BACKGROUND
the development of skeletal muscle affects meat production and growth rate in pigs.
the genetic mechanisms controlling these traits are being uncovered  <cit> .
in addition, the selection of meat quality based on molecular regulation is important for livestock breeding, and there have been many studies on skeletal muscle in different pig breeds  <cit> .
the total number of fibers is constant in the postnatal stage, and postnatal muscle growth is through hypertrophy of myofibers, or conversion of myofiber types  <cit> .
primary and secondary fibers emerge at distinct embryonic stages during the skeletal muscle development of pigs  <cit> .
primary myofiber formation occurs in the first wave of fiber generation, from about 35 days of gestation until around 60 days.
secondary myofibers appear at around 50– <dig> days based on the template of the primary myofiber surface.
the third wave involves the process of transition between slow-oxidative and fast-glycolytic fiber types, which occurs from birth to 60 days of age  <cit> .
muscle maturation is completed in the early postnatal period  <cit> .
therefore, we chose the critical skeletal muscle developmental stages across the prenatal to postnatal periods  for studying the regulatory mechanisms and expanding the molecular genetics of muscle fiber development.
many genes or factors participate in the process of muscle fiber formation, and muscle development is under complex genetic regulation.
it is known that the muscle regulatory factor  and myocyte-specific enhancer binding factor  <dig>  gene families play critical roles in regulating muscle fiber development.
mrf genes are muscle-specific transcription factors, and induce myoblast proliferation or fusion differentiation during distinct stages of myogenesis  <cit> .
mef <dig> regulates muscle-specific transcription during myogenesis and is activated during the development of the muscle  <cit> .
however, knowledge of the functional genetics of myogenesis is currently insufficient and incomprehensive.
monitoring the transcriptional profiling from the prenatal to postnatal developmental stages in pigs will act as an animal model that will not only uncover the regulatory mechanisms behind factors affecting muscle development  <cit> , but will also benefit the understanding of diseases affecting human muscle and muscular atrophy  <cit> .
with biotechnological advances in gene expression profiling, we have the ability to study large-scale differential regulation of genes genome wide, and to measure the transcriptional responses to certain complex biological conditions  <cit> .
previous studies have presented expression analysis of myogenesis using microarrays  <cit> , long sage  <cit> , and differential display rt -polymerase chain reaction   <cit> .
digital gene expression technology is sensitive for the detection of low-abundant transcripts and small changes in gene expression  <cit> , and has been applied extensively to compare the differences in transcription profiles of different tissues or breeds  <cit> .
we present a panorama of transcriptome-wide longissimus dorsi muscle development using digital gene expression with solexa/illumina’s genome analyzer platform to study the variation between the dynamic transcriptome profiles of tongcheng  and yorkshire  pig breeds.
previous studies have demonstrated differences in gene expression in pig longissimus dorsi muscle during fetal development, and some have shown breed-specific expression profiles  <cit> .
indeed, differences exist in muscle traits such as muscularity and muscle fiber type; muscle characteristics are different between pig breeds, and molecular biomarkers have been reported  <cit> .
however, the molecular genetic mechanisms of these breed-specific differences remain unclear, in terms of meat quality and visual phenotype.
in this study, we selected the tc pig, a characteristic indigenous pig breed from the hubei province of central china with slow growth and prolific reproduction  <cit> , and the yk pig, which is characterized by fast growth, low back fat, and a high lean meat percentage, to survey the differences in transcription.
previous studies have shown that indigenous swine breeds have slower growth rates and less lean meat percentages than exotic pigs  <cit> .
our study opens up the scope of knowledge of muscle-specific regulation and the microscopic molecular mechanisms.
our objective was to analyze the biological information regarding the transcriptional profiles of longissimus muscle between the two breeds and to further identify key gene regulatory networks and breed-specific pathways affecting skeletal muscle development in the pig.
RESULTS
histological section and fiber size
we evaluated the morphological differences of skeletal muscle development between tc and yk pigs, from the histological appearance through the prenatal and postnatal stages.
histological cross-section  showed that myoblasts developed quickly from  <dig> dpc , but there was no differentiation into primary fibers in either pig breed at  <dig> dpc, and the number and density of myoblasts in tc was more than that in yk.
the myoblasts differentiated gradually, with primary fibers emerging from  <dig> to  <dig> dpc, and could be seen in the histological section to be fully formed at  <dig> dpc.
primary fibers continued to expand and enlarge until  <dig> dpc.
meanwhile, the primary fiber bundles were separated by the connective tissues.
secondary fibers appeared at  <dig> dpc in yk pigs and formed until  <dig> dpc, but they emerged later in tc pigs.
secondary fibers could be seen clearly around the primary fibers at  <dig> dpc, and the secondary fibers increased gradually until  <dig> dpc.
the fiber bundles fused at around  <dig> dpc and appeared to form into the myotubes at  <dig> dpc; the primary and secondary fibers were not distinguishable at this period.
the intramuscular fat texture showed differences in the histological section in the latter period between tc and yk pigs.
these differences are associated with the different development of fat deposits in these two breeds.
the muscle fibers grew rapidly, and finally the muscular tube formed and constituted the muscle mass.
the phenotypes and sizes of the muscle fibers still varied based on the cross-sectional areas during the postnatal development stages  for both tc and yk pigs).
solexa sequencing and tag mapping
after filtering adaptor tags, empty tags, low-quality tags, and one-copy tags from the raw data,  <dig> , <dig> total clean tags were obtained in the tc breed, including  <dig> , <dig> distinctly clean tags.
<dig> , <dig> total clean tags and  <dig> , <dig> distinctly clean tags were obtained in the yk pig .
the total clean tags accounted for  <dig> % and  <dig> % of the raw tags in tc and yk, respectively.
the number of unknown distinct tags was  <dig>  and  <dig> , on average, in each sample from tc and yk pigs.
moreover, the heterogeneity and redundancy of the mrna were confirmed, and the results showed that the high copy number clean tags  accounted for  <dig> % of the total clean tags in tc and  <dig> % in yk, while the low copy number clean tags  accounted for less than 1% of the total clean tags in both breeds.
additional file 3: figure s <dig> illustrates the distribution of total clean tags and distinct clean tags of tc and yk.
the type of distinct tags were gradually stable, with the total number of sequence tags rising according to saturation analysis, and the percentage of genes identified was no longer increasing when the library size approached  <dig> million .
the number of tags mapping to genes was  <dig>  and  <dig>  in tc and yk, and the number of genes with unambiguous tag mapping was  <dig>  and  <dig> .
the number of unambiguous tags mapping to genes accounted for  <dig> % of all tags mapping to genes, and  <dig> % of the total clean tags for the tc breed, in contrast to  <dig> % and  <dig> % for the yk breed .
differential gene expression analysis and validation of solexa sequencing data
in this study, differentially expressed  genes at the  <dig> time points of skeletal muscle development of tc and yk pigs were investigated through transcriptome-wide solexa sequencing, and analyzed using the noiseq method.
in the tc libraries, a total of  <dig>  genes were found to be de; of these,  <dig>  genes were upregulated and  <dig>  genes were downregulated in  <dig> comparison libraries .
in the yk libraries, a total of  <dig>  de genes were identified, with a total of  <dig>  upregulated and  <dig>  downregulated genes in  <dig> comparison libraries during different muscle development stages.
these results suggest that the number of de genes changed significantly at 30– <dig> dpc, 55– <dig> dpc, and 90– <dig> dpc in the prenatal muscle development stages, and at 3– <dig> wpn in the postnatal stages.figure  <dig> number of de genes between the comparison libraries.
tc: tongcheng, yk: yorkshire.
before birth:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and 105 days post-coitus.
after birth:  <dig>   <dig>   <dig>  and 5 weeks post-natum.
a: tongcheng pigs, b: yorkshire pigs, c: the identical period comparison between tc and yk pigs.
five genes were chosen randomly for the purpose of validating the results of the solexa sequencing using real-time quantitative pcr .
tnnc <dig>  is a key gene encoding a protein that regulates striated muscle contraction  <cit> .
gdf <dig>  regulates cell growth and differentiation in both embryonic and adult tissues, and is related to skeletal muscle age  <cit> .
myot  encodes a cytoskeletal protein that is involved in myofibril assembly and actin binding in the muscle tissue  <cit> .
pgam <dig>  is a muscle-specific phosphoglycerate mutase and is involved in the glycolysis pathway for myoblast fusion  <cit> .
mylpf  is related to a structural constituent of muscle and calcium ion binding  <cit> .
in our study, rad <dig> homolog b was used as the reference gene because it showed consistent expression.
the data and spearman’s correlation coefficient analysis suggest that the solexa sequencing had a highly significant correlation with real-time qpcr , and the expression patterns of these genes were consistent between the two methods.figure  <dig> validation of solexa sequencing data with qpcr.
the vertical axis indicates the fold change of transcript abundance.
the horizontal axis indicates the samples for tc and yk pigs.
the r-value shows the spearman’s correlation between the two methods.
cluster analysis of de genes
gene expression patterns at the scale of the transcriptome were measured by systematic cluster analysis, to explore the similarities and compare the relationship between the different libraries in tc and yk pigs.
the results of hierarchical clustering indicate that the gene expression similarity could be classified into distinct groups .
tc <dig>  tc <dig>  and tc <dig> were clustered together, and tc <dig> and tc <dig> were of the same class; while yk <dig> and yk <dig> had similar expression patterns, and yk <dig> and yk <dig> were clustered into another class in the prenatal stage owing to the similarity of gene expression.
the gene expression patterns of  <dig>   <dig>  and 5 weeks of age were grouped together in both breeds.figure  <dig> hierarchical clustering analysis for all the de genes in tc and yk pig breeds, respectively.
a: tc , b: yk .
go analysis of de genes
the screened de genes were classified by gene ontology  based on their biological process and function in tc and yk pigs.
the go annotation results are presented in additional file 6: table s <dig> and additional file 7: table s <dig>  significant go categories were selected , and showed different extents of enrichment .
go analysis showed that biological processes related to muscle development were mainly enriched in different stages for both breeds; these genes were associated with go terms including myofibril, sarcomere, and myosin complex, which are involved in muscle component and structure.
muscle tissue morphogenesis and development, cell differentiation and development, regulation of muscle contraction and developmental process were enriched significantly in early embryo development.
more de genes were involved with functional processes in the postnatal stages, which were distributed between the proteasome complex, actin filament-based movement, intramolecular transferase activity, and lactate dehydrogenase activity.
meanwhile, the go terms of embryonic organ morphogenesis, neurogenesis, and development were also associated with many de genes.
although the go annotation and biological process categories were similar in many aspects involved in embryo and myogenesis development between tc and yk pigs, the amount of de genes involved in certain biological processes were quite different between the two breeds.figure  <dig> significant go terms of de genes  of each comparison library in tc and yk.
pathway enrichment analysis between tc and yk
to identify the biological pathways that were involved in muscle development, we mapped the de genes  to the reference canonical pathways in the kegg database .
in addition, we also separately gained pathways from  <dig> different comparison libraries in tc and yk pigs .
in the top  <dig> significantly enriched pathways, cardiac muscle contraction, parkinson’s disease, and citrate cycle  are shared between tc and yk pigs .
focal adhesion, protein digestion and absorption, gabaergic synapse, axon guidance, ecm-receptor interaction, mapk signaling pathway, arginine, and proline metabolism were found to be more associated with the de genes in tc pigs, while oxidative phosphorylation, huntington’s disease, ribosome biogenesis in eukaryotes, metabolic pathways, alzheimer’s disease, glycolysis/gluconeogenesis, and proteasome are closely related to yk pigs.
furthermore, the heat map results show that different pathways have distinct functional category enrichments between the  <dig> comparison libraries of the two breeds .
for instance, axon guidance shows significant enrichment in the early periods of muscle development in tc pigs, but not in yk pigs.
the citrate cycle  performs important roles close to birth in yk pigs, but not in tc pigs.
these differences provide some cues to survey the spatial and temporal expression of genes.table  <dig>
top  <dig> functional enrichment pathways in tongcheng pigs
path-id
path name
degs
fdr
enrichment
path-id
path name
degs
fdr
enrichment
discussion
previous studies have investigated the differences in gene expression patterns or molecular genetic mechanisms of skeletal muscle development between characteristically different pigs  <cit> .
our studies will be helpful to explore and elucidate the control mechanisms for muscle development and growth on a genome-wide scale.
moreover, tc pigs, a typical local breed in central china, have a developmental period with three time points for skeletal muscle  <cit> , which may limit studies on muscle development; comparative genomics could uncover the determining factors for the genetic basis of biological functions  <cit> .
the pig genomic sequence is very similar to the human genome, even though pigs and humans are divergent species  <cit> .
transcriptional profiling in our study may provide a reference for studies on human muscle tissue development and dysfunction.
muscle fiber development occurs in two waves, around  <dig> and  <dig> dpc, which involve the formation of primary and secondary fibers  <cit> .
in our study, primary and secondary fibers formed at approximately  <dig> and  <dig> dpc , and the muscle fiber diameter at birth was larger in the yk pig than the tc pig.
previous studies have also shown that the muscle fiber diameter of a chinese indigenous pig  is smaller than an exotic pig , and that myogenesis starts earlier but progresses more slowly in lt pigs than in lr pigs  <cit> .
likewise, duroc and pietrain pig breeds are also extremes, with myogenesis initiating earlier in duroc pigs than in pietrain pigs  <cit> .
the number of myoblasts in early tc embryos was more than that of early yk embryos, which may be the result of the proliferation and differentiation of myogenic precursor cells beginning earlier and developing more intensely in early tc embryos than in early yk embryos.
it is known that the number of muscle fibers determines the capacity for postnatal muscle fiber growth and hypertrophy.
importantly, postnatal muscle fiber hypertrophy is inversely correlated with the total number of fibers in the muscle  <cit> , which may be a reason for the slower growth rate and lower lean meat content for tc pigs than for conventional western pig breeds  <cit> .
in the histological cross-sections, muscle fiber development and differentiation were slower for tc pigs than for yk pigs.
the intramuscular fat texture showed differences in the later period of muscle development between yk and tc, which may be the consequence of different expression profiles of fatty acid metabolism in the two extreme muscularity breeds  <cit> .
gene expression profiles can be investigated easily and extensively using rna sequencing technology, with the results of these studies showing high levels of reproducibility in both technical and biological replicates  <cit> .
to investigate the transcription profiles of tc and yk pigs reliably and comprehensively, we used five muscle samples at each stage, and evaluated the sequencing quality of each library of  <dig> samples .
as large and comprehensive bio-information resources were obtained, transcripts at low abundance and novel transcripts could also be detected.
the distributions of total clean tags and distinct clean tags suggest that we obtained high-quality sequencing; and there were a high proportion of lower copy number tags , which showed many genes expressed at a low level.
we validated our results using real-time qpcr, which demonstrated that the quality of the solexa sequencing is reliable and trustworthy.
moreover, qpcr measures a single specific gene, while solexa sequencing involves massive parallel sequencing, and the abundance of the transcript can be affected by the test method and mrna samples storage.
more de genes were detected in the early period of muscle development , which reflects that the differences in the expression of muscle development genes are more intense in the period of primary fiber formation than in the period of secondary fiber formation  <cit> .
there were  <dig>  de genes in tc pigs and  <dig> de genes in yk pigs that were involved in myoblast differentiation at this stage, which indicates that tc pigs at the early stage have a more complex regulatory mechanism of initiation.
in addition, cluster analysis showed that there were differences in expression patterns between tc and yk pigs, and that the gene expression patterns at  <dig>   <dig>  and  <dig> wpn were more similar in yk than tc pigs .
<dig> dpc and  <dig> wpn  were more similar in tc pigs than yk pigs; however,  <dig> dpc was an exception.
the differences at the prenatal stage were greater than the postnatal differences in the two breeds overall.
these results show that muscle morphological cross-sections clearly varied from  <dig> to  <dig> dpc , and that the primary and secondary fibers were forming and changing during these stages, which is important for the growth and development of muscle fibers.
myosin heavy chain  composition determines the type of muscle fiber and its speed-related contractile properties, and myhc isoforms show different expression patterns in adult rodent muscle fibers  <cit> ; fast muscles have a higher content of myhc-iib  and myhc-iix  fibers, which have faster contractile speeds; while muscles with lower contractile speeds generally have a high content of myhc-i  and myhc-iia  fibers  <cit> .
myhc isoforms are the primary factor determining the speed-related contractile properties of muscle fibers.
our data analysis shows that myh <dig> and myh <dig> expression levels were higher after birth, and higher with aging in yk pigs than in tc pigs .
this indicates that, in the postnatal stage, the proportion of fast-glycolytic fiber types are varied and increased, and the muscle locomotor system has faster contractile speeds.
myh <dig> showed a peak at birth, and myh <dig> was expressed highly before and after birth with a higher level in tc pigs than yk pigs.
this result indicates that tc pigs have more myhc-i fiber, while yk pigs have more myhc-iib fiber.figure  <dig> gene expression patterns related to muscle fiber development.
the vertical axis indicates the normalized gene expression level in each stage on average.
the horizontal axis indicates the different developmental periods in tc and yk pigs; d indicates days prenatal and w indicates weeks of age.
we further analyzed the gene expression patterns related to muscle fiber development .
myog and myf <dig> are myogenic differentiation factors  <cit>  that participate in myoblast fusion and differentiation  <cit> .
in our study, the expression level of myog was high at  <dig> and  <dig> dpc in tc pigs and  <dig> and  <dig> dpc in yk pigs, but obviously decreased after  <dig> dpc.
myf <dig> was at a low level at  <dig> dpc, and then began to increase at  <dig> dpc in tc and yk pigs, which indicates that it may be involved in differentiation before and after birth.
the mef <dig> and mrf families coordinate the regulation of muscle-specific genes and myogenic differentiation.
mef2d deletion in mice has little effect on skeletal muscle development, but deletion of mef2c has been shown to cause defects in muscle formation  <cit> .
additionally, mef2c and mef2d also play a role in muscle regeneration of satellite cells  <cit> .
mef2c and mef2d have similar expression patterns in tc and yk breeds, but mef2c showed a higher expression level in our study.
myostatin  is an inhibitor of skeletal muscle growth  <cit> , and knockout mice show a dramatic increase in muscle mass  <cit> .
our data show high expression of mstn in two phases, 40– <dig> dpc and 70– <dig> dpc, to inhibit over-proliferation of myoblasts when the primary and secondary fibers are forming.
myoz <dig> encodes a structural protein that constitutes the z-lines in skeletal muscle.
the expression of myoz <dig> increases gradually with age in both breeds, but was higher in early tc embryos, and lower in later tc embryos and piglets, than in yk.
igf <dig> is a critical regulatory factor for growth and differentiation of mammalian muscle and fetal development  <cit> ; the sequencing results show high expression of igf <dig> across 40– <dig> dpc, and lower expression after birth in tc and yk pigs.
this suggests that igf <dig> plays an important role during the key stages of primary and secondary fiber growth and development in the prenatal stage.
pathway analysis showed that eno <dig> is involved in the glycolysis biological process, and participates in energy metabolism for muscle development  <cit> .
the eno <dig> expression level was higher in the postnatal stage than the prenatal stage in both breeds, but was higher in later yk embryos and piglets than in the tc breed.
this indicates that eno <dig> is important for muscle development in the later period, and mainly provides energy by glycolytic metabolism.
go terms were screened for significant enrichment according to functional, cellular, and biological annotation to investigate gene roles in the  <dig> stages of muscle development of tc and yk pigs.
many de genes were focused on the features associated with contractile fiber, myofibril, actin cytoskeleton, sarcomere, contractile fiber part, and myosin complex ; the results show muscle-specific differential expression across muscle growth for these genes.
interestingly, many de genes were also enriched for cellular components, which were distributed in the cytoplasm, cytoplasmic part, cellular, intracellular, and intracellular parts, which indicates that cellular signal transduction, intracellular communication, and transportation perform important roles in myogenesis.
these annotations give valuable insights into the regulatory pathways for studying specific processes and biological functions in skeletal muscle growth.
we also found that de genes had different levels of enrichment between the distinct comparison libraries ; many de genes were aligned with go terms related to muscle characteristics and diseases.
therefore, we selected de genes related to skeletal muscle development based on their go annotation;  <dig> genes were obtained,  <dig> of these in tc pigs and  <dig> in yk pigs .
this indicates a more complicated molecular regulatory mechanism for muscle development in tc pigs, and that more de genes were involved in muscle fiber growth , which is consistent with longsage sequencing results  <cit> .
we constructed gene regulatory interaction networks with the top  <dig> correlated genes  to explore the key candidate genes related to muscle development.
these potential gene interaction networks regulate the processes forming muscle fibers, and the interaction relationship is different between tc and yk pigs.
the genes cxcl <dig>  eif2b <dig>  psma <dig>  fbxo <dig>  and loc <dig> played vital roles in the muscle regulatory networks in the tc breed, while the genes sgcd, eng, thbd, cxcl <dig>  aqp <dig>  and btg <dig> performed key roles in the interactions in yk pigs.
we then carried out pathway analysis of the de genes and a heat map of the significant pathways  to shed light on functional regulation.
these analyses indicate that the pathway for parkinson’s disease is significant  in both breeds.
the pathways for huntington’s disease and alzheimer’s disease showed significant enrichment  in yk pigs but not in tc pigs; these may be critical target candidate genes for muscle diseases.figure  <dig> correlation networks from muscle development related to genes between two breeds.
a: tongcheng pigs, b: yorkshire pigs.
the solid and dotted lines indicate positive and negative correlation coefficients, respectively; and the line intensity denotes their strength.
each straight arrow, from tail to head, indicates the interaction direction.
the gene networks were obtained by the method “genenet” from the r package and display the top  <dig> largest absolute correlations.
CONCLUSIONS
we provide a new insight into transcriptional profiles of skeletal muscle development at  <dig> stages of tc and yk pigs using a genome-wide deep sequencing method.
the critical stage for the formation of primary and secondary fibers is from  <dig> to  <dig> dpc for both tongcheng and yorkshire pigs.
muscle development in early tc embryos is more intense than in yk embryos, and is more intense in yk piglets than in tc piglets from the natal day to 1-week postnatum; this suggests that muscle fiber formation in tc pigs is initiated earlier than in yk pigs , while the muscle maturation processes are more complicated in early yk piglets .
many de genes related to muscle development show phase-specific regulatory mechanism in myogenesis.
moreover, muscle contractile speeds and locomotion capacity are different in tc and yk pigs, which may be associated with more myhc-i fibers in tc pigs and more myhc-iib fibers in yk pigs.
the transition of energy metabolism between the oxidative phosphorylation and gluconeogenesis pathways affects the characteristics of the muscle fiber types.
furthermore, this study of skeletal muscle transcriptional profiles is helpful in understanding the differences between the genetic mechanisms in tc and yk pigs, and for future work exploring breed-specific changes in muscle development.

 the development of skeletal muscle affects meat production and growth rate in pigs. the genetic mechanisms controlling these traits are being uncovered. in addition, the selection of meat quality based on molecular regulation is important for livestock breeding, and there have been many studies on skeletal muscle in different pig breeds. the total number of fibers is constant in the postnatal stage, and postnatal muscle growth is through hypertrophy of myofibers, or conversion of myofiber types. primary and secondary fibers emerge at distinct embryonic stages during the skeletal muscle development of pigs. primary myofiber formation occurs in the first wave of fiber generation, from about 35 days of gestation until around 60 days. secondary myofibers appear at around 50– days based on the template of the primary myofiber surface. the third wave involves the process of transition between slow-oxidative and fast-glycolytic fiber types, which occurs from birth to 60 days of age. muscle maturation is completed in the early postnatal period. therefore, we chose the critical skeletal muscle developmental stages across the prenatal to postnatal periods for studying the regulatory mechanisms and expanding the molecular genetics of muscle fiber development. many genes or factors participate in the process of muscle fiber formation, and muscle development is under complex genetic regulation. it is known that the muscle regulatory factor and myocyte-specific enhancer binding factor gene families play critical roles in regulating muscle fiber development. mrf genes are muscle-specific transcription factors, and induce myoblast proliferation or fusion differentiation during distinct stages of myogenesis. mef regulates muscle-specific transcription during myogenesis and is activated during the development of the muscle. however, knowledge of the functional genetics of myogenesis is currently insufficient and incomprehensive. monitoring the transcriptional profiling from the prenatal to postnatal developmental stages in pigs will act as an animal model that will not only uncover the regulatory mechanisms behind factors affecting muscle development, but will also benefit the understanding of diseases affecting human muscle and muscular atrophy. with biotechnological advances in gene expression profiling, we have the ability to study large-scale differential regulation of genes genome wide, and to measure the transcriptional responses to certain complex biological conditions. previous studies have presented expression analysis of myogenesis using microarrays, long sage, and differential display rt -polymerase chain reaction. digital gene expression technology is sensitive for the detection of low-abundant transcripts and small changes in gene expression, and has been applied extensively to compare the differences in transcription profiles of different tissues or breeds. we present a panorama of transcriptome-wide longissimus dorsi muscle development using digital gene expression with solexa/illumina’s genome analyzer platform to study the variation between the dynamic transcriptome profiles of tongcheng and yorkshire pig breeds. previous studies have demonstrated differences in gene expression in pig longissimus dorsi muscle during fetal development, and some have shown breed-specific expression profiles. indeed, differences exist in muscle traits such as muscularity and muscle fiber type; muscle characteristics are different between pig breeds, and molecular biomarkers have been reported. however, the molecular genetic mechanisms of these breed-specific differences remain unclear, in terms of meat quality and visual phenotype. in this study, we selected the tc pig, a characteristic indigenous pig breed from the hubei province of central china with slow growth and prolific reproduction, and the yk pig, which is characterized by fast growth, low back fat, and a high lean meat percentage, to survey the differences in transcription. previous studies have shown that indigenous swine breeds have slower growth rates and less lean meat percentages than exotic pigs. our study opens up the scope of knowledge of muscle-specific regulation and the microscopic molecular mechanisms. our objective was to analyze the biological information regarding the transcriptional profiles of longissimus muscle between the two breeds and to further identify key gene regulatory networks and breed-specific pathways affecting skeletal muscle development in the pig.  histological section and fiber size we evaluated the morphological differences of skeletal muscle development between tc and yk pigs, from the histological appearance through the prenatal and postnatal stages. histological cross-section showed that myoblasts developed quickly from dpc, but there was no differentiation into primary fibers in either pig breed at dpc, and the number and density of myoblasts in tc was more than that in yk. the myoblasts differentiated gradually, with primary fibers emerging from to dpc, and could be seen in the histological section to be fully formed at dpc. primary fibers continued to expand and enlarge until dpc. meanwhile, the primary fiber bundles were separated by the connective tissues. secondary fibers appeared at dpc in yk pigs and formed until dpc, but they emerged later in tc pigs. secondary fibers could be seen clearly around the primary fibers at dpc, and the secondary fibers increased gradually until dpc. the fiber bundles fused at around dpc and appeared to form into the myotubes at dpc; the primary and secondary fibers were not distinguishable at this period. the intramuscular fat texture showed differences in the histological section in the latter period between tc and yk pigs. these differences are associated with the different development of fat deposits in these two breeds. the muscle fibers grew rapidly, and finally the muscular tube formed and constituted the muscle mass. the phenotypes and sizes of the muscle fibers still varied based on the cross-sectional areas during the postnatal development stages for both tc and yk pigs). solexa sequencing and tag mapping after filtering adaptor tags, empty tags, low-quality tags, and one-copy tags from the raw data,, total clean tags were obtained in the tc breed, including, distinctly clean tags., total clean tags and, distinctly clean tags were obtained in the yk pig. the total clean tags accounted for % and % of the raw tags in tc and yk, respectively. the number of unknown distinct tags was and, on average, in each sample from tc and yk pigs. moreover, the heterogeneity and redundancy of the mrna were confirmed, and the results showed that the high copy number clean tags accounted for % of the total clean tags in tc and % in yk, while the low copy number clean tags accounted for less than 1% of the total clean tags in both breeds. additional file 3: figure s illustrates the distribution of total clean tags and distinct clean tags of tc and yk. the type of distinct tags were gradually stable, with the total number of sequence tags rising according to saturation analysis, and the percentage of genes identified was no longer increasing when the library size approached million. the number of tags mapping to genes was and in tc and yk, and the number of genes with unambiguous tag mapping was and. the number of unambiguous tags mapping to genes accounted for % of all tags mapping to genes, and % of the total clean tags for the tc breed, in contrast to % and % for the yk breed. differential gene expression analysis and validation of solexa sequencing data in this study, differentially expressed genes at the time points of skeletal muscle development of tc and yk pigs were investigated through transcriptome-wide solexa sequencing, and analyzed using the noiseq method. in the tc libraries, a total of genes were found to be de; of these, genes were upregulated and genes were downregulated in comparison libraries. in the yk libraries, a total of de genes were identified, with a total of upregulated and downregulated genes in comparison libraries during different muscle development stages. these results suggest that the number of de genes changed significantly at 30– dpc, 55– dpc, and 90– dpc in the prenatal muscle development stages, and at 3– wpn in the postnatal stages.figure number of de genes between the comparison libraries. tc: tongcheng, yk: yorkshire. before birth: and 105 days post-coitus. after birth: and 5 weeks post-natum. a: tongcheng pigs, b: yorkshire pigs, c: the identical period comparison between tc and yk pigs. five genes were chosen randomly for the purpose of validating the results of the solexa sequencing using real-time quantitative pcr. tnnc is a key gene encoding a protein that regulates striated muscle contraction. gdf regulates cell growth and differentiation in both embryonic and adult tissues, and is related to skeletal muscle age. myot encodes a cytoskeletal protein that is involved in myofibril assembly and actin binding in the muscle tissue. pgam is a muscle-specific phosphoglycerate mutase and is involved in the glycolysis pathway for myoblast fusion. mylpf is related to a structural constituent of muscle and calcium ion binding. in our study, rad homolog b was used as the reference gene because it showed consistent expression. the data and spearman’s correlation coefficient analysis suggest that the solexa sequencing had a highly significant correlation with real-time qpcr, and the expression patterns of these genes were consistent between the two methods.figure validation of solexa sequencing data with qpcr. the vertical axis indicates the fold change of transcript abundance. the horizontal axis indicates the samples for tc and yk pigs. the r-value shows the spearman’s correlation between the two methods. cluster analysis of de genes gene expression patterns at the scale of the transcriptome were measured by systematic cluster analysis, to explore the similarities and compare the relationship between the different libraries in tc and yk pigs. the results of hierarchical clustering indicate that the gene expression similarity could be classified into distinct groups. tc tc and tc were clustered together, and tc and tc were of the same class; while yk and yk had similar expression patterns, and yk and yk were clustered into another class in the prenatal stage owing to the similarity of gene expression. the gene expression patterns of and 5 weeks of age were grouped together in both breeds.figure hierarchical clustering analysis for all the de genes in tc and yk pig breeds, respectively. a: tc, b: yk. go analysis of de genes the screened de genes were classified by gene ontology based on their biological process and function in tc and yk pigs. the go annotation results are presented in additional file 6: table s and additional file 7: table s significant go categories were selected, and showed different extents of enrichment. go analysis showed that biological processes related to muscle development were mainly enriched in different stages for both breeds; these genes were associated with go terms including myofibril, sarcomere, and myosin complex, which are involved in muscle component and structure. muscle tissue morphogenesis and development, cell differentiation and development, regulation of muscle contraction and developmental process were enriched significantly in early embryo development. more de genes were involved with functional processes in the postnatal stages, which were distributed between the proteasome complex, actin filament-based movement, intramolecular transferase activity, and lactate dehydrogenase activity. meanwhile, the go terms of embryonic organ morphogenesis, neurogenesis, and development were also associated with many de genes. although the go annotation and biological process categories were similar in many aspects involved in embryo and myogenesis development between tc and yk pigs, the amount of de genes involved in certain biological processes were quite different between the two breeds.figure significant go terms of de genes of each comparison library in tc and yk. pathway enrichment analysis between tc and yk to identify the biological pathways that were involved in muscle development, we mapped the de genes to the reference canonical pathways in the kegg database. in addition, we also separately gained pathways from different comparison libraries in tc and yk pigs. in the top significantly enriched pathways, cardiac muscle contraction, parkinson’s disease, and citrate cycle are shared between tc and yk pigs. focal adhesion, protein digestion and absorption, gabaergic synapse, axon guidance, ecm-receptor interaction, mapk signaling pathway, arginine, and proline metabolism were found to be more associated with the de genes in tc pigs, while oxidative phosphorylation, huntington’s disease, ribosome biogenesis in eukaryotes, metabolic pathways, alzheimer’s disease, glycolysis/gluconeogenesis, and proteasome are closely related to yk pigs. furthermore, the heat map results show that different pathways have distinct functional category enrichments between the comparison libraries of the two breeds. for instance, axon guidance shows significant enrichment in the early periods of muscle development in tc pigs, but not in yk pigs. the citrate cycle performs important roles close to birth in yk pigs, but not in tc pigs. these differences provide some cues to survey the spatial and temporal expression of genes.table top functional enrichment pathways in tongcheng pigs path-id path name degs fdr enrichment path-id path name degs fdr enrichment discussion previous studies have investigated the differences in gene expression patterns or molecular genetic mechanisms of skeletal muscle development between characteristically different pigs. our studies will be helpful to explore and elucidate the control mechanisms for muscle development and growth on a genome-wide scale. moreover, tc pigs, a typical local breed in central china, have a developmental period with three time points for skeletal muscle, which may limit studies on muscle development; comparative genomics could uncover the determining factors for the genetic basis of biological functions. the pig genomic sequence is very similar to the human genome, even though pigs and humans are divergent species. transcriptional profiling in our study may provide a reference for studies on human muscle tissue development and dysfunction. muscle fiber development occurs in two waves, around and dpc, which involve the formation of primary and secondary fibers. in our study, primary and secondary fibers formed at approximately and dpc, and the muscle fiber diameter at birth was larger in the yk pig than the tc pig. previous studies have also shown that the muscle fiber diameter of a chinese indigenous pig is smaller than an exotic pig, and that myogenesis starts earlier but progresses more slowly in lt pigs than in lr pigs. likewise, duroc and pietrain pig breeds are also extremes, with myogenesis initiating earlier in duroc pigs than in pietrain pigs. the number of myoblasts in early tc embryos was more than that of early yk embryos, which may be the result of the proliferation and differentiation of myogenic precursor cells beginning earlier and developing more intensely in early tc embryos than in early yk embryos. it is known that the number of muscle fibers determines the capacity for postnatal muscle fiber growth and hypertrophy. importantly, postnatal muscle fiber hypertrophy is inversely correlated with the total number of fibers in the muscle, which may be a reason for the slower growth rate and lower lean meat content for tc pigs than for conventional western pig breeds. in the histological cross-sections, muscle fiber development and differentiation were slower for tc pigs than for yk pigs. the intramuscular fat texture showed differences in the later period of muscle development between yk and tc, which may be the consequence of different expression profiles of fatty acid metabolism in the two extreme muscularity breeds. gene expression profiles can be investigated easily and extensively using rna sequencing technology, with the results of these studies showing high levels of reproducibility in both technical and biological replicates. to investigate the transcription profiles of tc and yk pigs reliably and comprehensively, we used five muscle samples at each stage, and evaluated the sequencing quality of each library of samples. as large and comprehensive bio-information resources were obtained, transcripts at low abundance and novel transcripts could also be detected. the distributions of total clean tags and distinct clean tags suggest that we obtained high-quality sequencing; and there were a high proportion of lower copy number tags, which showed many genes expressed at a low level. we validated our results using real-time qpcr, which demonstrated that the quality of the solexa sequencing is reliable and trustworthy. moreover, qpcr measures a single specific gene, while solexa sequencing involves massive parallel sequencing, and the abundance of the transcript can be affected by the test method and mrna samples storage. more de genes were detected in the early period of muscle development, which reflects that the differences in the expression of muscle development genes are more intense in the period of primary fiber formation than in the period of secondary fiber formation. there were de genes in tc pigs and de genes in yk pigs that were involved in myoblast differentiation at this stage, which indicates that tc pigs at the early stage have a more complex regulatory mechanism of initiation. in addition, cluster analysis showed that there were differences in expression patterns between tc and yk pigs, and that the gene expression patterns at and wpn were more similar in yk than tc pigs. dpc and wpn were more similar in tc pigs than yk pigs; however, dpc was an exception. the differences at the prenatal stage were greater than the postnatal differences in the two breeds overall. these results show that muscle morphological cross-sections clearly varied from to dpc, and that the primary and secondary fibers were forming and changing during these stages, which is important for the growth and development of muscle fibers. myosin heavy chain composition determines the type of muscle fiber and its speed-related contractile properties, and myhc isoforms show different expression patterns in adult rodent muscle fibers; fast muscles have a higher content of myhc-iib and myhc-iix fibers, which have faster contractile speeds; while muscles with lower contractile speeds generally have a high content of myhc-i and myhc-iia fibers. myhc isoforms are the primary factor determining the speed-related contractile properties of muscle fibers. our data analysis shows that myh and myh expression levels were higher after birth, and higher with aging in yk pigs than in tc pigs. this indicates that, in the postnatal stage, the proportion of fast-glycolytic fiber types are varied and increased, and the muscle locomotor system has faster contractile speeds. myh showed a peak at birth, and myh was expressed highly before and after birth with a higher level in tc pigs than yk pigs. this result indicates that tc pigs have more myhc-i fiber, while yk pigs have more myhc-iib fiber.figure gene expression patterns related to muscle fiber development. the vertical axis indicates the normalized gene expression level in each stage on average. the horizontal axis indicates the different developmental periods in tc and yk pigs; d indicates days prenatal and w indicates weeks of age. we further analyzed the gene expression patterns related to muscle fiber development. myog and myf are myogenic differentiation factors that participate in myoblast fusion and differentiation. in our study, the expression level of myog was high at and dpc in tc pigs and and dpc in yk pigs, but obviously decreased after dpc. myf was at a low level at dpc, and then began to increase at dpc in tc and yk pigs, which indicates that it may be involved in differentiation before and after birth. the mef and mrf families coordinate the regulation of muscle-specific genes and myogenic differentiation. mef2d deletion in mice has little effect on skeletal muscle development, but deletion of mef2c has been shown to cause defects in muscle formation. additionally, mef2c and mef2d also play a role in muscle regeneration of satellite cells. mef2c and mef2d have similar expression patterns in tc and yk breeds, but mef2c showed a higher expression level in our study. myostatin is an inhibitor of skeletal muscle growth, and knockout mice show a dramatic increase in muscle mass. our data show high expression of mstn in two phases, 40– dpc and 70– dpc, to inhibit over-proliferation of myoblasts when the primary and secondary fibers are forming. myoz encodes a structural protein that constitutes the z-lines in skeletal muscle. the expression of myoz increases gradually with age in both breeds, but was higher in early tc embryos, and lower in later tc embryos and piglets, than in yk. igf is a critical regulatory factor for growth and differentiation of mammalian muscle and fetal development; the sequencing results show high expression of igf across 40– dpc, and lower expression after birth in tc and yk pigs. this suggests that igf plays an important role during the key stages of primary and secondary fiber growth and development in the prenatal stage. pathway analysis showed that eno is involved in the glycolysis biological process, and participates in energy metabolism for muscle development. the eno expression level was higher in the postnatal stage than the prenatal stage in both breeds, but was higher in later yk embryos and piglets than in the tc breed. this indicates that eno is important for muscle development in the later period, and mainly provides energy by glycolytic metabolism. go terms were screened for significant enrichment according to functional, cellular, and biological annotation to investigate gene roles in the stages of muscle development of tc and yk pigs. many de genes were focused on the features associated with contractile fiber, myofibril, actin cytoskeleton, sarcomere, contractile fiber part, and myosin complex; the results show muscle-specific differential expression across muscle growth for these genes. interestingly, many de genes were also enriched for cellular components, which were distributed in the cytoplasm, cytoplasmic part, cellular, intracellular, and intracellular parts, which indicates that cellular signal transduction, intracellular communication, and transportation perform important roles in myogenesis. these annotations give valuable insights into the regulatory pathways for studying specific processes and biological functions in skeletal muscle growth. we also found that de genes had different levels of enrichment between the distinct comparison libraries; many de genes were aligned with go terms related to muscle characteristics and diseases. therefore, we selected de genes related to skeletal muscle development based on their go annotation; genes were obtained, of these in tc pigs and in yk pigs. this indicates a more complicated molecular regulatory mechanism for muscle development in tc pigs, and that more de genes were involved in muscle fiber growth, which is consistent with longsage sequencing results. we constructed gene regulatory interaction networks with the top correlated genes to explore the key candidate genes related to muscle development. these potential gene interaction networks regulate the processes forming muscle fibers, and the interaction relationship is different between tc and yk pigs. the genes cxcl eif2b psma fbxo and loc played vital roles in the muscle regulatory networks in the tc breed, while the genes sgcd, eng, thbd, cxcl aqp and btg performed key roles in the interactions in yk pigs. we then carried out pathway analysis of the de genes and a heat map of the significant pathways to shed light on functional regulation. these analyses indicate that the pathway for parkinson’s disease is significant in both breeds. the pathways for huntington’s disease and alzheimer’s disease showed significant enrichment in yk pigs but not in tc pigs; these may be critical target candidate genes for muscle diseases.figure correlation networks from muscle development related to genes between two breeds. a: tongcheng pigs, b: yorkshire pigs. the solid and dotted lines indicate positive and negative correlation coefficients, respectively; and the line intensity denotes their strength. each straight arrow, from tail to head, indicates the interaction direction. the gene networks were obtained by the method “genenet” from the r package and display the top largest absolute correlations.  we provide a new insight into transcriptional profiles of skeletal muscle development at stages of tc and yk pigs using a genome-wide deep sequencing method. the critical stage for the formation of primary and secondary fibers is from to dpc for both tongcheng and yorkshire pigs. muscle development in early tc embryos is more intense than in yk embryos, and is more intense in yk piglets than in tc piglets from the natal day to 1-week postnatum; this suggests that muscle fiber formation in tc pigs is initiated earlier than in yk pigs, while the muscle maturation processes are more complicated in early yk piglets. many de genes related to muscle development show phase-specific regulatory mechanism in myogenesis. moreover, muscle contractile speeds and locomotion capacity are different in tc and yk pigs, which may be associated with more myhc-i fibers in tc pigs and more myhc-iib fibers in yk pigs. the transition of energy metabolism between the oxidative phosphorylation and gluconeogenesis pathways affects the characteristics of the muscle fiber types. furthermore, this study of skeletal muscle transcriptional profiles is helpful in understanding the differences between the genetic mechanisms in tc and yk pigs, and for future work exploring breed-specific changes in muscle development. 
the transcripts were annotated in terms of gene ontology related to muscle development. we found that the genes cxcl eif2b psma fbxo and loc played vital roles in the muscle regulatory networks in the tc breed, whereas the genes sgcd, eng, thbd, aqp and btg played dominant roles in the yk breed. finally, the differentially expressed genes from our sequencing results were confirmed by real-time quantitative polymerase chain reaction. muscle morphological changes indicate the importance of primary fiber formation from to dpc, and secondary fiber formation from to dpc. cluster analysis showed different gene expression patterns between tc and yk pigs. this study detected many functional genes and showed differences in the molecular mechanisms of skeletal muscle development between tc and yk pigs. tc pigs showed slower muscle growth and more complicated genetic regulation than yk pigs. we present transcriptome profiles of skeletal muscle samples from tongcheng and yorkshire pigs at developmental periods using digital gene expression on solexa/illumina’s genome analyzer platform to investigate the differences in prenatal and postnatal skeletal muscle between the two breeds. our data provide a better understanding of skeletal muscle developmental differences and valuable information for improving pork quality. the growth and development of skeletal muscle directly impacts the quantity and quality of pork production. tongcheng and yorkshire pigsskeletal musclebioinformatic transcriptomehistology and histochemistryqpcrissue-copyright-statement© the author 2015 a heat map showed that significantly enriched pathways had stage-specific functional regulatory mechanisms.
 the growth and development of skeletal muscle directly impacts the quantity and quality of pork production. chinese indigenous pig breeds and exotic species vary greatly in terms of muscle production and performance traits. we present transcriptome profiles of skeletal muscle samples from tongcheng and yorkshire pigs at developmental periods using digital gene expression on solexa/illumina’s genome analyzer platform to investigate the differences in prenatal and postnatal skeletal muscle between the two breeds.  muscle morphological changes indicate the importance of primary fiber formation from to dpc, and secondary fiber formation from to dpc. we screened differentially expressed genes in tc and in yk. cluster analysis showed different gene expression patterns between tc and yk pigs. the transcripts were annotated in terms of gene ontology related to muscle development. we found that the genes cxcl eif2b psma fbxo and loc played vital roles in the muscle regulatory networks in the tc breed, whereas the genes sgcd, eng, thbd, aqp and btg played dominant roles in the yk breed. these genes showed breed-specific and development-dependent differential expression patterns. furthermore, genes were identified in myogenesis. a heat map showed that significantly enriched pathways had stage-specific functional regulatory mechanisms. finally, the differentially expressed genes from our sequencing results were confirmed by real-time quantitative polymerase chain reaction.  this study detected many functional genes and showed differences in the molecular mechanisms of skeletal muscle development between tc and yk pigs. tc pigs showed slower muscle growth and more complicated genetic regulation than yk pigs. many differentially expressed genes showed breed-specific expression patterns. our data provide a better understanding of skeletal muscle developmental differences and valuable information for improving pork quality. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tongcheng and yorkshire pigsskeletal musclebioinformatic transcriptomehistology and histochemistryqpcrissue-copyright-statement© the author 2015 