Identification And Differential Analysis Of MRNA Transcriptome During Goat Skeletal Muscle Development

Skeletal muscle growth and development of goat is regulated by various regulatory factors.To explore the key regulatory genes and regulate network on skeletal muscle development may help us to better understand the molecular mechanism of skeletal muscle development and is also benefit for the meat-type goat breeding.Here in this study,skeletal muscle tissue（Longzissimus dorsi muscle）samples were collected at these four developmental stages:three gestation stages（E45,E60,and E105）and one postnatal stage（B3）.By using the RNA sequencing（RNA-seq）approach,a large of novel mRNA transcripts,SNVs and INDELs were identified,and the differential expressed（DE）genes between the different developmental stages of goat skeletal muscle were also identified and annotated.At last,we further used Q-PCR to verify the mRNA expression from RNA-seq.The main results as follows:1.We performed largescale cDNA sequencing experiments in eleven libraries using the Illumina paired-end RNA-seq approach,resulting in a total of～131 Gb（gigabases）paired-end reads of the 125 nucleotide（nt）length.An average of 11.97 ± 1.87 Gb of raw reads was obtained per library.This yielded 131.62 Gb of sequence,and about 98.20%of the reads passed initial quality thresholds（Illumila protocol）and were deemed as clean reads for the subsequent analyses.Approximately 78.99-87.73%of the clean reads in all the libraries were mapped to the goat reference genome（NCBI,CHIR₁.0）by using TopHat2 software,and about 78.52%-86.60%of the clean reads have a unique genomic location.2.Using Cufflinks assembler,we obtained an average of 51,053（48,398-53266）transcripts for each sequencing library.On average,which covered 80.41%（75.88%-85.10%）of goat Refseq transcripts,and the transcripts with the coverage of 90%or more in each library account for 51.44%of the total transcripts.3.In all eleven sequencing libraries,we identified 21,658 reliable mRNA transcripts,including 6,200 known and 15,458 novel mRNA transripts.4.Based on transcriptome level and alignment results between each library and goat reference genome,we detected 1,004,499 SNVs and 81,787 INDELs in all sequencing libraries from different developmental stages of goat skeletal muscle.5.Differential expression analysis showed that 7,053 mRNAs（3,126 genes）that were differentially expressed in pairwise comparisons between goat skeletal muscle at the different development stages,including 3,273 known and 3,780 novel mRNA transripts.We did not detect any stage-specific expressed mRNAs among the four developmental stages,but 1,730 differentially expressed mRNAs that were detected in all four developmental stages.6.Analysis of functional enrichment indicated that differentially expressed mRNAs were annotated to 139 GO terms that were significantly enriched（P<0.05）,including 102 Biological Process GO terms,Molecular Function GO terms and Celluar Component GO terms,some of the terms were muscle development-related terms.Analysis of KEGG enrichment showed that differentially expressed mRNAs were enriched in 286 KEGG pathways,and some of the pathways were muscle development process-related signal pathways.7.The mRNA expression results from RNA-seq were verified by Q-PCR,we found the two quantitative results showed the same expression trend in different developmental stages,and have a good correlation（r = 0.96～0.99）with each other,indicating that the RNA-seq results are accurate and reliable,which can accurately reflect the status of transcriptome at different developmental stages.