Sequencing And Characterization Of The Mirnaome And Transcriptome Of Longissimus Dorsi Muscle Between Laiwu And Yorkshire Pigs

The domestic pig is not only an important agricultural animal used as a source of meatworldwide, but also considered as a suitable model organism for biomedical research. Due tothe difference of genteic background and artificial selection between the Chinese indigenouspigs and foreigin commercial pig breeds, there have different intrinsic features. For instance,Laiwu pig is a typical Chinese indigenous pig, which has good meat quality; however, thewestern pig breeds have the faster grow performance than Chinese native pig breeds. It hasbeen proved that the differentially expressed genes are associated with the difference betweenChinese indigenous pig and western pig. Therefore, it is important to study the genome-widedifferentially expressed genes of skeletal muscle between Chinese native pig and western pigbreeds, and to investigate the molecular mechanism of the difference.This experiment herein is based on the contrast of porcine skeletal muscles between theslow-growing Laiwu pig (LW) breed with better meat quality and fast-growing Yorkshire pig(YS) breed with poor meat quality. The RNA-seq technology was used to identify thedifferentially expressed miRNAs and genes of the skeletal muscle, longissimus dorsi muscle,for LW and YS pig breeds, and the identified differentially expressed miRNAs and geneswere enriched to the GO terms and KEGG pathways, which can analyze the functions of thedifferentially expressed genes. We also predicted the target genes of the differentiallyexpressed miRNAs, and constructed the regulated network between miRNAs and target genes.The miRNAs and genes expression results of the RNA-seq data were validated by the qRT-PCR, and the result shows the RNA-seq was more reliable. The results of our study arefollowing:(1) Small RNA sequencing of longissimus dorsi muscle between LW and YS pig breedsOn average, we obtained approximately13,493,607clean reads in Laiwu pig and12,938,257clean reads in Yorkshire pig in this study. Totally,265known miRNAs wereidentified, and among these,229miRNAs were expressed in all the six pigs. In addition,94novel mature miRNAs and95novel hairpins were detected in the muscle libraries. From the265known miRNAs and95novel miRNAs,25of these miRNAs were found significantlydifferentially expressed (q <0.05). Of these significantly expressed miRNAs,10miRNAs were significantly up-regulated and15miRNAs were down-regulated in Laiwu pig comparedwith Yorkshire pig; and ssc-miR-1was the most abundant miRNA in both two pig breeds.Among the significantly expressed miRNAs, ssc-miR-194b-5p has the largest fold-change(6.35-fold). Sixteen differentially expressed miRNAs selected from high throughputsequencing were confirmed by real-time RT-PCR, and the results indicated that theexpression patterns were consistent with sequencing results. Further analysis of gene ontologyand KEGG pathway showed that249pathways were enriched which involved4,972targetgenes.(2) Transcriptome analysis of longissimus dorsi muscle between LW and YS pig breedsRNA-seq was applied to sample transcripts of the longissimus dorsi muscle between thetwo pig breeds to gain insights into whole-genome transcription profiles in this study. Theresulting data set provided expression patterns for many annotated, predicted and novel piggenes. We found that alternative5’ first exon was the commonest type of alternative splicingevent in this study. Moreover,178significantly differentially expressed genes (q <0.05) wereidentified between the Laiwu and Yorkshire pigs, with98up-regulated and80down-regulated genes in Laiwu pig compared with Yorkshire pig. Gene expression results of theRNA-seq data were validated by qRT-PCR for twelve genes, revealing a strong correlationbetween the qRT-PCR and RNA-seq data (R2=0.93). In addition, pathway analysis revealedthat the differentially expressed genes played roles in metabolic processes, signalingpathways, and biological functions.(3) Correlation analysis of differentially expressed miRNAs and genesIn order to investigate the functions of the differentially expressed miRNAs and genes,we analyzed the correlations of the miRNAs and genes. The results show that10miRNAwere significantly expressed between LW and YS pig breeds (q <0.05). There were4up-regulated miRNAs and6down-regulated miRNAs. The correlation results suggested that the6down-regulated miRNAs could regulate20up-regulated differentially expressed genes, andthe4up-regulated miRNAs also could regulate20down-regulated differentially expressedgenes.In conclusion, the present study provides a comprehensive view of differences in themuscle miRNAome and transcriptome between two pig phenotypes. These results not onlycontribute to the understanding of the porcine miRNA transcriptome profiles andtranscriptome but also provide a valuable resource for future pig breeding research.