Document Type : Research Paper
Authors
Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
Abstract
Keywords
Main Subjects
Extended Abstract
Objective
The fertility of female cows is the main factor for the survival of dairy farming. The interval between calvings and the response to the first insemination are used as the most important fertility traits. Infertility and low fertility are important and widespread problems in domestic animals, and the main limitation of reproductive efficiency in mammalian species is embryonic mortality. This study was performed to determine the differentially expressed genes in fertility in Holstein cows using phenotypic data and next-generation sequencing (NGS) technology.
Materials and Methods
Corpus luteum samples from 18 Holstein cows at up to two parturitions were used in two high and low fertility groups, three samples from each group were pooled together and a total of three biological samples from each were submitted for whole genome sequencing. RNA sequencing was performed with Illumina Hiseq 2500 using paired-end reads, each read being 75 bp in length. The preparation and analysis steps were performed on the Galaxy platform. Quality control was performed with Galaxy FastQC. For low-quality samples, the Trimmomatic software was used to remove substandard samples. HISAT2 software was used to match the transcriptome reads to the cow reference genome. After mapping the transcripts to the reference genome, the expression level of each transcript was counted using the featureCounts softwar. Differential gene expression analysis was used for two treatment groups using DESeq2. Gene ontology using the DAVID database was performed for genes that showed significantly different expressions between the two treatments.
Results
Results from the Trimmomatic software showed that only 0.87% of the data was deleted and approximately 99.13% of the raw data was used for mapping to the reference genome. The results of the alignment showed that 93.48% of the transcriptome sequence mapped to the reference genome, 90.58% to one site, and only 2.90% to more than one site. The results of differential gene expression analysis in luteal tissue showed that 19 genes out of 13049 expressed transcripts had a significant difference in expression between high and low-fertility cows with an FDR-adjusted p-value (q-value) of less than 0.05. UBE3B, NIF3L1 and ORC2 genes were three functional marker genes that were more highly expressed in the corpus luteum of high-fertility cows. The UBE3B gene is involved in biological processes as a protein involved in the catabolic process of ubiquitin-dependent protein. NIF3L1 is involved in the biological process of neuronal differentiation, positive transcriptional regulation, DNA pattern, and negative transcriptional regulation by nucleic acid pattern. The ORC2 gene plays a role in the biological process of DNA replication. The genes KRT8, PHLDB3, PPT2, LOC787628, PPYR1, TOX, TP73, DHX8, KCNN1, CLEC6A, PXMP4, LRRC26, SLC34A3, and OR13C7 have been shown to be more highly expressed in the corpus luteum in low fertility cows. Among these genes, three genes PPT2, PPYR1 and CLEC6A were functional indicators. The PPT2 gene is responsible for the removal of palmitoyl groups from a macromolecule, the PPYR1 gene is involved in the biological process of cell surface receptor signaling pathway, neuropeptide signaling pathway, nutritional behavior and blood circulation and the CLC34A3 gene plays a role in the biological process of cellular phosphate ion homeostasis.
Conclusion
the loci UBE3B, NIF3L1 and ORC2, which were expressed more strongly in the corpus luteum of highly fertile cows, can be used as functional indicator genes in the genetic selection of dairy cows.
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