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.

Analysis of agouti signaling protein (ASIP) gene polymorphisms and association with coat color in Tibetan sheep (Ovis aries).

Tibetan sheep, an indigenous breed, have a wide variety of phenotypes and a colorful coat, which make this breed an interesting model for evaluating the effects of coat-color gene mutations on this phenotypic trait. The agouti signaling protein (ASIP) gene is a positional candidate gene, as was inferred based on previous study. In our research, ASIP gene copy numbers in genomic DNA were detected using a novel approach, and the exon 2 g.100-104 mutation and copy number variation (CNV) of ASIP were associated with coat color in 256 sheep collected from eight populations with different coat colors by high-resolution melting curve assay. We found that the relative copy numbers of ASIP ranged from one to eight in Tibetan sheep. All of the g.100-104 genotypes in the populations were in Hardy-Weinberg equilibrium, and there was no relationship between the g.100-104 genotype and coat color (P > 0.05). The single ASIP CNV allele was found to be almost entirely associated with solid-black coat color; however, not all solid-black sheep displayed the putative single ASIP CNV genotype. From our study, we speculate that the ASIP CNV is under great selective pressure and the single ASIP CNV allows selection for black coat color in Tibetan sheep, but this does not explain all black phenotypes in Tibetan sheep.

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.

Results of carbon ion radiotherapy for skin carcinomas in 45 patients.

UNLABELLED: BACKGROUND; Heavy ions represent the best tool for external radiotherapy (RT) of inoperable tumours. Heavy ion RT has been used in the treatment of various tumours, especially for radioresistant tumours mediated by hypoxia, localized near organs at risk. Most of these treatments are concentrated in deep-seated tumours such as those of the brain, head, lung, liver, rectum and urogenital organs, and treatment of skin carcinomas is limited. OBJECTIVES: To evaluate the outcome and toxicity after carbon ion RT for skin carcinomas at the Heavy Ion Research Facility in Lanzhou, China. METHODS: Between November 2006 and March 2009, 45 patients with skin carcinoma [squamous cell carcinoma (SCC) (n = 16), basal cell carcinoma (BCC) (n = 12), malignant melanoma (MM) (n = 7), Bowen disease (n = 8) and Paget disease (n = 2)] were treated with carbon ion RT within a clinical Phase I trial. Patients received total doses of 60-70 GyE for SCC and BCC, 61-75 GyE for MM, 60 GyE for Bowen disease and 42·5 GyE for Paget disease, administered in 6-11 fractions over 6-11 days, with a fraction dose of 7-10 GyE. RESULTS: The mean follow-up was 24 months, range 12-36 months. The actuarial local control rates at 1 and 3 years were 90·9% and 65·5% for SCC, 91·7% and 80·2% for BCC, 85·7% and 42·9% for MM, 90% and 90% for Bowen and Paget diseases, respectively. The actuarial 1- and 3-year overall survival rates for 45 patients were 88·9% and 86%, respectively. No severe side-effects greater than Common Toxicity Criteria grade 3 have been observed. CONCLUSIONS: The results demonstrated that heavy ion RT offers high local tumour control and progression-free survival rates without significant radiation-induced toxicity for patients with skin carcinomas.

Expression of modified low-density lipoprotein receptors by trabecular meshwork cells.

We examined the incorporation of fluoresceinated low-density lipoprotein (LDL) and acetylated or acetoacetylated low-density lipoprotein (A-LDL or AA-LDL) by a number of ocular cells in culture. All the cells investigated, including bovine, monkey, human trabecular meshwork cells, human corneal endothelial cells, human corneal stromal cells and human scleral cells, took up fluorescently labeled LDL. The bovine, monkey and human trabecular meshwork cells showed the strongest fluorescence reactions. In addition, we found that the trabecular meshwork cells became fluorescent after incubations with labeled A-LDL or AA-LDL. They were the only cell type examined that possessed this capacity. The fluorescence intensity was markedly diminished by adding to the incubation solution either fucoidin, a competitive inhibitor of modified LDL uptake, unlabeled A-LDL or AA-LDL. The trabecular meshwork cells in situ also became brightly labeled after exposure to fluoresceinated native LDL, A-LDL or AA-LDL. The uptake of modified LDL separated the trabecular meshwork cells from other types of ocular cells, which may be used to aid identification of trabecular meshwork cells in culture as well as in situ. This property also suggested that trabecular meshwork cells may have some functional similarities to macrophages.