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Summary We determined the genetic diversity and evolutionary relationships among 26 Chinese indigenous horse breeds and two introduced horse breeds by genotyping these animals for 27 microsatellite loci. The 26 Chinese horse breeds come from 12 different provinces. Two introduced horse breeds were the Mongolia B Horse from Mongolia and the Thoroughbred Horse from the UK. A total of 330 alleles were detected, and the expected heterozygosity ranged from 0.719 (Elenchuns) to 0.780 (Dali). The mean number of alleles among the horse breeds ranged from 6.74 (Hequ) to 8.81 (Debao). Although there were abundant genetic variations found, the genetic differentiation was low between the Chinese horses, which displayed only 2.4% of the total genetic variance among the different breeds. However, genetic differentiation (pairwise FST) among Chinese horses, although moderate, was still apparent and varied from 0.001 for the Guizou–Luoping pair to 0.064 for the Jingjiang–Elenchuns pair. The genetic differentiation patterns and genetic relationships among Chinese horse breeds were also consistent with their geographical distribution. The Thoroughbred and Mongolia B breeds could be discerned as two distinct breeds, but the Mongolia B horse in particular suffered genetic admixture with Chinese horses. The Chinese breeds could be divided into five major groups, i.e. the south or along the Yangtze river group (Bose, Debao, Wenshan, Lichuan, Jianchang, Guizhou, Luoping, Jinjiang and Dali), the Qinghai-Tibet Plateau group (Chaidamu, Hequ, Datong, Yushu, Tibet Grassland and Tibet Valley), the Northeast of China group (Elenchuns, Jilin and Heihe), the Northwest of China group (Kazakh, Yili and Yanqi) and the Inner Mongolia group (Mongolia A, Sanhe, Xinihe,Wuzhumuqin and Sengeng). This grouping pattern was further supported by principal component analysis and structure analysis.

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.

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.

The desmoglein 4 (DSG4) gene is a potential candidate in the search for genes that may affect wool traits, because of its function. This study aimed to screen for polymorphisms in partial exon 16 and 3ꞌUTR of the sheep desmoglein 4 DSG4 gene, and to test its possible association with wool length and crimp associated with fur. Overall, 326 sheep were scanned via single-strand conformational polymorphism assay, through three pairs of primers. The breeds included Tan, Han, and TanxHan from China, Polled Dorset from Australia, and Suffolk from Britain genotypes AA, BB, and AB for primer2 and genotypes DD, EE, and DE for primer3 were detected in native breeds. Six SNPs and 3-bp insertion/deletions were found in exon 16, of which 4 lead to amino acid substitutions. In addition, 1 SNP was found in 3ꞌUTR. The DSG4 genotype was found to be strongly associated with all wool traits that were considered in this study (P0.01). Sheep with the genotype MM had a higher least square mean compared to sheep with the genotype WW or WM with respect to birth scapular wool length (P0.01), crimp number of birth scapular wool crimp (P0.01), crimp number of weaning scapular wool crimp (P0.01), and crimp number of weaning rump wool crimp (P0.01, P0.05). In conclusion, our study is the first to demonstrate that the DSG4 gene may be a candidate, or major gene, which influences important wool traits.