Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation.

Understanding the genetic mechanisms of phenotypic variation in hybrids between domestic animals and their wild relatives may aid germplasm innovation. Here, we report the high-quality genome assemblies of a male Pamir argali (O ammon polii, 2n = 56), a female Tibetan sheep (O aries, 2n = 54), and a male hybrid of Pamir argali and domestic sheep, and the high-throughput sequencing of 425 ovine animals, including the hybrids of argali and domestic sheep. We detected genomic synteny between Chromosome 2 of sheep and two acrocentric chromosomes of argali. We revealed consistent satellite repeats around the chromosome breakpoints, which could have resulted in chromosome fusion. We observed many more hybrids with karyotype 2n = 54 than with 2n = 55, which could be explained by the selfish centromeres, the possible decreased rate of normal/balanced sperm, and the increased incidence of early pregnancy loss in the aneuploid ewes or rams. We identified genes and variants associated with important morphological and production traits (e.g., body weight, cannon circumference, hip height, and tail length) that show significant variations. We revealed a strong selective signature at the mutation (c.334C > A, p.G112W) in TBXT and confirmed its association with tail length among sheep populations of wide geographic and genetic origins. We produced an intercross population of 110 F2 offspring with varied number of vertebrae and validated the causal mutation by whole-genome association analysis. We verified its function using CRISPR-Cas9 genome editing. Our results provide insights into chromosomal speciation and phenotypic evolution and a foundation of genetic variants for the breeding of sheep and other animals.

CRISPR/Cas9-mediated loss of FGF5 function increases wool staple length in sheep.

Fibroblast growth factor 5 (FGF5) regulates hair length in humans and a variety of other animals. To investigate whether FGF5 has similar effects in sheep, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to generate loss-of-function mutations with the FGF5 gene in Chinese Merino sheep. A total of 16 lambs were identified with genetic mutations within the targeting locus: 13 lambs had biallelic modifications and three lambs had monoallelic modifications. Characterization of the modifications revealed that 13 were frameshift mutations that led to premature termination, whereas the other three were in-frame deletions. Thus, CRISPR/Cas9 efficiently generated loss-of-function mutations in the sheep FGF5 gene. We then investigated the effect of loss of FGF5 function on wool traits in 12 lambs and found that wool staple length and stretched length of genetically modified (GM) yearling sheep were significantly longer compared with that of wild-type (WT) control animals. The greasy fleece weight of GM yearling sheep was also significantly greater compared with that of WT sheep. Moreover, the mean fiber diameter in GM sheep showed no significant difference compared with WT sheep, suggesting that the increase in greasy fleece weight was likely attributed to the increase in wool length. The results of this study suggest that CRISPR/Cas9-mediated loss of FGF5 activity could promote wool growth and, consequently, increase wool length and yield.

Addition of alpha-tocopherol to culture medium improves the quality and cryosurvival of nuclear-transferred ovine embryos.

The aim of this study was to reconstruct and cryopreserve somatic cell nuclear transferred (SCNT) ovine embryos and evaluate the effect of alpha-tocopherol on blastocyst development and subsequent cryosurvival of the SCNT embryos. The alpha-tocopherol (100 microg/ml) was added into culture medium for the SCNT embryos, the yield and total cell numbers of blastocysts were determined and the apoptosis incidences of the blastocysts were evaluated using the TUNEL assay. The blastocysts from the alpha-tocopherol and untreated groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. The results showed that there were no significant differences in blastocyst yield (26.3 vs. 22.3%) and total cell number (68.2 vs. 64.3) between the alpha-tocopherol and untreated groups. However, addition of alpha-tocopherol to the culture medium significantly decreased the apoptotic cell number (3.4 vs. 5.5) and significantly increased the cryosurvival of SCNT blastocysts (66.8 vs. 50.7%). In conclusion, addition of alpha-tocopherol to SCNT embryo culture medium was beneficial for improving embryo quality by decreasing the apoptotic blastocyst cell number and improving the tolerance of the embryos to cryopreservation.

[Density fluctuation of microfilariae and the role of residual infection source in filariasis transmission after its interruption].

OBJECTIVE: To investigate the density fluctuation of microfilariae, persistence of microfilaremia and possible new infection due to residual microfilaremia in areas with filariasis transmission interrupted. METHODS: The observation site was made in a village of Jishou City, Hunan Province. Inhabitants were regularly examined by thick blood smear and the density fluctuation of residual microfilaremia in known and newly-found cases were followed up. With a consent from the cases with residual microfilaremia, no treatment was given until they naturally turned negative. Antifilarial antibody level was detected by IFAT and a test kit for filariasis-special IgG4. Culex quinquefociatus was dissected to determine the natural infection rate and density of III stage filarial larvae in transmission season. The identified cases were followed-up by interviews and physical examinations to see if clinical manifestations appeared. RESULTS: Blood examination was carried out for all inhabitants for 10 times, 4 cases with microfilaremia, including 3 cases found at the beginning of the project and one newly infected case, were discovered after the interruption of filariasis transmission in the 19-year period. Among the 4 cases followed up, one case naturally turned negative within 7 years, one case became negative in the 9th year but returned positive in the 12th year, and then naturally turned negative in the 13th year. The 3rd case turned negative in the 14th year and was again positive in the 19th and the 20th years, and became negative through diethylcarbamazine (DEC) treatment in the 21st year. The new case was found to have microfilaremia in the 16th year and kept positive for 5 years until DEC treatment. Serological tests (IFAT and special IgG4) revealed no new positive cases. The natural infection rate and larvae density in Culex quinquefasciatus decreased annually. Conclusion The persistence period of residual microfilaremia in individual cases might last for more than 20 years after filariasis transmission has been interrupted.