Identification And Bioinformatics Analysis Of MiRNAs In Beef Cattle Intermuscular Fat

The intermuscular fat deposition and distribution in beef cattle,usually being categerized into different degree of marbling,is an important index for evaluation of high-grade beef.Micro RNAs(mi RNAs)are a group of small noncoding RNAs,playing regulatory roles in many aspects of development in animals and plants by post-transcriptional regulation of target genes,thereby affecting the production performance of animals and plants.Currently,although identification and functional analysis of mi RNAs closely related to intermuscular fat development have been well studied,researches on differences of mi RNAs among major beef cattle breeds or differentially expressed mi RNAs attributed to different genders are still scarce.Therefore,screen and identification of mi RNAs potentially regulating intermuscular fat deposition will better our understanding of molecular mechanisms underlying intermuscular fat deposition regulated by mi RNAs,and will provide molecular basis for breeding and utilization of excellent beef cattle.To explore differentially expressed mi RNAs and their potential roles in fat deposition in beef cattle of different breeds or genders,and lay foundation for clarification of such mi RNAs’ role in the regulation of bovine intermuscular fat deposition,the present study was conducted to screen and identify mi RNAs potentially involving in the regulation of fat deposition in beef cattle.The research mainly includes two parts.The first part was to screen and identify mi RNAs of different beef cattle breeds.Six-month-old bull calves of Angus,Simmental and Wagyu,with 3 of each breeds,were respectively selected,raised at the same nutrition level and management conditions,and fattened to 24 months of age,the small RNAs extracted from intramuscular fat in aformentioned three breeds were sequenced using high-throughput sequencing methods.(1)the distribution and sequence characteristics of small RNAs in intramuscular fat of three breeds were analyzed;(2)the transcriptomic characteristics of mi RNAs in intramuscular fat of three breeds were analyzed,and the expression profile and genomic information of such mi RNAs of three breeds were then obtained,which represents a reference for subsequent functional analysis;(3)By pairwise comparison,we revealed differences of mi RNAs expressed in intermuscular fat among three cattle breeds,and quantitative real-time PCR also validated sequencing results of five randomly selected mi RNAs.The second part was to screen and identify differentially expressed mi RNAs involving in intermuscular fat deposition in beef cattle of different genders.One bull calf and five heifers of F1 crossbred Wagyu-Wannan cattle were selected at the same age and subjected to slaughter at 24 months of age,and mi RNAs in intramuscular fat were then arrayed with Affymetrix micro RNA 3.0 gene chip.Differential expression of mi RNAs in intermuscular fat from bull calves and heifers was determined.The results from gene chip were verified by quantitative real-time PCR,and the target gene of differentially expressed mi RNAs were also predicted.The detail results and conclusions are as follows:(1)The mi RNAs libraries of intramuscular fat tissue from Angus,Simmental and Wagyu beef bulls were successfully constructed.In three libraries,we obtained 11873367,11588623 and 11843883 clean reads,which accounted for 99.41%,97.04% and 99.19% of total libraries,respectively.The small RNAs fragments in three libraries were dominantly distributed in 20~24nt,and the fragments of 22 nt length accounted for 49.17%,45.47% and 50.41% of the fragments of all length,respectively.(2)There are 9135368 total clean reads that can be matched to the bovine genome of Angus cattle,accounting for 76.94% of the total,and only 128405 unique reads which can be matched to the bovine genome,accounting for 45.41% of the total unique reads;There are 9168676 total clean reads that can be matched to the bovine genome of Simmental cattle,accounting for 79.12% of the total,and only 112138 unique reads which can be matched to the bovine genome,accounting for 30.27% of the total;There are 9385045 of total clean reads which can be matched to the bovine genome of Wagyu cattle,accounting for 79.24% of the total,and only 85957 unique reads that can be matched to the bovine genome,accounting for total 34.48% of the total unique reads.(3)In total clean reads,mi RNAs represented 70.63%,62.98%,and 71.22% of three small RNAs libraries,respectively.(4)Comparison of common and specific sequence of total clean reads and unique reads in three libraries was acomplished by using pairwise comparison method.As for clean reads,Angus shared 97.46% in common with Simmental,while had 98.11% in common with Wagyu,and Simmental shared 97.77 % in common with Wagyu.As for unique reads,Angus engaged 17.82% in common with Wagyu,while Angus and Simmental only shared 15.49 % common unique sequence.(5)In the intramuscular fat libraries of Angus,Simmental and Wagyu,we detected 347,347 and 319 mi RNAs,respectively;55,48 and 53 mi RNA-5p,respectively;51,50 and 48 mi RNA-3p,respectively.Using Mireap software,we predicted 55 potential mi RNAs in Angus library,30 potential mi RNAs in Simmental library and 43 potential mi RNAs in Wagyu library.(6)In mi RNAs libraries of intramuscular fat in Angus and Simmental breeds,we found 129 differentially expressed mi RNAs,of which 49 up-regulated and 80 downregulated;In mi RNAs libraries of intramuscular fat in Angus and Wagyu,we observed 103 differentially expressed mi RNAs,of which 24 up-regulated and 79 down-regulated;In mi RNAs libraries of intramuscular fat in Simmental and Wagyu,we revealed 55 differentially expressed mi RNAs,of which 12 up-regulated and 43 down-regulated;(7)bta-mi R-1281 and bta-mi R-148 b were differentially expressed mi RNAs involving in fat deposition in F1 Wagyu-Wannan bull calves and heifers.(8)Using Target Scan software and Pic Tar software,189 possible common target genes of mi R-148 b were predicted.(9)Of note,SOX5 gene was predicted as one potential candidate target gene of mi R-148 b,shown already that it is an important regulation gene in animal fat deposition.Therefore,if mi R-148 b regulates intermuscular fat deposition of beef cattle through SOX5 requires further study.