The complete mitochondrial genome sequence of the dwarf blue sheep, Pseudois schaeferi haltenorth in China.

The dwarf blue sheep (Pseudois schaeferi haltenorth) belongs the subfamily Caprinae, which is distributed in Sichuan, Tibet, Yunnan, and Qinghai in China. In this study, the complete mitochondrial genome of Pseudois schaeferi haltenorth was sequenced. The mitogenome was 16 741 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region (D-loop region). As in other mammals, most mitochondrial genes are encoded on the heavy strand, except for ND6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the Pseudois schaeferi haltenorth is 33.54% A, 26.37% T, 26.91% C, and 13.18% G, A + T (59.91%) was higher than G + C (40.09%). The phylogenetic relationships was analyzed using the complete mitogenome sequence, results show that P. schaeferi haltenorth should be a different species differ from the Genus pseudois hodgson. These information provide useful data for further study on the protection of genetic resources and the taxonomy of Caprinae.

The complete mitochondrial genome sequence of the wild Huoba Tibetan sheep of the Qinghai-Tibetan Plateau in China.

The wild Huoba Tibetan sheep belongs to the subfamily Caprinae, which distributes in Huoba Town of Tibet Autonomous Region, China. In the present work, we report the complete mitochondrial genome sequence of wild Huoba Tibetan sheep for the first time. The total length of the mitogenome is 16 621 bp, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a non-coding control region (D-loop region). As in other mammals, most mitochondrial genes are encoded on the heavy strand. Its overall base composition is A: 33.64%, T: 27.32%, C: 25.90%, and G: 13.14%, A + T (61.96%) was higher than G + C (39.04%). The phylogenetic relationships was analyzed using the complete mitogenome sequence, results show that wild Huoba Tibetan sheep should be a different species differ from the Ovis aries. These information provide an important data for further study on protection of genetic resources and the taxonomy of Caprinae.

De novo assembly and characterization of skin transcriptome using RNAseq in sheep (Ovis aries).

Wool is produced via synthetic processes of wool follicles, which are embedded in the skin of sheep. The development of new-generation sequencing and RNA sequencing provides new approaches that may elucidate the molecular regulation mechanism of wool follicle development and facilitate enhanced selection for wool traits through gene-assisted selection or targeted gene manipulation. We performed de novo transcriptome sequencing of skin using the Illumina Hiseq 2000 sequencing system in sheep (Ovis aries). Transcriptome de novo assembly was carried out via short-read assembly programs, including SOAPdenovo and ESTScan. The protein function, clusters of orthologous group function, gene ontology function, metabolic pathway analysis, and protein coding region prediction of unigenes were annotated by BLASTx, BLAST2GO, and ESTScan. More than 26,266,670 clean reads were collected and assembled into 79,741 unigene sequences, with a final assembly length of 35,447,962 nucleotides. A total of 22,164 unigenes were annotated, accounting for 36.27% of the total number of unigenes, which were divided into 25 classes belonging to 218 signaling pathways. Among them, there were 17 signal paths related to hair follicle development. Based on mass sequencing data of sheepskin obtained by RNA-Seq, many unigenes were identified and annotated, which provides an excellent platform for future sheep genetic and functional genomic research. The data could be used for improving wool quality and as a model for human hair follicle development or disease prevention.

Analysis of geographic and pairwise distances among sheep populations.

This study investigated geographic and pairwise distances among seven Chinese local and four introduced sheep populations via analysis of 26 microsatellite DNA markers. Genetic polymorphism was rich, and the following was discovered: 348 alleles in total were detected, the average allele number was 13.38, the polymorphism information content (PIC) of loci ranged from 0.717 to 0.788, the number of effective alleles ranged from 7.046 to 7.489, and the observed heterozygosity ranged from 0.700 to 0.768 for the practical sample, and from 0.712 to 0.794 for expected heterozygosity. The Wright's F-statistic of subpopulations within the total (FST) was 0.128, the genetic differentiation coefficient (GST) was 0.115, and the average gene flow (Nm) was 1.703. The phylogenetic trees based on the neighbor-joining method by Nei's genetic distance (DA) and Nei's standard genetic distance (DS) were similar. Sheep populations clustered into group 1 (Ta, M, L, H, O, G, and Q breeds) and group 2 (PD, WS, B, and T breeds). These results will have an important value applied and directive significance for sheep breeding in the future.

Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep.

Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gel-based methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.

Transcription levels of sirtuin family in neural stem cells and brain tissues of adult mice.

Neural stem cells (NSCs) has been used as a well-known model to investigate apoptosis, differentiation, maintenance of stem cells status, and therapy of neurological disease. The C17.2 NSCs line was produced after v-myc transformation of neural progenitor from mouse cerebellar cortex. Sirtuin family plays important roles involved in neuronal differentiation, genomic stability, lifespan, cell survival. However, little is known about gene expression variation of sirtuin family in C17.2 NSCs, primary NSCs, and different brain tissues in adult mice. Here, we confirmed that the mRNA expression levels of sirt2, 3, 4, 5, and 7 in E14.5 NSCs were significantly higher than in C17.2 NSCs, whereas that sirt 6 displayed an opposing mode. Moreover, a higher mRNA level of sirtuin family was observed in the adult mouse brain compared to C17.2 NSCs. In addition, histone deacetylase (HDAC) inhibitors nicotinamide and Trichostatin A (TSA) were used to explore differential changes at the transcriptional level of sirtuins. Results indicated that the expression of sirt1, sirt5 and sirt6 was significant downregulated by nicotinamide treatment. Whereas, a significant downregulation in sirt1 and sirt3 and a significant upregulation in sirt2, sirt4, sirt6, and sirt7 were observed in the treatment of TSA. Thus our studies indicate different sirtuin mRNA expression profiles between C17.2 NSCs, E14.5 NSCs and brain tissues, suggesting the transcriptional regulation of sirtuin family could be mediated by different histone acetylation.

Survival of genetically engineered Escherichia coli in natural soil and river water.

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased.