Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos
Transcriptional Profiling Identifies Location- Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos
Rong-Ping Zhang 0 1 2
He-He Liu 0 1 2
Jun-Ying Liu 0 1 2
Ji-Wei Hu 0 1 2
Xi-Ping Yan 0 1 2
Ding-Min- Cheng Wang 0 1 2
Liang Li 0 1 2
Ji-Wen Wang 0 1 2
0 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University , Ya'an 625014 , China
1 Funding: This study was supported by the National Waterfowl Industrial Technology System (No. CARS- 43-6 , Beijing, China), the National Natural Science Foundation of China (No.31301964, Beijing , China) , the Breeding of Multiple Crossbreeding System in Waterfowl (2011NZ00998 , Sichuan, China) , Innovation Research Project (13TD0034 , Sichuan
2 Editor: Marinus F.W. te Pas, Wageningen UR Livestock Research , NETHERLANDS
Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected Pvalue<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our understanding of the location-specific and breed-specific genes and pathways but also provides some candidate molecular targets for increasing muscle products and meat quality by genetic control.
China), and the Modernization Industry Chain
(2014NZ0030-1, Sichuan, China). The funders had
no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Myogenesis is a highly complex physiology process that involves myogenic progenitor
proliferation, myoblast proliferation and differentiation, and the formation of none-nuclei and
multi-nuclei myotubes and eventually mature muscle. As such, myogenesis is highly regulated
by numerous signature pathways and genes [
]. Embryo myogenesis is pivotal for muscle
production in adult livestock because the myofiber number is determined during the embryonic
stage for most animals and does not increase in the postnatal period. Rather muscle mass gain
in adult livestock mainly depends on increasing the length and thickness of existing
myofibers, a process referred to as hypertrophy [
]. In addition, muscle stem (satellite) cells also
play a vital role in muscle development in adults, specifically in muscle regeneration during
muscle injury, overload [
] and exercise[
]. Satellite cells originate from somites in the
embryo stage and reside between the basement membrane and the myofiber sarcolemma in
adults . Unfortunately, the number and function of satellite cells will inevitably continually
decrease with age [
]. Therefore, the number of myofibers established in the embryo stage is
the critical determinant of muscle production in livestock. A previous study using microarray
hybridization reported that, in turkeys, a higher number of differentially expressed genes
occurred early in development (day 18 of the embryonic stage) than at 1 day and 16 weeks
after birth, suggesting that the phenotypic differences in adults between the two turkey lines
may largely be determined during embryonic myogenesis [
]. Other studies have
demonstrated that muscle growth is predominantly determined during prenatal skeletal muscle
]. Thus, evidence indicates that the embryonic stage is an important period
in the research of muscle development, and a better understanding of the genes and pathways
involved is necessary.
Waterfowl breeding plays an important and unique role in agricultural development.
However, compared to human, mouse or chicken, studies of the myogenesis mechanisms in duck, a
non-model species, are incomplete and are still in their infancy. Most of these studies in duck
have mainly focused on comprehensively investigating the expression patterns of a few crucial
regulative genes. The key genes six1 [
], Pax3/7 [
], MRFs (MyoD, MyoG and MRF4) [
], mTOR and S6k  regulate myoblast proliferation and myofiber hypertrophy. These
studies identified expression differences of such genes between pectoral and leg muscles in
Peking duck have been identified. In addition, previous studies have shown that the carcass
and meat quality of duck are influenced by breed and sex [
]. These results indicate that
the numerous biological and genetic differences between skeletal muscles depend on their
anatomical location and breed.
In this study, we used several individuals from two native duck breeds, Heiwu duck and
Peking duck, to identify changes in gene expression which may be responsible for the
differences in muscle development between locations and breeds. We observed phenotypic
differences between pectoral and leg muscle and between the same muscle type from both breeds
(for detailed data, see Fig 1). To further investigate these differences, a highly effective and
accurate digital gene expression (DGE) technology was used to obtain abundant sequences at
the transcript level. In 2013, Huang et al. released the draft genome sequence of Anas
Platyrhynchos acquired using Illumine technology [
], which equips us to better study myogenesis
of duck using DGE technology. This study will help us to identify differentially expressed genes
related to pectoral muscle and leg muscle myogenesis in two duck breeds. While our data was
collected from a relatively small sample set, this work will likely still be helpful for
understanding the molecular basis of the different muscle development capabilities of the leg and pectoral
muscle of Peking duck and Heiwu duck, providing further knowledge and new clues for
investigation of muscle development mechanisms, and improving duck breeding research.
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Fig 1. Skeletal muscle variations in leg muscle (LM) and pectoral muscle (PM) between Heiwu duck (H) and Peking duck (P) during development.
A. The increase in the weight of four skeletal muscles from post-hatching week 1 (W1) to week 16 (W14). B. The changing rate of skeletal muscle weight to
carcass weight from post-hatching week 1 (W1) to week 16 (W14). C. The variation in the weight of the four skeletal muscles in embryo from embryonic day
15 (E15) to day 28 (E28). D. The changing rate of skeletal muscle weight to carcass weight from embryonic day 15 (E15) to day 28 (E28). At each time point
(W1, W4, W10, etc), statistically significant differences are indicated by different letters (a, b, c, etc). At a given time point, any samples that are not
significantly different are labeled with the same letter.
Materials and Methods
All ducks were obtained from the Sichuan Agriculture University Waterfowl Breeding
Experimental Farm, Sichuan, China. This study was carried out according to Beijing Animal Welfare
Committee (Beijing, China) and approved by the institutional Animal Care and Use
Committee of Sichuan Agriculture University (Permit Number: DKY B20121405). All surgery was
performed under sodium pentobarbital anesthesia, and all efforts were made to minimize
Peking duck (Anas platyrhynchos domestica) (average weight 80 g) and Heiwu duck (average
weight 75 g) eggs were incubated under the same conditions, 37±0.5°C and 86–87% humidity.
After incubation for 15 days at the Sichuan Agriculture University Waterfowl Breeding
Experimental Farm, leg muscles (LM) and pectoral muscles (PM) were collected from the Heiwu
duck (H) and Peking duck (P) specimens, separately, five individual ducks were used per
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breed. All of the muscles were frozen in liquid nitrogen and stored at -80°C prior to
During the embryo stage, LM and PM of the H and P specimens were collected at 15, 20, 25
and 28 days of incubation, five individual ducks were used per breed at each time point. All of
the samples were weighed immediately after isolation from the embryo.
Post-hatching, the duck breeds were raised under the same conditions. For both breeds, six
ducks (three male and three female) were weighed and slaughtered at every week until 16
weeks. Then, the LM and PM of six ducks were isolated and weighed separately. These
procedures were approved by the Beijing Animal Welfare Committee (Beijing, China).
RNA preparation, Illumina RNA-sequencing
The total RNA from four group muscles (H-LM, H-PM, P-LM and P-PM) was extracted using
Trizol reagent (Takara, China) according to the manufacturer’s instructions. Five individuals’
RNA samples per group were equally mixed to generate an RNA pool. The RNA purity was
checked using the NanoPhotometer1 spectrophotometer (IMPLEN, CA, USA). The RNA
integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system
(Agilent Technologies, CA, USA).
Sequencing libraries were generated using the NEBNext1 Ultra™ RNA Library Prep Kit for
Illumina1 (NEB, USA) following the manufacturer’s recommendations, and index codes were
added to attribute sequences to each sample. Briefly, mRNA was purified from the total RNA
using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent
captions under elevated temperature in the NEBNext First Strand Synthesis Reaction Buffer (5×).
First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse
Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using
DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via
exonuclease/polymerase activities. After the adenylation of the 3’ ends of DNA fragments,
NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. To
preferentially select cDNA fragments of 150~200 bp in length, the library fragments were
purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μl USER Enzyme
(NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by
5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA
polymerase, Universal PCR primers and Index (X) Primer. Finally, the PCR products were purified
(AMPure XP system) and the library quality was assessed on the Agilent Bioanalyzer 2100
DGE read annotation
To identify the gene expression patterns in the skeletal muscle of the Peking duck and Heiwu
duck specimens, all of the clean reads were annotated by mapping to the sequenced genome of
] using the TopHat v2.0.9 software. For gene expression analysis, HTSeq v0.5.4p3 was
used to count the read numbers that were mapped to each gene. Then, RPKM (Reads Per Kilo
bases per Million reads) was used to calculate and normalize the number of expression tags. A
DEG analysis of two locations/breeds was performed using the DESeq R package (1.10.1).
DESeq provides statistical routines for determining the differential expression in digital gene
expression data using a model that was based on the negative binomial distribution. Genes
with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed. The
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented
by the GOseq R package, in which gene length bias was corrected. GO terms with a corrected
P-value < 0.05 were considered significantly enriched by differentially expressed genes. We
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used KOBAS software to test the statistical enrichment of differentially expressed genes in
KEGG pathways. All of the sequence data were submitted to the GEO database, and the GEO
accession number is GSE65628.
Quantitative real-time PCR confirmation
A total of 15 genes were chosen randomly and detected by quantitative real-time PCR
(RT-PCR) to confirm the accurate DGE. All primers (S1 Table) were designed using Primer
5.0 software and synthesized by BGI Company (China). Sample RNAs (1 μg) were
reversetranscribed to cDNA using a reverse-transcription system (Takara, Dalian, China), three
individuals’ RNA were used per group at E15, and RT-PCR for each sample was conducted in
triplicate. The reaction was run using the IQTM5 System (Bio-Rad, Hercules, CA), and the data
were analyzed by the 2-ΔΔCt method using β-actin and GAPDH as internal reference genes. A
statistical analysis was performed with GLM processes and t-test using SAS 8.0 software (SAS
Institute Inc., Cary, NC).
Phenotypic analysis of skeletal muscle in ducks
In order to identify breed-specific mechanisms that may contribute to the differences in muscle
development capability, two phenotypically different duck breeds were examined. We began
by comparing two different muscle groups within each breed. As shown in Experiment 1 (Fig
1A), in both breeds skeletal muscle has a significant weight difference between LM and PM, at
all measurement time points except W10 in the Heiwu duck. To take into account the carcass
weight of each duck, we calculated the ratio of LM and PM weight to carcass weight and found
that every ratio was significantly different between locations, and this was true of both breeds
(Fig 1B). When comparing across breeds in Experiment 2, we found that Peking duck has a
higher skeletal muscle weight than Heiwu duck in the postnatal period. However, the ratio of
LM weight to carcass weight was significantly different between the duck breeds only at W1,
while the ratio of PM weight to carcass weight differed significantly at W10 and W14 (Fig 1B).
These data indicate that Peking duck and Heiwu duck are two phenotypically extreme duck
breeds, and these differences are more obvious in PM than in LM.
We then went on to examine skeletal development in embryos. In Experiment 1, the muscle
weight was significantly different between locations for both breeds during the embryo period,
with the exception of the Heiwu duck at E20 (Fig 1C). Comparing across breeds in Experiment
2, LM weight, unlike PM weight, was significantly different between the Peking duck and the
Heiwu duck, except at E25. However, the ratio of LM or PM to embryo weight was not
significantly different between the Peking duck and the Heiwu duck, with a sole exception. The only
ratio to show a significant difference between the breeds was the LM:embryo weight ratio at
E15 (Fig 1D). These weight data from both either in the embryo or at and the post-hatching
periods indicate phenotypic differences between muscle locations and breeds.
Analysis and alignment of the digital gene expression (DGE) profile
In this study, four DGE-read libraries (H-LM, H-PM, P-LM and P-PM) were constructed
using LM and PM tissues from two phenotypically extreme duck breeds (H and P). The
statistics of the DGE reads are shown in Table 1. More than 13 million raw reads were generated for
each library. After filtering the adapter reads, more than 10% of the N (uncertain base
information) reads and low-quality reads and more than 97% of the raw reads were clean reads for
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Analysis of the level of gene expression
To identify genes that were differentially expressed in the muscle tissues of the duck breeds, we
compared pairs of DGE profiles in the four libraries (Experiment 1: H-PM vs H-LM and P-PM
vs P-LM, Experiment 2: P-PM vs H-PM and P-LM vs H-LM) to analyze gene expression
variations. |log.2fold-change|>0.5 and P-value <0.05 were used as the thresholds for significant
differential gene expression (Fig 2A). In Experiment 1, a total of 336 genes were differentially
expressed between LM and PM. Of these, 246 genes were significantly affected in the H breed,
and 258 genes were significantly affected in the P breed (Fig 2B). Many fewer genes were found
to be significantly different between breeds in Experiment 2. Only 8 genes were differentially
expressed between the two duck breeds (Fig 2C). All of the DEGs are listed in the S1 Excel file.
As expected, the majority of gene expression changes occurred between LM and PM, and these
data indicate that muscle location rather than breed differentiation is mainly responsible for
muscle disposition differences (S1 Fig).
Validation of DEGs by RT-PCR
To confirm the differentially expressed genes from the RNA-Seq data, 15 genes (ACTA1,
ACTC1, ANGPT2, APOBEC0, EEF1A1, ENO1, FBLN5, LOC101792412, HOXA6, LAMB2,
PENK, RET, RPSS35, RSPO3, and SEMA3C) were chosen randomly and measured by
realtime PCR. Only EEFIA1 and LOC101792412 were common between Experiment 1 and
Experiment 2. The RT-PCR expression profiles of the fifteen genes closely resemble the expression
pattern obtained from the DGE results (Fig 3), except that some genes have a different relative
expression level on DGE at a specific muscle tissue. The RT-PCR expression profiles therefore
indicate the reliability of the RNA-Seq data.
DEGs involved in myogenesis and muscle metabolism
Studies have showed that many genes are involved in myogenesis and muscle metabolism.
Some key genes related to skeletal muscle development are listed in Table 2. Our data reveal
that two myosin heavy-chain isoforms (MYH7b and MYH15) and three myosin light-chain
isoforms (MYL1, MYL3 and MYL10) were highly expressed in either LM or PM with the same
breed. However, only MYH7b was common between Experiment 1 and Experiment 2 and was
up-regulated in the P breed compared to the H breed. We also observed that some important
genes that have been implicated in collagen fibril organization and biosynthetic processes, such
as COL11A1, COL12A1, COL14A1, COL1A2, COL5A2 and COL9A1, were down-regulated in
PM compared to LM. Of troponins and tropomyosins, TNNI2, TNNT3, TPM2 and TPM3
exhibited similar profiles with high expression in LM. Additionally, angiopoietin 2 (ANGPT2)
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Fig 2. The distribution of DEGs in each library. A: The numbers of differentially expressed genes in each comparison. The up-regulated genes are shown
in red, whereas the down-regulated genes are shown in green. “A” was the control group, and “B” was the experimental group in “A vs B”. B. The distribution
of DEGs that were found to be commonly and specifically expressed between locations in Experiment 1. C. The distribution of DEGs that were found to be
commonly and specifically expressed between breeds in Experiment 2.
and platelet-derived growth factor (PDGH), which are involved in the PI3K/AKT signaling
pathway, were up-regulated in PM. In Experiment 2, DEGs were mainly concentrated in
ribosome proteins (RPS6, RPS23, RPL36, etc.).
Functional and pathway analysis of DEGs
After identifying all of the genes differentially expressed between the two breeds, we annotated
the sequences using the Gene Ontology (GO) database (http://www.geneontology.org/) to
investigate changes in the patterns of gene expression between locations and breeds. In the
H-PM vs H-LM library, 1563 sequences could be classified into 47 secondary level categories
(correct P-value <0.001) (Fig 4A). In the P-PM vs P-LM library, 1927 sequences could be
categorized into 60 functional groups (Fig 4B). In Experiment 1, “Multicellular organismal process”
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Fig 3. RT-PCR validates the correction of RNA-Seq. The relative expression levels were calculated using β-actin and GAPDH as the internal controls.
RPKM: the number of reads per kilo bases per million reads (RPKM).
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NO: there is not sequence number for the gene in NCBI.
and “single-multicellular organism process”, “extracellular region” and “calcium ion binding”
were found to be dominant in the three corresponding categories. This experiment
demonstrated that skeletal muscle location differences are conserved between the Peking duck breed
and the Heiwu duck breed. On the other hand, in Experiment 2, 99 and 128 sequences were
classified into 12 and 13 secondary functional categories, respectively (Fig 4C and 4D). Only
the category “translation” was significantly classified into “biological process” (correct p-value
<0.001). The GO analysis showed that the DEGs identified in this study span a broad range of
functions and cellular processes.of the identified genes are involved in various processes.
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Fig 4. Histogram presents gene ontology classification. The left axis indicates the percentage of the
specific category of genes in the main category. The right axis indicates the number of genes in a category. A,
B, C and D represent the H-PM vs H-LM.GO, P-PM vs P-LM.GO, P-LM vs H-LM.GO, and P-PM vs H-PM.GO,
The KEGG data were used to better identify the active biological pathways in the DGE
libraries; only significantly enriched genes were displayed in this study (P-value <0.05). In the
H-LM vs H-PM libraries, differentially expressed genes were significantly assigned to 10
pathways (Table 3). A total of 11 pathways were significantly enriched in the P-LM vs P-PM library.
In Experiment 1, Focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor
interaction are the top three pathways. Comparing across breeds in Experiment 2, however, only the
ribosome pathway was detected as having differentially expressed components in both muscle
locations (Table 3).
Although some DEGs between skeletal muscles have recently been identified in several species,
such as pigs [
], mice , turkey [
], sheep [
], and dog [
], the molecular mechanism
underlying muscle development in duck remains unclear. Here, we analyzed the DEGs between
duck breeds with different growth rates using RNA-Seq technology. P and H specimens with
the same embryo day and different growth rates were used as our study animals. In poultry, the
form of secondary myofibers, which accounts for the majority of skeletal muscle fibers,
happened from E8 to E16 stage [
]. Meanwhile, our phenotypic data shown that leg muscle
weight: embryo weight ratio is only significantly different between H and P breeds at E15 (Fig
1D). Gu et al’s research indicated that from E13 to E19 is the fastest growth stage of Peking
duck pectoral muscle in embryo [
]. Based on these acknowledge, we believe E15 is the proper
day to investigate DEGs between locations and breeds. The goal of the current study was to
identify global genes and pathways affecting duck skeletal muscle deposition between locations
This study was specifically designed to identify DEGs between two duck breeds and two
muscle locations within a breed rather than assessing differences between individuals.
Therefore data analysis was performed on data collected from mixed sample pools comprised of
tissues from 5 individuals. This design allowed us to analyze genetic data from 5 individuals of
each duck breed at once, thereby providing us with a cost-effective way to minimize the
possibility of identifying individual-specific DEGs rather than the desired breed-specific DEGs.
Confirmation of the RNA-seq results with RT-PCR experiments provides greater confidence in
DEGs identified. We acknowledge that this experimental design, while commonly employed,
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does not yield biological or technical replicates and all conclusions were drawn with this in
Analysis of library data
According to the muscle weight data, it is clear that dramatic phenotypic differences exist
between LM and PM and between P and H. One phenotypic example is that PM has a higher
degree of protein metabolism (high expression level of mTOR, S6K, FoxO1, MuRFbx and
MAFbx), mainly in response to a higher growth rate, than LM in the Peking duck [
phenotypic differences may be associated with differentially expressed genes between the two
locations or the two breeds. As a powerful tool, RNA-Seq was used to index these DEGs
between LM and PM in both of the ducks. A total of 15 genes were randomly selected to verify
the accuracy and repeatability of the sequence data using RT-PCR. Although all 15 genes have
the same expression pattern between RT-PCR and DGE data, some genes have a different
expression level between RT-PCR and DGE at a specific muscle tissue. This may be due to that
the 2-ΔΔCt analysis has less accurate than RNA-Seq technology, especially for lowly expressed
gene. Compared with RT-PCR, the fold changes of these gene expressions were larger when
measured by RNA-Seq. In addition, 4 of 15 genes were low-expressed genes, including FBLN5,
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PENK, RET and RSPO3 (0.76<RPKM<46.28), as verified using RT-PCR. This result is
probably because the RNA-Seq was more sensitive in determining gene expression levels, particularly
for low-abundance transcripts [
]; the same phenomenon has also been reported in other
The identification of DEGs between leg muscle and pectoral muscle tissues from 2 different
duck breeds indicates that location differences accounted for more of the genetic profile
differences than did breed differences. This result also confirmed that different locations shared
many more myogenetic genes and pathways than did different breeds. The functional
annotation analysis also presented similar GO biological process terms and KEGG pathways between
locations but not between breeds, whereas DEGs matched to only 1 GO biological process
(“translation”) and 2 KEGG pathways (“ribosome” and “Complement and coagulation
Function of DEGs implicated in muscle development
Muscle fibers are the basic elements for muscle deposition. In different mammalian breeds,
skeletal muscles contain four myosin heavy-chain (MyHC) isoforms, slow/β-, 2a-, 2b- and
2xMyHC, and three major myosin light-chain (MLC) isoforms, the “slow” MLC1s and the “fast”
MLC1f and MLC3f [
]. Based on the expression of the four MyHC gene isoforms, muscle
fibers are characterized into four different fiber types: I, IIA, IIB and IIX . In this study,
MYH7b was identified as a differentially expressed gene in Experiment 1 and 2. MYH15 was
only identified in Experiment 1 (between LM and PM) and down-regulated in PM. On the
other hand, three MYL genes (MYL1, MYL3 and MYL10) were found to be DEGs in
Experiment 1, and MYL10 was also up-regulated in P-PM compared to H-PM. All of these variances
in the gene profile can provide explanation for the differences in the fiber types in skeletal
muscle for different locations and breeds. A previous study identified MYH3 and MYH8 as
differentially expressed genes between intact and castrated cattle [
], indicating that these two
genes are major genes for muscle fiber properties. We believe that these genes (MYH7b,
MYH15, MYL1, MYL3 and MYL10) partially contribute to the difference in skeletal muscle
deposition between locations and duck breeds.
The thin filament regulatory proteins troponin and tropomyosin are responsible for striated
muscle contractions according to the effect of the intracellular Ca2+ concentration. Troponin
consists of three subunits [
]: the Ca2+-binding troponin C (TNNC), the inhibitory troponin I
(TNNI) and the tropomyosin-binding troponin T (TNNT), which interact strongly with each
other. Previous studies have suggested that human mutations in TNNT3, TNNI2, and TPM2
increase the contractility of fast-twitch muscle fibers and cause distal arthrogryposis (DAs)
]. During myogenesis in vitro, Troponin I and slow MYBPC isoforms (MYBPC1)
had a predominant expression in proliferating human mononucleated myoblasts and
myotubes , and MYBPC1 is also a novel gene that is responsible for DA1 [
]. In the current
study, these genes (TNNI2, TNNT3, TPM2/3 and MYBPC1) displayed the same profile, being
down-regulated in PM. Another down-regulated gene in PM, FBN1, is a member of the
homologous molecules family and regulates the structure and function of microfibers and elastic
fibers, which provide an extracellular reservoir for inactive growth factor [
]. Mutations in
FBN1 cause an autosomal dominant connective tissue disorder, Marfan syndrome (MFS),
which displays variable manifestations in the skeletal, cardiovascular and ocular systems [
]. As the only protein that has been unambiguously implicated in determining limb-type
morphologies, Pitx1 is necessary for the normal initiation of hind limb outgrowth as a result of
the regulation of Tbx4 expression . Tbx4, which is exclusively expressed in the hind limb,
plays a crucial role in hind limb bud initiation [
]. Those genes that were down-regulated in
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PM indicate that duck LM had a higher growth rate than PM at embryonic day 15, indicating
that the leg muscle has an earlier developmental origin than does the pectoral muscle.
CTNNB1, which is a primary mediator of the WNT/β-catenin signaling pathway, is
responsible for skeletal myogenesis involving hypertrophy [
]. A previous study demonstrated that
the CTNNB1 gene had an increased expression in Pietrain fetuses compared to that in Duroc
fetuses , but this gene expression as detected by qRT-PCR did not confirm the results
observed with the microarray. In the present study, we found only CTNNB1 to have a
location-special expression pattern. For its breed-specific expression pattern, further studies may
Function of some pathways related to muscle development
Based on the functional annotation analysis of DEGs from different muscle tissues, we
identified the predominant differentially expressed genes as being related to focal adhesion, the
PI3K-Akt signaling pathway and ECM-receptor interactions for location-specific DEGs in
Experiment 1 and as being related to ribosomes for breed-specific DEGs in Experiment 2.
Focal adhesions are integrin-based structures that determine the adhesive behavior of cells
in response to cell migration, growth, and differentiation [
]. Fibronectin (FN) and
vitronectin (VN) are two common and vital components. We found that FN and VN are
locationspecific genes and that VN is also a breed-specific gene in PM. Burridge’s review (1988)
mentioned that in avian cells, integrin is concentrated even within those focal adhesions lacking FN
and indicated that VN is probably another ECM component that binds to integrin . In this
study, we found that FN1 and VN had contrasting expression patterns in Experiment 1 for
Peking ducks, indicating that their role in skeletal muscle growth warrants further study. The
study by Timmons et al (2005) demonstrated that FN1 increased the expression of the Laminin
gene family (LAMA4, LAMB1, and LAMC1) by endurance exercise training [
]. LAMA2 and
LAMC1 play distinct roles in myogenesis [
]. Mutation in the LAMA2 gene causes
merosindeficient congenital muscular dystrophy (MDC1A) [
]. The differential expression of LAMB1
has been observed during pig muscle development [
], and LAMB1 is a positive factor in
the activation of myofiber formation [
]. These DGEs displayed a different expression pattern
in Experiment 1, suggesting that the focal adhesion pathway contributes to the difference in
skeletal muscle development between locations and that these DGEs could be the main
candidate gene for this difference.
PI3K is pivotal in growth factor-, insulin- and G protein-mediated signal transduction and
is involved in adhesion and migration regulation. In addition, the PI3K/AKT pathway plays an
important role in the process of myotube differentiation [
], which is mediated by the
dystrophin-glycoprotein complex (DGC). The disruption of DGC induced apoptosis in muscle
cell cultures by decreasinged phosphorylation of AKT and its downstream effector GSK-3β
. The inhibition of PI3K by specific inhibitors reduces adhesion and migration in a variety
of cell types [
]. Down-regulated Akt activation by overexpressed SHIP-2 causes cell-cycle
]. In addition, many studies have demonstrated that the AKT/mTOR (mammalian
target of rapamycin) signaling pathway is activated during hypertrophy [
] and improves the
increase in muscle mass according to the increase in muscle fiber size [
]. Although we did
not detect PI3K and AKT as DEGs between locations and breeds, some genes involved in the
PI3K/AKT pathway caught our attention. Down-regulated LAMA2, LAMC1, TNNI2 and
TNNT3 and up-regulated angiopoietin 2 (ANGPT2) and PDGFC/D are enriched in the PI3K/
AKT pathway. ANGPT2 expression increases significantly during myoblast differentiation
into myotubes and promotes skeletal myoblast survival and differentiation though the
activation of the PI3K/AKT and Erk1/2 pathways [
]. The PDGF family consists of four members:
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PDGF-A, PDGF-B, PDGF-C and PDGF-D, and the last two members (PDGF-C and PDGF-D)
were recently discovered and were found to be DEGs in Experiment 1 in the present study. The
review by Reigstad (2005) et al showed that both PDGF-C and PDGF-D are involved in various
malfunctions: PDGF-C seems to play an important role in Ewing family sarcomas, whereas
PDGF-D is related to lung, prostate and ovarian cancers [
]. Until now, information about
the roles of PDGF-C and PDGF-D in skeletal muscle development has been limited; our
finding that PDGF-C and PDGF-D are location-specific genes in skeletal muscle indicates that
these two genes may play a special role in skeletal muscle development and require further
ECM-receptor interactions play a profound role in major cellular programs, including
migration, growth, difference and survival [
], and play an important role in myogenesis [
Collagen (types I, III, IV, and V) [
] and fibronectin  are the main constituents of
ECM in skeletal muscle tissue. Collagen type V is probably already suggested to be involved in
the sequence of events leading to myoblast differentiation [
]. In this study, a total of six
collagen members (COL11A1, COL12A1, COL14A1, COL1A2, COL5A2 and COL9A1) were
differently expressed in Experiment 1 and Experiment 2. Previous studies have also reported that
many other collagen family numbers have different expression patterns during skeletal muscle
]. Taken together, these findings indicate that collagen gene expression
variability plays an important role in affecting muscle development.
Interestingly, some pathways in Experiment 1 are highly related to cardiac development,
including the hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy and cardiac
muscle contraction pathways, which indicates that these DGEs in skeletal muscle also have a link
with cardiac development. This is consistent with previous studies wherein genetic
manipulation conducted for many years resulted in increased muscle production while promoting
dysfunction of cardiac development [
]. However, the mechanism linking muscle development
and cardiac disease remains incomplete and requires further study. Yet another difference we
identified between LM and PM is that protein digestion and absorption pathway is also
significant between locations, demonstrating that leg and pectoral muscle already display differential
protein metabolism during the embryo stage. This variation in protein metabolism could,
conceivably, be the main reason for muscle mass gain difference after post-hatching [
Breed-specific DEGs identified in Experiment 2 are all pertaining to ribosome assembly and
function. This is interesting, as ribosomes are platforms upon which to perform protein
synthesis and thereby promote skeletal muscle deposition. Many ribosomal proteins (RPS6, RPS23,
RPL36, RPSA, RPL8, etc.) and Elongation factor 1 (eEF1) (eEF1A1, eEEF1A2 and eEF1D) were
detected by RNA-Seq in the present study, especially in Experiment 2. eEF1 is responsible for
transferring aminoacyl-tRNA to the empty A-site on the ribosome [
]. In line with a previous
study reporting that protein metabolism is the main reason for promoting duck skeletal muscle
], these findings indicate that protein synthesis may also be the main
determinant for differences in skeletal muscle development between duck breeds.
Taken together, all of the data from the present study demonstrate obvious differences in
muscle weight and gene expression between leg and pectoral muscles and between Heiwu duck and
Peking duck. It is intriguing that were more location-specific genes identified than
breed-specific genes. The GO results suggested that location-specific functional gene groups are
conserved between two duck breeds, and include a much broader range of functions than do the
breed-specific functional groups. This study suggests that focal adhesion, the PI3K-Akt
signaling pathway and ECM-receptor interaction may be the main molecular networks that are
14 / 18
responsible for muscle development differences between leg and pectoral muscles.
Furthermore, our findings suggest that ribosomes could be the main molecular driver of the differences
between the two duck breeds. Thus, this study successfully identified candidate genes and
pathways involved in the differences in muscle growth between leg and pectoral muscles and
between Heiwu duck and Peking duck, and it might provide the basis for future experiments
that focus on these candidate genes, their proteins products and their functions in duck skeletal
S1 Excel. List of differentially expressed genes in each library comparison. DEGs between
pairs of libraries are shown (H-PM vs H-LM, P-PM vs P-LM, P-LM vs H-LM and P-PM vs
S1 Table. The primer information for RT-PCR.
S1 Fig. Cluster analysis of differentially expressed genes according to Pearson’s correlation
This study was supported by the National Waterfowl Industrial Technology System (No.
CARS-43-6, Beijing, China), the National Natural Science Foundation of China (No.31301964,
Beijing, China), the Breeding of Multiple Crossbreeding System in Waterfowl (2011NZ00998,
Sichuan, China), Innovation Research Project (13TD0034, Sichuan, China) and the
Modernization Industry Chain (2014NZ0030-1, Sichuan, China). We thank Christina Rosenberger for
a careful reading of this manuscript.
Conceived and designed the experiments: RPZ JWW HHL. Performed the experiments: RPZ
JYL JWH XPY DMCW LL. Analyzed the data: RPZ HHL. Contributed reagents/materials/
analysis tools: RPZ JYL JWH XPY DMCW LL. Wrote the paper: RPZ JWW HHL.
15 / 18
16 / 18
17 / 18
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