BACKGROUND
genome-wide transcriptional studies have revealed that large regions of eukaryotic genomes are transcribed into a heterogeneous population of non-coding rnas .
generally, ncrnas shorter than  <dig> nucleotides are usually described as small/short ncrna, such as micrornas , piwi-interacting rnas , small interfering rnas , and classical ncrnas such as ribosomal rnas , transfer rnas , and small nucleolar rnas , whereas ncrnas longer than  <dig> nucleotides are described as long ncrnas .
in the past few years, an increasing number of lncrnas have been discovered in mammal, including homo sapiens  <cit> , mus musculus , bos taurus  <cit> , sus scrofa , and ovis aries  <cit> .
and accordingly unveiled that lncrnas play critical roles in biological processes like transcriptional regulation , epigenetic modification , development , cell differentiation , as well as in some diseases .
as important economic animals worldwide, domestic goats  are raised mainly for meat production.
thus, unveiling the molecular mechanisms underneath skeletal muscle formation and development is of vital interest.
muscle development is a complex process that requires the concerted expression and interaction of multiple factors  <cit> .
several recent studies have indicated that lncrnas play crucial roles in myogenic differentiation and myogenesis .
nevertheless, currently the majority strategy for exploring molecular mechanisms underlying skeletal muscle growth and development in mammals  is detecting the expression and functions of coding genes like the mrf   <cit> , mef <dig>   <cit>  families, and the paired box proteins  <cit> , though high-throughput sequencing technologies is also employed to profile expression of mirna and mrna expression in goat  <cit> .
therefore, information about skeletal muscle development-related lncrnas is still limited especially in goats.
here, we report the systematic identification and characterization of lncrnas in fetal and postnatal goat skeletal muscle using an illumina hiseq  <dig> platform.
a total of  <dig> lncrna transcripts were identified and  <dig> of these transcripts were significantly differentially expressed in pairwise comparisons between rna libraries of skeletal muscle at the different development stages.
to the best of our knowledge, no other report on muscle lncrnas and their biological functions in goat is currently available.
our results will provide a useful resource for better understanding the regulatory functions of lncrnas in goat and for annotating the goat genome, as well as contribute to better comprehending skeletal muscle development in mammals.
RESULTS
overview of rna sequencing
to identify lncrnas expressed in goat skeletal muscle development, we constructed  <dig> cdna libraries  from goat longissimus dorsi muscle samples at four developmental stages: three gestation stages at  <dig>   <dig>  and 105 days of gestation , and one postnatal stage .
three biological replicates for e <dig>  e <dig>  and b <dig>  and two biological replicates for e <dig> were used.
the libraries were sequenced using an illumina hiseq  <dig> platform and 125 bp paired-end reads were generated.
a total of  <dig> , <dig>  raw reads were generated in all  <dig> libraries.
after discarding adaptor sequences and low-quality reads, we obtained  <dig> , <dig>  clean reads.
the percentage of clean reads in each library ranged from  <dig>  to  <dig>  % .
we mapped the clean reads to the goat reference genome sequence .
approximately  <dig> – <dig>  % of the clean reads in all the libraries were mapped to the goat reference genome .
the mapped sequences in each library were assembled and a total of  <dig>  unique assembled transcripts were obtained.
identification of lncrnas in goat skeletal muscle
we developed a highly stringent filtering pipeline to discard transcripts that did not have all the characteristics of lncrnas .
our pipeline yielded  <dig> lncrna transcripts, including  <dig> intergenic lncrnas  and  <dig> anti-sense lncrnas  , these transcripts corresponded to  <dig> lncrna genes, an average of  <dig>  transcripts per lncrna locus.
we found that the lncrna transcripts were distributed in all chromosomes except the y chromosome .
the illumina rna-seq also produced  <dig>  protein-coding transcripts with an average length of 1978 bp and  <dig>  exons, which was longer than the lncrna genes, which averaged 1296 bp in length and  <dig>  exons.
however, the exon size in the protein-coding genes was smaller than the exon size in the lncrna genes  .
we also found that protein transcripts with two and three exons accounted for  <dig>  % of all the protein-coding genes, which was much lower than the percentage of lncrna genes with two and three exons .fig.
<dig> identification pipeline for lncrnas.
each step is described in detail in the methods section
fig.
<dig> comparison of the features of goat lncrnas and protein-coding genes.
a exon size distribution of goat lncrnas and protein-coding genes.
b exon numbers per transcript of goat lncrnas and protein-coding genes
identification of differentially expressed lncrnas
the expression levels of the lncrna transcripts were estimated by fpkm  using cuffdiff.
we identified  <dig> lncrna transcripts that were differentially expressed during muscle development ; the number of down-regulated lncrnas was higher than the number of up-regulated lncrnas during development.
the expression patterns of differentially expressed lncrnas were measured by systematic cluster analysis, to explore the similarities and to compare the relationships between the different libraries .
the replicates for each developmental stage clustered together, and e <dig> and e <dig> formed one group and e <dig> and b <dig> formed another group.
to further analyze the interactions among the differentially expressed lncrnas, we constructed a venn diagram using the  <dig>   <dig>   <dig>  and  <dig> lncrnas that were differentially expressed in e <dig>  e <dig>  e <dig>  and b <dig>  respectively.
we did not detect any stage-specific differentially expressed lncrnas among the four developmental stages, but we identified  <dig> differentially expressed lncrnas that were detected in all four developmental stages .fig.
<dig> numbers of up-regulated and down-regulated lncrnas in goat skeletal muscle at four developmental stages
fig.
<dig> analyses of differentially expressed lncrnas in the rna-seq libraries.
a hierarchical clustering analysis of lncrna expression profiles from  <dig> libraries with  <dig> differentially expressed lncrnas.
data are expressed as fpkm.
red: relatively high expression; green: relatively low expression.
b venn diagram showing the differentially expressed lncrnas at the four developmental stages
enrichment analysis of nearest neighbor genes of the lncrnas
to investigate the possible functions of the lncrnas, we predicted the potential targets of lncrnas in cis-regulatory relationships.
we searched for protein-coding genes 10-kb upstream and downstream of all the identified lncrnas.
we found  <dig> lncrnas that were transcribed close to   <dig> protein-coding neighbors .
gene ontology   <cit>  analysis of the cis lncrna targets was performed to explore their functions.
we found  <dig> go terms that were significantly enriched , and  <dig> of these terms were associated with regulation of gene expression.
for example, the top  <dig> enriched terms included nucleotide binding, regulation of rna metabolic process, dna-dependent regulation of transcription, transcription regulator activity, and transcription factor activity .
these results suggest that one of the principal roles of lncrnas may be transcriptional regulation of gene expression.
interestingly, we also found genes, including rbp <dig>  pln, mylk <dig>  rara, cacnb <dig>  nr2f <dig>  cdk <dig> and pitx <dig>  that were annotated with muscle development-related go term, striated muscle tissue development .
these results suggest that muscle development may be regulated by the action of lncrnas on these neighboring protein-coding genes.
pathway analysis  <cit>  showed that the  <dig> candidate cis target genes were enriched in  <dig> kyoto encyclopedia of genes and genomes  pathways, several of which were related to muscle development such as insulin signaling pathway, mapk signaling pathway, tgf-beta signaling pathway, and ppar signaling pathway .
enrichment analysis of co-expressed genes of lncrnas
we also predicted the potential targets of lncrnas in trans-regulatory relationships using co-expression analysis.
a total of  <dig>  interaction relationships  were detected between  <dig> lncrna transcripts and  <dig>  protein-coding transcripts that corresponded to  <dig> protein-coding genes in the goat reference genome .
functional analysis showed that the co-expressed genes were enriched in  <dig> go terms  that encompassed a variety of biological processes .
importantly, some of the terms were muscle development-related terms, including muscle cell development , striated muscle cell development , muscle contraction , and muscle system process .
in addition, the co-expressed genes were enriched in  <dig> kegg pathways, several of which were related to muscle development, including tgf-beta signaling, mapk signaling, and ppar signaling pathways .
these findings indicate that lncrnas also regulate trans target genes.
validation of differentially expressed lncrnas
we randomly selected six differentially expressed lncrnas and examined their expression patterns at four developmental stages by qpcr.
the results confirmed that the six lncrnas were expressed at all four development stages  and showed differential expression at different stages.
in addition, the qpcr confirmed that the expression patterns of the six lncrnas were consistent with their expression levels calculated from the rna-seq data.
all our results show that our pipeline was highly strict in identifying putative lncrnas, and indicate that most of the identified lncrnas were truly expressed in vivo.fig.
<dig> validation of six differentially expressed lncrnas by qpcr.
data are the mean ± sem
discussion
the identification and characterization of goat lncrnas, particularly in fetal skeletal muscle development, have been very limited compared with lncrnas in human  <cit>  and other model organisms  <cit> .
in goat skeletal muscle, the main focus has been on genes and mirnas rather than on lncrnas  <cit> .
in the present study, we identified a total of  <dig> multiple exon lncrnas in fetal and postnatal goat skeletal muscle.
to the best of our knowledge, this is the first report to systematically identify lncrnas from rna-seq data during goat skeletal muscle development.
non-coding and protein-coding genes are distinguished by their potential coding capability.
in this study, we developed a highly stringent filtering pipeline  to minimize the selection of false positive lncrnas, which aimed to remove transcripts with evidence of protein-coding potential.
we identified  <dig> putative lncrnas with high confidence across four development stages of goat skeletal muscle.
in agreement with similar studies on different organisms, the identified putative lncrnas had fewer exon numbers, shorter transcript lengths, and lower expression levels than protein-coding genes  <cit> .
the number of putative lncrnas detected in this study was more than that reported in previous studies in cattle and goat  <cit> .
six randomly selected differentially expressed lncrna transcripts were validated using qpcr, and the results were consistent with the results from the rna-seq data.
together, these results confirmed that the identified lncrnas were of high quality.
lncrnas are a group of endogenous rnas that function as regulators of gene expression, and are involved in developmental and physiological processes  <cit> .
we detected  <dig> putative lncrnas that were differentially expressed in pairwise comparisons between goat skeletal muscle at the different development stages.
these lncrnas may have specific biological roles in early muscle development in fetal goat.
skeletal muscle development from the fetal to the adult stage consists of a series of exquisitely regulated and orchestrated changes in the expression of many genes  <cit> .
in recent years, the roles of some lncrnas in muscle biology have been reported.
for example, the long intergenic ncrna muscle differentiation linc-md <dig> was the first muscle-specific lncrna to be identified  <cit> .
linc-md <dig> is required for appropriate muscle differentiation, at least in part because it regulates the levels of myocyte enhancer factor 2c  and mastermind-like protein  <dig>  by sponging endogenous mir- <dig> and mir- <dig> in the cytoplasm of muscle cells  <cit> .
in addition, the substantial disintegration of linc-md <dig> in primary myoblasts of patients with duchenne muscular dystrophy suggests that it is likely involved in the pathogenesis of this muscle disorder  <cit> .
another study revealed that the lncrna, lncmyod, regulates skeletal muscle differentiation by blocking imp2-mediated mrna translation  <cit> .
therefore, the differentially expressed lncrnas reported here can be considered as important novel regulators of goat skeletal muscle biology.
unlike mirnas or proteins, the functions of ncrnas cannot currently be inferred from their sequence or structure; therefore, in this study, we predicted the potential function of the detected lncrnas using cis and trans methods.
the cis nature of a lncrna refers to its ability to act on a neighboring gene on the same allele from which it is transcribed; thus, this type of lncrna commonly forms a feedback loop that regulates itself and its neighboring genes.
in the cis prediction, we searched for coding genes 10-kb upstream and downstream of all the identified lncrnas.
go and kegg analyses of the neighboring protein-coding genes revealed that major enriched pathways were associated with transition metal ion binding, nucleotide binding, zinc ion binding, regulation of rna metabolic process, regulation of transcription, and transcription regulator activity.
these results indicate the possible role of lncrnas in transcriptional regulation of gene expression.
interestingly, we found some of the cis target protein-coding genes were involved in skeletal muscle tissue development  , implying that the corresponding lncrnas play regulatory roles in skeletal muscle development.
several recent studies also indicated that lncrnas were involved in cis-regulatory activity in muscle development; for example, the lncrna dum  upstream binding muscle lncrna) was identified in skeletal myoblast cells  <cit> .
dum promotes myoblast differentiation and damage-induced muscle regeneration by silencing its neighboring gene, dppa <dig>  in cis through recruiting dnmt <dig>  dnmt3a, and dnmt3b  <cit> .
similarly, a chip-seq study of the yin yang  <dig>  transcription factor, an important component of the pcg complex that negatively regulates myogenesis, identified a number of lncrnas regulated by yy <dig>   <cit> .
among the yams, yam- <dig> displayed a cis effect on the expression of neighboring genes, including one that encodes mir- <dig>  which targets and represses wnt7b in skeletal muscle.
many lncrnas can also function in trans mode to target gene loci distant from where the lncrnas are transcribed  <cit> .
in the co-expression analysis, we detected  <dig> lncrna transcripts that were related to protein-coding genes based on the expression correlation coefficient .
go enrichment analysis found that the enriched go terms referred mainly to development process, transcriptional regulation, and biosynthetic process.
furthermore, a cluster of lncrnas in the co-expression analysis often targeted protein-coding genes that were expressed specifically in muscle and were involved in muscle development .
this is an interesting observation, which indicates the functional complexity of lncrnas and is worth investigating further.
mousavi et al.
<cit>  found two lncrnas in two enhancer regions of the myod gene that they named ddrrna and cerna, where drr indicates distal regulatory regions and ce indicates core enhancer.
the study showed that cerna facilitated the occupancy of rna polymerase ii in cis by increasing chromatin accessibility, stimulating the expression of myod, while ddrrna functions in trans to promote the expression of myogenin, a key member of the myogenic transcription factor family.
more recently, mueller et al.
<cit>  identified a lncrna transcribed upstream of myod named munc , and demonstrated that one of the spliced isoforms of munc was drrrna.
consistent with the results of mousavi et al.
<cit> , experimentally decreasing munc expression blocked myoblast differentiation, further highlighting the role of enhancer-associated lncrnas during myogenesis  <cit> .
these results suggest that lncrnas exhibit regulatory function through cis-acting or trans-acting mechanisms in skeletal muscle biology and diseases.
all the studies mentioned above have demonstrated that lncrnas are an integral part of the regulatory network of muscle biology.
the present study provides evidence for the role of lncrnas in skeletal muscle development in goat, which is a starting point for understanding the regulatory mechanisms in which they are involved.
the identification of the lncrnas has greatly improved the annotation of the goat reference genome.
further, we believe that the putative lncrnas may contribute to a better understanding of the biological basis of regulatory interactions amongst mrna, mirna, and lncrna.
CONCLUSIONS
we elucidated skeletal muscle lncrna profiles of fetal and postnatal goats by rna-seq analysis and identified and characterized caprine lncrnas that may be involved in skeletal muscle development in goat.
this study provides a catalog of goat muscle lncrnas that will help in understanding their regulatory roles in goat muscle development.
in future studies, we plan to investigate the functions of some of these lncrnas to provide basic information that will add to the understanding of the regulatory mechanisms associated with goat muscle development at the molecular level.

 genome-wide transcriptional studies have revealed that large regions of eukaryotic genomes are transcribed into a heterogeneous population of non-coding rnas. generally, ncrnas shorter than nucleotides are usually described as small/short ncrna, such as micrornas, piwi-interacting rnas, small interfering rnas, and classical ncrnas such as ribosomal rnas, transfer rnas, and small nucleolar rnas, whereas ncrnas longer than nucleotides are described as long ncrnas. in the past few years, an increasing number of lncrnas have been discovered in mammal, including homo sapiens, mus musculus, bos taurus, sus scrofa, and ovis aries. and accordingly unveiled that lncrnas play critical roles in biological processes like transcriptional regulation, epigenetic modification, development, cell differentiation, as well as in some diseases. as important economic animals worldwide, domestic goats are raised mainly for meat production. thus, unveiling the molecular mechanisms underneath skeletal muscle formation and development is of vital interest. muscle development is a complex process that requires the concerted expression and interaction of multiple factors. several recent studies have indicated that lncrnas play crucial roles in myogenic differentiation and myogenesis. nevertheless, currently the majority strategy for exploring molecular mechanisms underlying skeletal muscle growth and development in mammals is detecting the expression and functions of coding genes like the mrf, mef families, and the paired box proteins, though high-throughput sequencing technologies is also employed to profile expression of mirna and mrna expression in goat. therefore, information about skeletal muscle development-related lncrnas is still limited especially in goats. here, we report the systematic identification and characterization of lncrnas in fetal and postnatal goat skeletal muscle using an illumina hiseq platform. a total of lncrna transcripts were identified and of these transcripts were significantly differentially expressed in pairwise comparisons between rna libraries of skeletal muscle at the different development stages. to the best of our knowledge, no other report on muscle lncrnas and their biological functions in goat is currently available. our results will provide a useful resource for better understanding the regulatory functions of lncrnas in goat and for annotating the goat genome, as well as contribute to better comprehending skeletal muscle development in mammals.  overview of rna sequencing to identify lncrnas expressed in goat skeletal muscle development, we constructed cdna libraries from goat longissimus dorsi muscle samples at four developmental stages: three gestation stages at and 105 days of gestation, and one postnatal stage. three biological replicates for e e and b and two biological replicates for e were used. the libraries were sequenced using an illumina hiseq platform and 125 bp paired-end reads were generated. a total of, raw reads were generated in all libraries. after discarding adaptor sequences and low-quality reads, we obtained, clean reads. the percentage of clean reads in each library ranged from to  %. we mapped the clean reads to the goat reference genome sequence. approximately –  % of the clean reads in all the libraries were mapped to the goat reference genome. the mapped sequences in each library were assembled and a total of unique assembled transcripts were obtained. identification of lncrnas in goat skeletal muscle we developed a highly stringent filtering pipeline to discard transcripts that did not have all the characteristics of lncrnas. our pipeline yielded lncrna transcripts, including intergenic lncrnas and anti-sense lncrnas, these transcripts corresponded to lncrna genes, an average of transcripts per lncrna locus. we found that the lncrna transcripts were distributed in all chromosomes except the y chromosome. the illumina rna-seq also produced protein-coding transcripts with an average length of 1978 bp and exons, which was longer than the lncrna genes, which averaged 1296 bp in length and exons. however, the exon size in the protein-coding genes was smaller than the exon size in the lncrna genes. we also found that protein transcripts with two and three exons accounted for  % of all the protein-coding genes, which was much lower than the percentage of lncrna genes with two and three exons.fig. identification pipeline for lncrnas. each step is described in detail in the methods section fig. comparison of the features of goat lncrnas and protein-coding genes. a exon size distribution of goat lncrnas and protein-coding genes. b exon numbers per transcript of goat lncrnas and protein-coding genes identification of differentially expressed lncrnas the expression levels of the lncrna transcripts were estimated by fpkm using cuffdiff. we identified lncrna transcripts that were differentially expressed during muscle development; the number of down-regulated lncrnas was higher than the number of up-regulated lncrnas during development. the expression patterns of differentially expressed lncrnas were measured by systematic cluster analysis, to explore the similarities and to compare the relationships between the different libraries. the replicates for each developmental stage clustered together, and e and e formed one group and e and b formed another group. to further analyze the interactions among the differentially expressed lncrnas, we constructed a venn diagram using the and lncrnas that were differentially expressed in e e e and b respectively. we did not detect any stage-specific differentially expressed lncrnas among the four developmental stages, but we identified differentially expressed lncrnas that were detected in all four developmental stages.fig. numbers of up-regulated and down-regulated lncrnas in goat skeletal muscle at four developmental stages fig. analyses of differentially expressed lncrnas in the rna-seq libraries. a hierarchical clustering analysis of lncrna expression profiles from libraries with differentially expressed lncrnas. data are expressed as fpkm. red: relatively high expression; green: relatively low expression. b venn diagram showing the differentially expressed lncrnas at the four developmental stages enrichment analysis of nearest neighbor genes of the lncrnas to investigate the possible functions of the lncrnas, we predicted the potential targets of lncrnas in cis-regulatory relationships. we searched for protein-coding genes 10-kb upstream and downstream of all the identified lncrnas. we found lncrnas that were transcribed close to protein-coding neighbors. gene ontology analysis of the cis lncrna targets was performed to explore their functions. we found go terms that were significantly enriched, and of these terms were associated with regulation of gene expression. for example, the top enriched terms included nucleotide binding, regulation of rna metabolic process, dna-dependent regulation of transcription, transcription regulator activity, and transcription factor activity. these results suggest that one of the principal roles of lncrnas may be transcriptional regulation of gene expression. interestingly, we also found genes, including rbp pln, mylk rara, cacnb nr2f cdk and pitx that were annotated with muscle development-related go term, striated muscle tissue development. these results suggest that muscle development may be regulated by the action of lncrnas on these neighboring protein-coding genes. pathway analysis showed that the candidate cis target genes were enriched in kyoto encyclopedia of genes and genomes pathways, several of which were related to muscle development such as insulin signaling pathway, mapk signaling pathway, tgf-beta signaling pathway, and ppar signaling pathway. enrichment analysis of co-expressed genes of lncrnas we also predicted the potential targets of lncrnas in trans-regulatory relationships using co-expression analysis. a total of interaction relationships were detected between lncrna transcripts and protein-coding transcripts that corresponded to protein-coding genes in the goat reference genome. functional analysis showed that the co-expressed genes were enriched in go terms that encompassed a variety of biological processes. importantly, some of the terms were muscle development-related terms, including muscle cell development, striated muscle cell development, muscle contraction, and muscle system process. in addition, the co-expressed genes were enriched in kegg pathways, several of which were related to muscle development, including tgf-beta signaling, mapk signaling, and ppar signaling pathways. these findings indicate that lncrnas also regulate trans target genes. validation of differentially expressed lncrnas we randomly selected six differentially expressed lncrnas and examined their expression patterns at four developmental stages by qpcr. the results confirmed that the six lncrnas were expressed at all four development stages and showed differential expression at different stages. in addition, the qpcr confirmed that the expression patterns of the six lncrnas were consistent with their expression levels calculated from the rna-seq data. all our results show that our pipeline was highly strict in identifying putative lncrnas, and indicate that most of the identified lncrnas were truly expressed in vivo.fig. validation of six differentially expressed lncrnas by qpcr. data are the mean ± sem discussion the identification and characterization of goat lncrnas, particularly in fetal skeletal muscle development, have been very limited compared with lncrnas in human and other model organisms. in goat skeletal muscle, the main focus has been on genes and mirnas rather than on lncrnas. in the present study, we identified a total of multiple exon lncrnas in fetal and postnatal goat skeletal muscle. to the best of our knowledge, this is the first report to systematically identify lncrnas from rna-seq data during goat skeletal muscle development. non-coding and protein-coding genes are distinguished by their potential coding capability. in this study, we developed a highly stringent filtering pipeline to minimize the selection of false positive lncrnas, which aimed to remove transcripts with evidence of protein-coding potential. we identified putative lncrnas with high confidence across four development stages of goat skeletal muscle. in agreement with similar studies on different organisms, the identified putative lncrnas had fewer exon numbers, shorter transcript lengths, and lower expression levels than protein-coding genes. the number of putative lncrnas detected in this study was more than that reported in previous studies in cattle and goat. six randomly selected differentially expressed lncrna transcripts were validated using qpcr, and the results were consistent with the results from the rna-seq data. together, these results confirmed that the identified lncrnas were of high quality. lncrnas are a group of endogenous rnas that function as regulators of gene expression, and are involved in developmental and physiological processes. we detected putative lncrnas that were differentially expressed in pairwise comparisons between goat skeletal muscle at the different development stages. these lncrnas may have specific biological roles in early muscle development in fetal goat. skeletal muscle development from the fetal to the adult stage consists of a series of exquisitely regulated and orchestrated changes in the expression of many genes. in recent years, the roles of some lncrnas in muscle biology have been reported. for example, the long intergenic ncrna muscle differentiation linc-md was the first muscle-specific lncrna to be identified. linc-md is required for appropriate muscle differentiation, at least in part because it regulates the levels of myocyte enhancer factor 2c and mastermind-like protein by sponging endogenous mir- and mir- in the cytoplasm of muscle cells. in addition, the substantial disintegration of linc-md in primary myoblasts of patients with duchenne muscular dystrophy suggests that it is likely involved in the pathogenesis of this muscle disorder. another study revealed that the lncrna, lncmyod, regulates skeletal muscle differentiation by blocking imp2-mediated mrna translation. therefore, the differentially expressed lncrnas reported here can be considered as important novel regulators of goat skeletal muscle biology. unlike mirnas or proteins, the functions of ncrnas cannot currently be inferred from their sequence or structure; therefore, in this study, we predicted the potential function of the detected lncrnas using cis and trans methods. the cis nature of a lncrna refers to its ability to act on a neighboring gene on the same allele from which it is transcribed; thus, this type of lncrna commonly forms a feedback loop that regulates itself and its neighboring genes. in the cis prediction, we searched for coding genes 10-kb upstream and downstream of all the identified lncrnas. go and kegg analyses of the neighboring protein-coding genes revealed that major enriched pathways were associated with transition metal ion binding, nucleotide binding, zinc ion binding, regulation of rna metabolic process, regulation of transcription, and transcription regulator activity. these results indicate the possible role of lncrnas in transcriptional regulation of gene expression. interestingly, we found some of the cis target protein-coding genes were involved in skeletal muscle tissue development, implying that the corresponding lncrnas play regulatory roles in skeletal muscle development. several recent studies also indicated that lncrnas were involved in cis-regulatory activity in muscle development; for example, the lncrna dum upstream binding muscle lncrna) was identified in skeletal myoblast cells. dum promotes myoblast differentiation and damage-induced muscle regeneration by silencing its neighboring gene, dppa in cis through recruiting dnmt dnmt3a, and dnmt3b. similarly, a chip-seq study of the yin yang transcription factor, an important component of the pcg complex that negatively regulates myogenesis, identified a number of lncrnas regulated by yy. among the yams, yam- displayed a cis effect on the expression of neighboring genes, including one that encodes mir- which targets and represses wnt7b in skeletal muscle. many lncrnas can also function in trans mode to target gene loci distant from where the lncrnas are transcribed. in the co-expression analysis, we detected lncrna transcripts that were related to protein-coding genes based on the expression correlation coefficient. go enrichment analysis found that the enriched go terms referred mainly to development process, transcriptional regulation, and biosynthetic process. furthermore, a cluster of lncrnas in the co-expression analysis often targeted protein-coding genes that were expressed specifically in muscle and were involved in muscle development. this is an interesting observation, which indicates the functional complexity of lncrnas and is worth investigating further. mousavi et al. found two lncrnas in two enhancer regions of the myod gene that they named ddrrna and cerna, where drr indicates distal regulatory regions and ce indicates core enhancer. the study showed that cerna facilitated the occupancy of rna polymerase ii in cis by increasing chromatin accessibility, stimulating the expression of myod, while ddrrna functions in trans to promote the expression of myogenin, a key member of the myogenic transcription factor family. more recently, mueller et al. identified a lncrna transcribed upstream of myod named munc, and demonstrated that one of the spliced isoforms of munc was drrrna. consistent with the results of mousavi et al., experimentally decreasing munc expression blocked myoblast differentiation, further highlighting the role of enhancer-associated lncrnas during myogenesis. these results suggest that lncrnas exhibit regulatory function through cis-acting or trans-acting mechanisms in skeletal muscle biology and diseases. all the studies mentioned above have demonstrated that lncrnas are an integral part of the regulatory network of muscle biology. the present study provides evidence for the role of lncrnas in skeletal muscle development in goat, which is a starting point for understanding the regulatory mechanisms in which they are involved. the identification of the lncrnas has greatly improved the annotation of the goat reference genome. further, we believe that the putative lncrnas may contribute to a better understanding of the biological basis of regulatory interactions amongst mrna, mirna, and lncrna.  we elucidated skeletal muscle lncrna profiles of fetal and postnatal goats by rna-seq analysis and identified and characterized caprine lncrnas that may be involved in skeletal muscle development in goat. this study provides a catalog of goat muscle lncrnas that will help in understanding their regulatory roles in goat muscle development. in future studies, we plan to investigate the functions of some of these lncrnas to provide basic information that will add to the understanding of the regulatory mechanisms associated with goat muscle development at the molecular level. 
these co-expressed mrnas were enriched in development-related biological processes such as muscle system processes, regulation of cell growth, muscle cell development, regulation of transcription, and embryonic morphogenesis. notably, in pairwise comparisons between the libraries of skeletal muscle at the different development stages, a total of transcripts were differentially expressed which were validated by qpcr using randomly selected six lncrna genes. additionally, pearson’s correlation coefficients of co-expression levels suggested lncrnas and mrnas were possibly in trans-regulatory relationships. the identified goat lncrnas shared some characteristics, such as fewer exons and shorter length, with the lncrnas in other mammals. we also found lncrnas genes were neighbored protein-coding genes and functionally enriched in transcriptional regulation and development-related processes, indicating they may be in cis-regulatory relationships. however, there are few reports on the characterization of lncrnas covering all the development stages of skeletal muscle in livestock. among them, lncrna transcripts corresponding to lncrna genes were identified, including intergenic lncrnas and anti-sense lncrnas. rna libraries constructed from developing longissimus dorsi muscle of fetal and postnatal goat were sequenced. large numbers of lncrnas have been identified in mouse, rat, and human, and some of them have been shown to play important roles in muscle development and myogenesis. this study provides a catalog of goat muscle-related lncrnas, and will contribute to a fuller understanding of the molecular mechanism underpinning muscle development in mammals. muscle developmentlncrnagoattranscriptomecis-actingtrans-actingdifferential expressionsichuan province science and technology support program2014nz 2015nz 16zc2849zhan siyuan issue-copyright-statement© the author 2016
 long non-coding rnas have been studied extensively over the past few years. large numbers of lncrnas have been identified in mouse, rat, and human, and some of them have been shown to play important roles in muscle development and myogenesis. however, there are few reports on the characterization of lncrnas covering all the development stages of skeletal muscle in livestock.  rna libraries constructed from developing longissimus dorsi muscle of fetal and postnatal goat were sequenced. a total of, clean reads were generated. among them, lncrna transcripts corresponding to lncrna genes were identified, including intergenic lncrnas and anti-sense lncrnas. notably, in pairwise comparisons between the libraries of skeletal muscle at the different development stages, a total of transcripts were differentially expressed which were validated by qpcr using randomly selected six lncrna genes. the identified goat lncrnas shared some characteristics, such as fewer exons and shorter length, with the lncrnas in other mammals. we also found lncrnas genes were neighbored protein-coding genes and functionally enriched in transcriptional regulation and development-related processes, indicating they may be in cis-regulatory relationships. additionally, pearson’s correlation coefficients of co-expression levels suggested lncrnas and mrnas were possibly in trans-regulatory relationships. these co-expressed mrnas were enriched in development-related biological processes such as muscle system processes, regulation of cell growth, muscle cell development, regulation of transcription, and embryonic morphogenesis.  this study provides a catalog of goat muscle-related lncrnas, and will contribute to a fuller understanding of the molecular mechanism underpinning muscle development in mammals. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords muscle developmentlncrnagoattranscriptomecis-actingtrans-actingdifferential expressionsichuan province science and technology support program2014nz 2015nz 16zc2849zhan siyuan issue-copyright-statement© the author 2016 