Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology

Abstract

Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs/CGs with RNA-seq data identified KCNIP4, CCSER1, DPP6, MAP3K5 and GHR CGs with highest SNPs hit loci in all three breeds and developmental ages. However, CAST CG with more than 100 SNPs hits were observed only in Polish HF and Hereford breeds.These findings are important for identification and construction of novel tissue specific SNP-db and breed specific SNP-db dataset by screening of putative SNPs according to QTL db and candidate genes for bovine growth and reproduction traits, one can develop genomic selection strategies for growth and reproductive traits.

Fig 1. Venn diagram showing the number of SNP segregating within three cattle breeds.

Fig 2. The phylogenetic relationship among RNA-seq samples using Maximum likelihood method.

An unrooted phylogeny tree of 18 bulls’ samples representing CP01-CP06 (Hereford), CP07-CP12 (Polish Red) and CP13-CP18 (Polish-HF). All nodes were robust at 100% bootstrap support. The scale bar denotes substitutions per site.

Fig 3. KASP SNP genotyping assay of BTA25_ 33248237 locus showing the data for single KASP assays on a single cluster plot.

Fig 4. KASP SNP genotyping assay of BTA16_ 81545856 locus showing the data for single KASP assays on a single cluster plot.

Fig 5. KASP SNP genotyping assay of BTA21_ 68782355 locus showing the data for single KASP assays on a single cluster plot.

Fig 6. KASP SNP genotyping assay of BTA23_ 9540557 locus showing the data for single KASP assays on a single cluster plot.

Fig 7. KASP SNP genotyping assay of BTA10_ 102942684 locus showing the data for single KASP assays on a single cluster plot.

Fig 8. KASP SNP genotyping assay of BTA25_ 37378666 locus showing the data for single KASP assays on a single cluster plot.

Fig 9. KASP SNP genotyping assay of BTA4_ 77555734 locus showing the data for single KASP assays on a single cluster plot.

Fig 10. KASP SNP genotyping assay of BTA19_ 27086284 locus showing the data for single KASP assays on a single cluster plot.