RNA-seq based SNP discovery in gluteus medius muscle of Polish Landrace pigs

Abstract

Background: Single nucleotide polymorphisms (SNPs) are the well-known molecular markers in genetics and breeding studies applied to veterinary sciences and livestock production. Advancement of next generation sequencing (NGS) provides a high-throughput means of potential putative SNP discovery. The aim of the study was to identify the putative genetic variants in gluteus medius muscle transcriptome of Polish Landrace pigs.Methods. RNA-seq based NGS experiment was performed on Polish Landrace pigs fed with omega-6 and omega-3 polyunsaturated fatty acids (and normal diets. Isolation of total RNA from gluteus medius muscle was performed PUFAs dietary of Polish Landrace pigs. The RNA-seq libraries were constructed by mRNA enrichment, mRNA fragmentation, second strand cDNA synthesis, adaptor ligation, size selection and PCR amplification using the illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego CA, USA), followed by NGS sequencing on MiSeq illumina platform. The quality control of raw RNA-seq data was performed using the Trimmomatic and FastQC tools. High QC pairedend RNA-seq data of gluteus medius muscle transcriptome were mapped to the reference genome Sus scrofa v.10.2. Finally, the SNPs discovery was performed using GATK and SAMtools bioinformatics SNPs caller tools. Results:The Fastq RNA-seq data generated from two pooled paired-end libraries (151bp) of gluteus medius muscle tissue of Polish Landrace pigs were submitted to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra). Study identified a total of 50.5 million pairedend reads (32.5 million low PUFAs dietary group and 18 million reads high PUFAs dietary group) of gluteus medius muscle transcriptome of Polish Landrace pigs. SNP discovery identified a total of 35436 homozygous and 28644 heterozygous cSNPs in gluteus medius muscle transcriptomes representing both dietary groups of Polish Landrace pig. Moreover, a total of 25187 and 5488 cSNP were identified as synonymous SNPs, and 18005 and 4780 cSNP were identified as nonsynonymous SNPs. Finally, single nucleotide variation (SNV) representing substitutions of all four possibilities (A,T,G,C) were identified ranging 2935 to 3227 SNVs (high PUFAs) and 3528 to 3882 SNVs (low PUFAs) for the heterozygous cSNPs and 2712 to 4058 (high PUFAs) and 4169 to 5692 SNVs (low PUFAs) for the heterozygous SNPs in gluteus medius muscle transcriptomes of Polish Landrace pigs. Conclusions.Study concluded that identification of cSNPs dataset representing the gluteus medius muscle transcriptome of Polish Landrace pigs fed with a control diet (low) and pigs fed with a PUFAs diet (high) may be helpful to develop a new set of genetic markers specific to Polish Landrace pig breed. Such cSNP markers eventually can be utilized in genome-wide association studies (GWAS) and to finally implement on marker assisted selection (MAS) and genomics selection (GS) program in active breeding population of Polish Landrace pigs in Poland.

RNA-seq based SNP discovery in liver transcriptome of Polish Landrace pigs

“…Related studies showed that consumption of PUFAs and other fatty acids diets were genetically regulated the growth performance and immune system in pigs [23]. Our mapping results identified a total of 25.3 million paired-end reads (13.5 million in high PUFAs and 11.8 million low PUFAs group) in the liver transcriptome of Polish Landrace pig, in comparison to 50.5 million paired-end reads (18 million in high PUFAs and 32.5 million low PUFAs group) in gluteus medius muscle transcriptome of Polish Landrace pig [24]. In an another study on the domestic Nero Siciliano male pig in Brazil [25], 346.8 million paired reads were identified and after QC check 99.03% of the reads were successfully mapped to the reference genome.…”

“…Finally, with regards to the same or alternative SNPs alleles encoding amino acids regions, 27141 synonymous cSNPs and 5989 non-synonymous cSNPs were identified in high PUFAs dietary group of liver transcriptome, in comparison to 18701 synonymous and 4389 non-synonymous cSNPs, respectively in gluteus medius muscle transcriptome [24]. Whereas, 15128 synonymous cSNPs and 3900 non-synonymous cSNPs were identified in the low PUFAs dietary groups liver transcriptome in comparison to 24491 synonymous and 5879 non-synonymous cSNPs, respectively in gluteus medius muscle transcriptome of Polish Landrace pigs [24].…”

“…Moreover, they mapped the RNA-seq data a male domestic Nero Siciliano pig on pig reference genome Sus scrofa11.1 and identified over 11 million SNPs, including 6,747 genetic variants at a rate of 1 variant every ~276 bases, and among these variants 1,132 were novel to the dbSNP151 database [25]. Our SNP detection results identified 25909 and 23290 cS-NPs in the high and low PUFAs dietary group of liver transcriptome, in comparison to 27404 and 36676 cSNPs in the high and low PUFAs dietary group of gluteus medius muscle transcriptome [24]. Finally, with regards to the same or alternative SNPs alleles encoding amino acids regions, 27141 synonymous cSNPs and 5989 non-synonymous cSNPs were identified in high PUFAs dietary group of liver transcriptome, in comparison to 18701 synonymous and 4389 non-synonymous cSNPs, respectively in gluteus medius muscle transcriptome [24].…”

“…Research methods: The detailed laboratory procedures of RNA extractions [26], library preparations, and MiSeq sequencing, as well as detailed bioinformatics analysis procedures [27][28][29][30][31], including analysis of the RNA-seq data [27][28][29], transcriptome mapping, and SNP detection [30][31] were described in the Pierzchala et al (2019) paper of this issue [24].…”