Transcriptome analysis of genetic mechanism of growth curve inflection point using a pig model

Animal growth curves play an important role for animal breeders to optimize feeding and management strategies (De Lange et al., 2001 [1]; Brossard et al., 2009 [2]; Strathe et al., 2010 [3]). However, the genetic mechanism of the phenotypic difference between the inflection point and noninflection points of the growth curve remains unclear. Here, we report the differentially expressed gene pattern in pig longissimus dorsi among three typical time points of the growth curve, inflection point (IP), before inflection point (BIP) and after inflection point (AIP). The whole genome RNA-seq data was deposited at GenBank under the accession number PRJNA2284587. The RNA-seq libraries generated 117 million reads of 5.89 gigabases in length. Totals of 21,331, 20,996 and 20,139 expressed transcripts were identified in IP, UIP and AIP, respectively. Furthermore, we identified 757 differentially expressed genes (DEGs) between IP and UIP, and 271 DEGs between AIP and IP. Function enrichment analysis of DEGs found that the highly expressed genes in IP were mainly enriched in energy metabolism, global transcriptional activity and bone development intensity. This study contributes to reveal the genetic mechanism of growth curve inflection point.


Direct link to deposited data
The RNA-seq raw data has been uploaded in GEO database under the accession number GSE69113 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE69113).

Experimental design, materials and methods
One Chinese native mountain-type pig breed, Liangshan pig, was taken as the animal model in the study (Shen et al., 2014 [4]). A total of 275 female Liangshan pigs were raised from birth to 250 days old to collect the growth traits (feed conversion rate, daily feed intake and average daily gain). The growth curve was fitted by three different nonlinear models. The inflection point analysis of the growth curve suggested that the Liangshan pig reached the maximum growth rate at day 193.40. Therefore, we selected other two symmetric non-inflection points (143 days for BIP and 243 days for AIP) to explore the transcriptome diversity of muscle development. The longissimus dorsi muscle was harvested from 9 Liangshan pigs (3 pigs for each time point), and used for transcriptome analysis.
For RNA-Seq library preparation, total RNA was extracted from longissimus dorsi using TRIzol (Invitrogen, CA, USA) and further purified with RNeasy column (Qiagen, USA) according to the manufacturer's protocol. The total RNA was isolated poly (A) mRNA by poly-T oligo attached magnetic beads (Thermo-Fisher). Following purification, the mRNA was fragmented into small pieces using divalent cations under an elevated temperature. Then the cleaved RNA fragments were constructed into the final cDNA library in accordance with the protocol for the Illumina RNA ligation based method (Illumina, San Diego, USA). A reverse transcription followed by PCR was used to create cDNA constructs. The average insert size for the single-end libraries was 300 bp (± 50 bp). Then the single end sequencing (50 bp) was

Genomics Data j o u r n a l h o m e p a g e : h t t p : / / w w w . j o u r n a l s . e l s e v i e r . c o m / g e n o m i c s -d a t a /
performed on an Illumina Hiseq2000 platform. For data analysis, the raw data containing adaptor sequences, reads with low quality sequences and unknown nucleotides N were filtered to obtain clean reads with 50 nt in length. Clean reads were then conducted for quality assessment (data shown in Table 1). These include the classification of total and distinct reads and show their percentage in the library, analyze saturation of the library and correlation analysis of biological replicates. All clean reads were mapped to the transcript sequence by bowtie (1.0.0); only 1 bp mismatch was allowed. For monitoring the mapping events on both strands, both the sense and complementary antisense sequences were included in the data collection (data shown in Table 2). The number of perfect clean reads corresponding to each gene was calculated and normalized to the number of Reads Per Kilobase of an exon model per Million mapped reads (RPKM). Based on the expression levels, the significant DEGs (differentially expressed genes) among different samples were identified with p-value ≤ 0.05 and log 2 fold-change|log 2 FC| ≥ 1. Raw and normalized data were accessible on public database: An enrichment analysis of DEGs found the immune system related genes were in the BIP stage. The energy metabolism rate, global transcriptional activity and bone development intensity were highly expressed in the inflection point period. Superior meat quality was developed in the AIP stage. The raw and normalized data of our study were accessible on public database: GEO submission number GSE69113.

Conflicts of interest
The authors declare no conflicts of interest.