T90 versus L90, p = 0.08166) and carbohydrate metabolism (12.50% versus 0% for T90 versus L90, p = 0.08166) were up-regulated in Tongcheng pigs (Figure 5c). We found that 48 unique tags had an abundance of at least 10 copies in one of the libraries and there was at least a 2.0-fold difference in expression (p < 0.01) between T90 and L90 (Additional data file 5). Within this group, genes related to muscle contraction were up-regulated in T90: FKBP1A (FK506 binding protein 1A, 12 kDa), VDAC3 (voltage-dependent anion channel 3), TNNT1 (troponin T type 1 (skeletal, slow)), RTN4 (reticulon 4), TPM2 (tropomyosin 2 (beta)), MYH2 (myosin, heavy polypeptide 2, skeletal muscle, adult), ACTN2 (actinin, alpha 2), RYR1 and TNNT3 (troponin T type 3). The expression of SDHD (succinate dehydrogenase complex, subunit D, integral membrane protein), FMOD (fibromodulin), GNAS and CD63 was higher in L90. Most conspicuously, the transcript for noncoding RNA, TncRNA, was also upregulated in T90. The transcript for RPL29 and a novel transcript corresponding to LongSAGE tag 'GGCGCAGGCGTGGGGGC', which fitted the criteria selected for both T33 versus L33 and T65 versus L65, were also up-regulated in L90.

Longer cDNA sequences obtained from the novel SAGE tags

On average, 30% of the unique tags that we screened did not match any known sequence, particularly tags with lower copy numbers. These novel tags might, therefore, represent uncharacterized genes or transcripts. To convert novel tags into their corresponding cDNA fragments, the generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification (GLGI) was carried out. A total of 113 longer cDNA sequences were experimentally obtained from 67 novel unique tags (Table 5). These ESTs ranged from 35-382 base-pairs (bp; mean 121 bp) in length. However, 100 sequences still matched no known sequence in the NCBI database. Six polyadenylation signals are frequently found in human transcripts 29. Of these, 'AATAAA' and 'ATTAAA' had the highest frequencies among the unidentified genes (AATAAA, 50; ATTAAA, 24; AATAAT, 6; AATTA, 11; CATAAA, 5; AGTAAA, 5). Moreover, a total of 12 cDNA ends among these sequences contained two or three CATG sites, perhaps because of incomplete digestion at the 3'-most CATG consensus site by the anchor enzyme 'NlaIII'.

Table 5

cDNA sequence isolated by GLGI from novel LongSAGE tags

LongSAGE tag

Product size (bp)*

No. of UniGene matches

Abundance

T33

T65

T90

L33

L65

L90

CCCCATTGTACTTGAAC

116,81,92

CCCATTGTACTCGAACT

117,214

CCCATTGTACCTGAACT

134,402,116,151,73

CCCATTGTACTGGAACT

106,74

CCCATTGTACTTGAGCT

74,92,114

CCCATTGTACCTTGAAC

121

CCCATTGTACTTGACTT

115

CCCATTGTACTTGAACC

136,116

CCCATTGTACTTGGACT

102,128

CCCATTGTACTTGCACT

118

ACAGGAACTCCTTGCCT

104

TCTGGAGAAGTGGGGAG

CAGTTCTCCCACCTTAT

GGGCGTCTAAATGTGAA

89,115,121

TTCTTGATCTCTTCCTG

119,127

GCCACATCCTTTCTCCC

189,161,116,98,145

GACGAGATGGAGTTCAC

94,107,100

GATCGGGACATTGGGGC

87,81

GAGAAAACGAAGACAAG

136,65

GAAAATTGCCCCCCCCC

113,77

GACTAGCAATTTCGGTT

55,68

GGGCTGCTTTTTGTCAC

58,156

TGCTAGATTGGAGTGGG

93,124,150

AAGCTGTGGTTTGATCC

AGACAGACAGTTGCTGG

97,161

AGCATCCCAAACAAACA

79,116

ATGGGCCGTTAATAAAG

CAAACTCTTGCCCCGAT

1,458

CAGCAGGTGCTCAATAA

106

32,297

CCAACACTCATAGCAAT

71,161,100

CCCATTGGACTTGAACT

247

CCCATTGTACTTGAACT

134

448

130

577

501

448

515

GAACGCCTAATAAAGCA

106

32,126

CCTGACCCCACACGCCT

AGTAAACGGGCTGCTCC

134