Sheep are among the major economically important livestock species worldwide because the animals produce milk, wool, skin, and meat. In the present study, the Illumina OvineSNP50 BeadChip was used to investigate genetic diversity and genome selection among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds from the United States. After quality-control filtering of SNPs (single nucleotide polymorphisms), we used 48,026 SNPs, including 46,850 SNPs on autosomes that were in Hardy-Weinberg equilibrium and 1,176 SNPs on chromosome × for analysis. Phylogenetic analysis based on all 46,850 SNPs clearly separated Suffolk from Rambouillet, Columbia, Polypay, and Targhee, which was not surprising as Rambouillet contributed to the synthesis of the later three breeds. Based on pair-wise estimates of F ST, significant genetic differentiation appeared between Suffolk and Rambouillet (F ST = 0.1621), while Rambouillet and Targhee had the closest relationship (F ST = 0.0681). A scan of the genome revealed 45 and 41 differentially selected regions (DSRs) between Suffolk and Rambouillet and among Rambouillet-related breed populations, respectively. Our data indicated that regions 13 and 24 between Suffolk and Rambouillet might be good candidates for evaluating breed differences. Furthermore, ovine genome v3.1 assembly was used as reference to link functionally known homologous genes to economically important traits covered by these differentially selected regions. In brief, our present study provides a comprehensive genome-wide view on within- and between-breed genetic differentiation, biodiversity, and evolution among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds. These results may provide new guidance for the synthesis of new breeds with different breeding objectives.
Differential display RT-PCR (DDRT-PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage-specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT-PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real-time PCR. The result of quantitative differential screening was found to be in agreement with DDRT-PCR banding patterns. In the second approach, a blastocyst-stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin-3 and fibronectin. As revealed by real-time PCR, the mRNA level of galectin-3 was three times higher in the blastocyst stage than in the morula stage. DDRT-PCR and SSH are both powerful tools for the identification of stage-specific expressed gene in preimplantation bovine embryos. Real-time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest.
To study the mRNA transcript profiles of some potential candidate developmental genes during bovine oocyte and blastocyst stages, RNA amplification procedures, cDNA microarray of 82 target genes spotted onto glass slide and real-time polymerase chain reaction (PCR) were used. Messenger RNAs were isolated from in vitro-produced bovine matured oocytes and blastocysts. Using equal amounts of input mRNAs but different cycles of amplifications, cDNAs were produced and served as template for RNA amplification by the in vitro transcriptions. After amplification, the RNA yields transcribed from cDNAs of different cycles were evaluated both by hybridization on the cDNA microarrays and by using real-time PCR techniques. The analyses indicated best results from lower amplification cycle templates with consistent signals at hybridization. Generally, the RNA yield was directly proportional to the amplification cycle but inversely related with signal consistency at repeated hybridizations. Using the protocols established, equal amounts of amplified RNA from matured oocytes and blastocysts were hybridized to the array. Analyses of replicated hybridizations indicated that 35 transcripts were differentially expressed. Most of these were not described in previous bovine embryo studies. Independent analyses of 23 transcripts with real-time PCR and unamplified RNA confirmed the results of 22 genes. Moreover, the functional analyses showed various roles related to development. Hence, it is possible to conclude that the genes identified here are potential candidates for characterizing developmental competence, and that the methods established can be used for large-scale gene expression analysis with more comprehensive arrays.
The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.
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