Comparison of characteristics of long noncoding RNA in Hanwoo according to sex

Article information

Asian-Australas J Anim Sci.. 2020;33(5):696-703
Publication date (electronic) : 2019 October 22
doi : https://doi.org/10.5713/ajas.18.0533
1Subtropical Livestock Research Institute, National Institute of Animal Science, RDA, Jeju 63242, Korea
2Department of Animal Biotechnology, College of Agricultural and Life Sciences, Chonbuk National University, Jeonju 54896, Korea
3The Animal Molecular Genetics & Breeding Center, Chonbuk National University, Jeonju, 54896, Korea
a

These two authors contribute equally to this work.

*Corresponding Authors: Donghyun Shin, Tel: +82-63-270-5931, Fax: +82-63-270-5937, E-mail: sdh1214@gmail.com. Jae-Don Oh, Tel: +82-63-270-5931, Fax: +82-63-270-5937, E-mail: oh5ow@naver.com
Received 2018 July 16; Revised 2018 September 19; Accepted 2019 October 14.

Abstract

Objective

Cattle were some of the first animals domesticated by humans for the production of milk, meat, etc. Long noncoding RNA (lncRNA) is defined as longer than 200 bp in non-protein coding transcripts. lncRNA is known to function in regulating gene expression and is currently being studied in a variety of livestock including cattle. The purpose of this study is to analyze the characteristics of lncRNA according to sex in Hanwoo cattle.

Methods

This study was conducted using the skeletal muscles of 9 Hanwoo cattle include bulls, steers and cows. RNA was extracted from skeletal muscle of Hanwoo. Sequencing was conducted using Illumina HiSeq2000 and mapped to the Bovine Taurus genome. The expression levels of lncRNAs were measured by DEGseq and quantitative trait loci (QTL) data base was used to identify QTLs associated with lncRNA. The python script was used to match the nearby genes

Results

In this study, the expression patterns of transcripts of bulls, steers and cows were identified. And we identified significantly differentially expressed lncRNAs in bulls, steers and cows. In addition, characteristics of lncRNA which express differentially in muscles according to the sex of Hanwoo were identified. As a result, we found differentially expressed lncRNAs according to sex were related to shear force and body weight.

Conclusion

This study was classified and characterized lncRNA which differentially expressed by sex in Hanwoo cattle. We believe that the characterization of lncRNA by sex of Hanwoo will be helpful for future studies of the physiological mechanisms of Hanwoo cattle.

INTRODUCTION

Cattle were some of the first animals to be domesticated by humans for agricultural purposes. About 10,000 years ago, the ancestors of cattle were tamed for the purpose of providing milk, meat, and leather to humans [1]. In 2006, a human genome sequencing center announced the sequence of the cattle genome [2]. Utilizing the genome-wide single nucleotide polymorphism panel for cattle allows for the use of quantitative trait loci (QTL) and prediction of the genetic merit of an animal without using phenotype and pedigree records [3]. Genetic studies using livestock have shown that most of the genetic variants associated with complex phenotypic traits are located outside the protein coding region [4].

Analysis of intracellular transcripts has revealed that 50% of the transcribed genomes were not aligned with known protein coding regions. Many of these have been proven to have protein coding potential [5]. Non-protein coding transcripts can potentially be noncoding RNA (ncRNA) [6]. Long noncoding RNA (lncRNA) is a molecule longer than 200 bp in a non-protein-coding transcript, or one longer than 2 kb with a coding potential of an amino acid sequence less than 100 bp [7]. LncRNA is a relatively new class of RNA molecules that are less well-characterized than microRNA (miRNA). It has been characterized only by some functional lncRNA and has been shown to regulate all levels of the gene-regulated expression pathway [8]. Previous studies have confirmed that lncRNA plays an important role in a variety of key biological processes, including translational control, RNA splicing, and chromatin modification [9]. In addition, functional studies of lncRNAs have shown that they play an important role in basic biological processes such as dose compensation, transcriptional regulation, and epigenetic regulation [1012]. The number of studies on lncRNA has continued to increase and many databases have been constructed that include lncRNA data for domesticated animals [13]. At the time of the announcement, 12,103 pig lncRNAs, 8,923 chicken lncRNAs, and 8,250 cattle lncRNAs were included in the ALDB (domestic-animal lncRNA database) database [14].

This study was conducted to compare the expression of lncRNA in bulls, steers and cows as well as the characteristics of each sex. Samples from muscles tissues were used for each sex, and the expression of lncRNA by tissue was analyzed according to sex. We also analyzed QTL associated with lncRNA, which is significantly expressed.

MATERIALS AND METHODS

Sample preparation and RNA-seq snalysis

All analysis was conducted with data reprocessed from the law data from a previous study [15]. The animals and sample preparation were as follows: A total of nine (three bulls, three steers and three cows) Hanwoo cattle (Bos taurus coreanae) were used in this study. They were fed the same diet and managed at the same location, Hanwoo Experimental Station in National Institute of Animal Science, throughout the experiment. The average (±standard deviation) carcass weight of the cattle at slaughter was 430.2 (±40.66) kg. Immediately after slaughter, muscle tissues were separated and sampled. All of the tissue samples were immediately frozen using liquid nitrogen and stored at −80°C until analysis. All animal use, care, and experimental protocols for this experiment were reviewed and preapproved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (number 2010-042). Total RNAs of tissues were isolated using TRIzol (Invitrogen, Karlsruhe, Germany) and an RNeasy RNA purification kit with DNase treatment (Qiagen, Hilden, Germany). The messenger RNA (mRNA) was isolated from the total RNA using oligo-dT beads and was reverse transcribed into double-stranded cDNA fragments. Constructing and sequencing of the RNA-seq library for each sample was carried out based on the protocols of Illumina HiSeq2000 in order to generate 101 pair-end reads. The qualities of the RNA-seq reads from all of the tissues were checked using FastQC.

Analysis of lncRNA discovery

Filtering was conducted in order to remove the low-quality sequences. The filtered sequences were mapped to Bovine Taurus genome (bosTau6) using STAR v2.4.0b [16]. Expression levels were calculated using Cufflinks v2.2.1. Bovine gene information was used to measure expression levels [17]. Multi read correction and frag bias correct options were additionally used to increase the accuracy of expression measurement, and other options were used as defaults. The bovine lncRNA analysis was used to conduct mapping with reference to annotated bos taurus ensemble ID results. We refer to the lncRNA list of cattle identified in the study of Koufariotis et al [18]. We matched the ensemble ID of the transcripts we found and identified lncRNA in the muscle of Hanwoo (ensembl.org).

Statistical analysis

Differences in the expression level of each tissue were expressed by Heatmap using R package gplots (v3.0.1). LncRNA principal component analysis (PCA) plots were used to identify the differences between bulls, steers and cows muscle tissues using Mev (http://mev.tm4.org/). The DEGseq R package was used to identify differential expression of lncRNA between the bulls, steers and cows muscle tissues. Significant lncRNAs were identified using cut-off of: |fold difference| ≥1 and p-value ≤0.05. Sex-specific differentially expressed lncRNAs statistics analysis were using Prism 5 program (San Diego, CA, USA). The Venn diagram was used to identify the lncRNA assemblages that were extracted between the bulls, steers and cows. The Venn diagram was displayed using InteractiVenn (http://www.interactivenn.net/).

Quantitative trait locus analysis

QTL regions for comparative analysis with lncRNAs were identified from the cattle QTL Database (http://www.animalgenome.org/cgi-bin/QTLdb/BT/index). In these cattle, the QTL associated with the quality and productivity of the meat was selected. The position of the selected QTL was compared with the lncRNA expressed in sex.

Nearby gene analysis

The location of lncRNA was identified by ensembl biomart (ensembl.org/biomart) using transcript ID. The databases used included ensemble genes 92 cow genes (UMD3.1). We found for by nearby gene predicted to be affected by lncRNA. The position information of lncRNA in the genome and the gene position information of the bovine were matched using Python script. Transcription directions of lncRNA and nearby genes were used transcript information by ensembl (ensembl.org).

RESULTS AND DISCUSSION

Information of RNA sequencing

All analyses were conducted by reprocessing data from previous studies (Lee et al [15]). The RNA-seq raw data information is as follows: 34.2 Mb of raw readings were averaged on the muscle. More than 99.5% readings were retained after being filtered by quality control, and over 95.9% of these were mapped to the reference genome. The average length of the lncRNA was 866 bp, the minimum length was 257 bp, and the maximum length was 1,911 bp.

Expression patterns of lncRNA in Hanwoo cattle by sex

A hierarchical clustering analysis of the transcripts of sex transcripts in muscle tissue was performed in Hanwoo cattle. Clustering analysis of steer expressions did not show any significant differences (Figure 1). Clustering analysis using PCA analysis showed that the distances between the three groups differed by sex. The distance between the bull and the steer was relatively close to the distance to the cow (Figure 2). An analysis of the differential expression of lncRNA in each sex was conducted and found seven lncRNAs in bulls, nine lncRNAs in steers, and five lncRNAs in cows (Table 1, Figure 3A). The results of Venn diagram analysis for the found lncRNA revealed that 5 lncRNAs were found commonly in bulls and steers. We found one common lncRNA that was differentially expressed in bulls and cows. It was also identified that there was no overlapped lncRNA in the steers and cows (Figure 3B).

Figure 1

Comparison of sex transcripts expression in Hanwoo muscles. Expression of transcripts by muscle tissue of bull, steer and cow were identified. Expression of each sex was expressed as z-score, with red as negative and blue as positive.

Figure 2

lncRNA principal component analysis plot of bulls, steers and cows in muscle tissues. Clustering analysis was conducted to determine the differences between the three sex. The distance between bull and steer is relatively close compared to cow. Red color is bull, blue color is steer and green color is cow.

List of differentially expressed lncRNA of Hanwoo cattle in muscle

Figure 3

Number of lncRNA found between each sex. (A) Number of lncRNAs by sex in muscle. (B) Venn diagram showing the co-expression of lncRNAs in bulls, steers and cows. Many of the lncRNAs from Bull and Steer were found to overlap. Red color is bull, blue color is steer and green color is cow.

LncRNA-related bovine QTL analysis

Functional traits are defined as characteristics of animals that increase efficiency by reducing the cost of breeding [19]. Livestock species have long been selected to improve their characteristics that are associated with economic benefits. These traits are generally affected by genetic and environmental factors [20]. The detection of genetic markers closely linked to loci affecting quantitative traits (QTL) will provide a tool for the application of marker-assisted selection and is a prerequisite for the detailed molecular genetic analysis of traits [21]. Previous studies have identified 7188 lncRNAs from cows and have identified 2753 loci on chromosomal regions of livestock QTL related to muscle development. The function of QTL was also related to intramuscular fat, lean meat yield, longissimus muscle area, shear force [22].

In this study, we conducted QTL analysis according to sex in Hanwoo muscle. QTL loci related to meat quality and productivity of cattle were selected with reference to a cattle QTL database. We identified 33 QTLs with overlapping loci with lncRNAs. Ten QTLs were identified in the bulls, 16 QTLs were identified in the steers, and seven QTLs were identified in the cows. In all sexes, lncRNA was found to be mainly associated with shear force QTL (Figure 4, Table 2).

Figure 4

Number of quantitative trait loci (QTLs) associated with Hanwoo cattle economic traits by sex in muscle. (A) Number of QTL loci associated with lncRNA by each sex. (B) QTL locus associated with lncRNA found in bull. (C) QTL locus associated with lncRNA found in steer. (D) QTL locus associated with lncRNA found in cow. Compared to other sex, many QTL loci were identified in steer. All three sex were found to be associated with shear force. Red color is bull, blue color is steer and green color is cow.

List of QTLs associated with Hanwoo cattle economic traits

LncRNA related genes identified

Most lncRNAs have lower conservation and expression levels than mRNA [23]. It has been reported that lncRNA can regulate the expression of adjacent protein coding genes through transcriptional activation and inhibition [24,25]. LncRNAs can be targeted by miRNAs and it has been reported that they can regulate the expression of mRNA [26,27]. The miRNAs consist of a length of about 22 nucleotides and function like silencing RNA and regulating gene expression after transcription [28,29]. In this study, three mRNAs were found to overlap with three lncRNAs. In addition, we identified and classified the transcriptional direction of lncRNA and mRNA (Table 3).

Information on lncRNAs with nearby genes

CONCLUSION

This study profiled lncRNA, which differentially expresses the difference of sex in Hanwoo cattle. We identified the expression patterns of transcripts in each sex by comparing the bull, steer and cow strains and identified 21 lncRNAs. We identified lncRNA related to meat quality and productivity according to sex in Hanwoo through QTL analysis. Many lncRNAs were found to overlap with QTL loci associated with shear force and body weight. We also identified adjacent genes that overlap with the three lncRNAs. It will be helpful to study the genetic characteristics of Hanwoo in the future.

Supplementary Data

Notes

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

This This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A2C1089807) and the Next-Generation BioGreen 21 Program (PJ01315101, PJ01321901), Rural Development Administration, Republic of Korea.

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Article information Continued

Figure 1

Comparison of sex transcripts expression in Hanwoo muscles. Expression of transcripts by muscle tissue of bull, steer and cow were identified. Expression of each sex was expressed as z-score, with red as negative and blue as positive.

Figure 2

lncRNA principal component analysis plot of bulls, steers and cows in muscle tissues. Clustering analysis was conducted to determine the differences between the three sex. The distance between bull and steer is relatively close compared to cow. Red color is bull, blue color is steer and green color is cow.

Figure 3

Number of lncRNA found between each sex. (A) Number of lncRNAs by sex in muscle. (B) Venn diagram showing the co-expression of lncRNAs in bulls, steers and cows. Many of the lncRNAs from Bull and Steer were found to overlap. Red color is bull, blue color is steer and green color is cow.

Figure 4

Number of quantitative trait loci (QTLs) associated with Hanwoo cattle economic traits by sex in muscle. (A) Number of QTL loci associated with lncRNA by each sex. (B) QTL locus associated with lncRNA found in bull. (C) QTL locus associated with lncRNA found in steer. (D) QTL locus associated with lncRNA found in cow. Compared to other sex, many QTL loci were identified in steer. All three sex were found to be associated with shear force. Red color is bull, blue color is steer and green color is cow.

Table 1

List of differentially expressed lncRNA of Hanwoo cattle in muscle

Sex Transcript ID Loci Length log2FC p-value
Bull ENSBTAT00000064621 14:20738814-20740407 1,594 −2.27 1.2E-33
ENSBTAT00000003304 6:62506125-62506524 400 −1.63 3.4E-02
ENSBTAT00000065672 23:34219298-34219825 528 −1.58 1.7E-44
ENSBTAT00000009243 5:104077163-104077952 790 −1.20 1.2E-02
ENSBTAT00000017165 8:31871390-31872132 743 −1.15 3.4E-04
ENSBTAT00000033843 3:19259499-19260488 990 −1.13 1.4E-05
ENSBTAT00000005333 6:71053202-71053458 257 1.41 1.7E-02
Steer ENSBTAT00000064288 3:27428793-27430601 1,809 −1.04 4.6E-03
ENSBTAT00000048956 8:11064112-11064777 666 −1.00 7.0E-06
ENSBTAT00000064497 26:14093095-14093609 515 1.04 2.1E-41
ENSBTAT00000009243 5:104077163-104077952 790 1.06 1.6E-02
ENSBTAT00000017165 8:31871390-31872132 743 1.26 2.2E-05
ENSBTAT00000064839 15:74915626-74916613 988 1.28 1.1E-03
ENSBTAT00000033843 3:19259499-19260488 990 1.36 1.7E-08
ENSBTAT00000065672 23:34219298-34219825 528 1.46 2.6E-48
ENSBTAT00000064621 14:20738814-20740407 1,594 1.75 1.8E-29
Cow ENSBTAT00000005333 6:71053202-71053458 257 −2.09 7.0E-03
ENSBTAT00000010270 10:76992202-76994112 1,911 −1.48 7.9E-06
ENSBTAT00000045699 1:84324177-84325181 1,005 −1.02 5.0E-12
ENSBTAT00000044622 29:40214778-40215227 450 −1.07 3.2E-57
ENSBTAT00000027478 4:58724265-58724900 636 1.19 1.3E-11

Table 2

List of QTLs associated with Hanwoo cattle economic traits

QTL ID Chr Peak Reference lncRNA Transcript ID
Bull
 Age at first calving 140097 14 - Mota RR ENSBTAT00000064621
 Body weight (weaning) 24749 3 20.34 Mahdi Saatchi ENSBTAT00000033843
24711 3 28.72 ENSBTAT00000033843
 Longissimus muscle area 126448 8 - de Oliveira Silva RM ENSBTAT00000017165
 Metabolic body weight 140487 14 - Hardie LC ENSBTAT00000064621
 Residual feed intake 56461 14 31.48 Saatchi M ENSBTAT00000064621
 Shear force 20764 6 71.21 McClure MC ENSBTAT00000005333
ENSBTAT00000003304
 Subcutaneous fat 20703 14 5.62 Veneroni-Gouveia G ENSBTAT00000064621
 Udder swelling score 106718 6 78.95 Michenet A ENSBTAT00000005333
Steer
 Age at first calving 140097 14 - Mota RR ENSBTAT00000064621
 Average daily gain 22798 15 97.09 Peters SO ENSBTAT00000064839
 Body weight (weaning) 24749 3 20.34 Mahdi Saatchi ENSBTAT00000033843
24711 3 28.72 ENSBTAT00000033843
ENSBTAT00000064288
 Calving ease 106459 26 20.05 Michenet A ENSBTAT00000064497
 Docosapentaenoic acid content 31772 3 29.66 Cesar AS ENSBTAT00000064288
 Longissimus muscle area 126448 8 - de Oliveira Silva RM ENSBTAT00000017165
126451 15 - ENSBTAT00000064839
 Metabolic body weight 140487 14 - Hardie LC ENSBTAT00000064621
 Omega-3 unsaturated fatty acid content 31782 3 29.66 Cesar AS ENSBTAT00000064288
 Residual feed intake 56461 14 31.48 Saatchi M ENSBTAT00000064621
 Shear force 20822 26 22.45 McClure MC ENSBTAT00000064497
20824 26 40.66 ENSBTAT00000064497
20823 26 31.95 ENSBTAT00000064497
20703 14 5.62 Veneroni-Gouveia G ENSBTAT00000064621
Cow
 Body weight (yearling) 22770 1 109.62 Peters SO ENSBTAT00000045699
 Margaric acid content 19759 29 46.64 Saatchi M ENSBTAT00000044622
 Shear force 20833 29 56.05 McClure MC ENSBTAT00000044622
20764 6 71.21 ENSBTAT00000005333
 Trans-6/9-C18:1 fatty acid content 20504 1 80.99 Saatchi M ENSBTAT00000045699
 Udder swelling score 106760 29 54.42 Michenet A ENSBTAT00000044622
106718 6 78.95 ENSBTAT00000005333

QTLs, quantitative trait loci.

Table 3

Information on lncRNAs with nearby genes

LncRNA transcript ID Gene ID Gene symbol Loci LncRNA strand Gene strand
ENSBTAT00000033843 ENSBTAG00000017566 TUFT1 3:19,238,265–19,289,315 Forward Reverse
ENSBTAT00000045699 ENSBTAG00000010394 MCF2L2 1:84,324,970–84,525,526 Reverse Forward
ENSBTAT00000064288 ENSBTAG00000000664 SLC22A15 3:27,378,643–27,481,821 Reverse Reverse

Three lncRNAs were identified to overlap with other genes in position. The transcription directions of the nearby genes and lncRNA were compared.

TUFT1, tuftelin 1; MCF2L2, MCF.2 cell line derived transforming sequence-like 2; SLC22A15, solute carrier family 22 member 15.