Single-Cell RNA-Sequencing Provides Insight into Skeletal Muscle Evolution during the Selection of Muscle Characteristics.

Skeletal muscle comprises a large, heterogeneous assortment of cell populations that interact to maintain muscle homeostasis, but little is known about the mechanism that controls myogenic development in response to artificial selection. Different pig (Sus scrofa) breeds exhibit distinct muscle phenotypes resulting from domestication and selective breeding. Using unbiased single-cell transcriptomic sequencing analysis (scRNA-seq), the impact of artificial selection on cell profiles is investigated in neonatal skeletal muscle of pigs. This work provides panoramic muscle-resident cell profiles and identifies novel and breed-specific cells, mapping them on pseudotime trajectories. Artificial selection has elicited significant changes in muscle-resident cell profiles, while conserving signs of generational environmental challenges. These results suggest that fibro-adipogenic progenitors serve as a cellular interaction hub and that specific transcription factors identified here may serve as candidate target regulons for the pursuit of a specific muscle phenotype. Furthermore, a cross-species comparison of humans, mice, and pigs illustrates the conservation and divergence of mammalian muscle ontology. The findings of this study reveal shifts in cellular heterogeneity, novel cell subpopulations, and their interactions that may greatly facilitate the understanding of the mechanism underlying divergent muscle phenotypes arising from artificial selection.

Insights into the genetic diversity of indigenous goats and their conservation priorities.

Objective: An experiment was conducted to evaluate genetic diversity of 26 Chinese indigenous goats by 30 microsatellite markers, and then to define conservation priorities to set up the protection programs according to the weight given to within- and between-breed genetic diversity. Methods: Twenty-six representative populations of Chinese indigenous goats, 1351 total, were sampled from different geographic regions of China. Within-breed genetic diversity and marker polymorphism were estimated calculating the mean number of alleles (MNA), observed heterozygosities (HO), expected heterozygosities (He), fixation index (FIS), effective number of alleles (NE) and allelic richness (Rt). Conservation priorities were analyzed by statistical methods. Results: A relatively high level of genetic diversity was found in twenty-four populations, the exceptions were in the Daiyun and Fuqing goat populations. Within-breed kinship coefficients matrix identified seven highly inbred breeds which should be concerned. Of these, six breeds will get a negative contribution to heterozygosity when the method was based on proportional contribution to heterozygosity. Based on Weitzman or Piyasatian and Kinghorn methods, the breeds distant from others i.e. Inner Mongolia Cashmere goat, Chengdu Brown goat and Leizhou goat will obtain a high ranking. Evidence from Caballero and Toro and Fabuel et al. method prioritized Jining Gray goat, Liaoning Cashmere goat and Inner Mongolia Cashmere goat, that in agreement with results from Kinship-based methods. Conclusion: Conservation priorities had planned according to multiple methods. Our results suggest Inner Mongolia Cashmere goat (most methods), Jining Gray goat and Liaoning Cashmere goat (high contribution to heterozygosity and total diversity) should be prioritized based on most methods. Furthermore, Daiyun goat and Shannan White goat also should be prioritized based on consideration of effective population size. However, if one breed could survive in changing conditions all the time, the straightforward approach is to increase its utilization and attraction for production via mining breed germplasm characteristic.

Genome-wide analysis reveals adaptation to high altitudes in Tibetan sheep.

Tibetan sheep have lived on the Tibetan Plateau for thousands of years; however, the process and consequences of adaptation to this extreme environment have not been elucidated for important livestock such as sheep. Here, seven sheep breeds, representing both highland and lowland breeds from different areas of China, were genotyped for a genome-wide collection of single-nucleotide polymorphisms (SNPs). The FST and XP-EHH approaches were used to identify regions harbouring local positive selection between these highland and lowland breeds, and 236 genes were identified. We detected selection events spanning genes involved in angiogenesis, energy production and erythropoiesis. In particular, several candidate genes were associated with high-altitude hypoxia, including EPAS1, CRYAA, LONP1, NF1, DPP4, SOD1, PPARG and SOCS2. EPAS1 plays a crucial role in hypoxia adaption; therefore, we investigated the exon sequences of EPAS1 and identified 12 mutations. Analysis of the relationship between blood-related phenotypes and EPAS1 genotypes in additional highland sheep revealed that a homozygous mutation at a relatively conserved site in the EPAS1 3' untranslated region was associated with increased mean corpuscular haemoglobin concentration and mean corpuscular volume. Taken together, our results provide evidence of the genetic diversity of highland sheep and indicate potential high-altitude hypoxia adaptation mechanisms, including the role of EPAS1 in adaptation.

Genome-wide analysis reveals population structure and selection in Chinese indigenous sheep breeds.

BACKGROUND: Traditionally, Chinese indigenous sheep were classified geographically and morphologically into three groups: Mongolian, Kazakh and Tibetan. Herein, we aimed to evaluate the population structure and genome selection among 140 individuals from ten representative Chinese indigenous sheep breeds: Ujimqin, Hu, Tong, Large-Tailed Han and Lop breed (Mongolian group); Duolang and Kazakh (Kazakh group); and Diqing, Plateau-type Tibetan, and Valley-type Tibetan breed (Tibetan group). RESULTS: We analyzed the population using principal component analysis (PCA), STRUCTURE and a Neighbor-Joining (NJ)-tree. In PCA plot, the Tibetan and Mongolian groups were clustered as expected; however, Duolang and Kazakh (Kazakh group) were segregated. STRUCTURE analyses suggested two subpopulations: one from North China (Kazakh and Mongolian groups) and the other from the Southwest (Tibetan group). In the NJ-tree, the Tibetan group formed an independent branch and the Kazakh and Mongolian groups were mixed. We then used the d i statistic approach to reveal selection in Chinese indigenous sheep breeds. Among the 599 genome sequence windows analyzed, sixteen (2.7%) exhibited signatures of selection in four or more breeds. We detected three strong selection windows involving three functional genes: RXFP2, PPP1CC and PDGFD. PDGFD, one of the four subfamilies of PDGF, which promotes proliferation and inhibits differentiation of preadipocytes, was significantly selected in fat type breeds by the Rsb (across pairs of populations) approach. Two consecutive selection regions in Duolang sheep were obviously different to other breeds. One region was in OAR2 including three genes (NPR2, SPAG8 and HINT2) the influence growth traits. The other region was in OAR 6 including four genes (PKD2, SPP1, MEPE, and IBSP) associated with a milk production quantitative trait locus. We also identified known candidate genes such as BMPR1B, MSRB3, and three genes (KIT, MC1R, and FRY) that influence lambing percentage, ear size and coat phenotypes, respectively. CONCLUSIONS: Based on the results presented here, we propose that Chinese native sheep can be divided into two genetic groups: the thin type (Tibetan group), and the fat type (Mongolian and Kazakh group). We also identified important genes that drive valuable phenotypes in Chinese indigenous sheep, especially PDGFD, which may influence fat deposition in fat type sheep.

Genetic structure of Chinese indigenous goats and the special geographical structure in the Southwest China as a geographic barrier driving the fragmentation of a large population.

BACKGROUND: China has numerous native domestic goat breeds, however, extensive studies are focused on the genetic diversity within the fewer breeds and limited regions, the population demographic history and origin of Chinese goats are still unclear. The roles of geographical structure have not been analyzed in Chinese goat domestic process. In this study, the genetic relationships of Chinese indigenous goat populations were evaluated using 30 microsatellite markers. METHODOLOGY/PRINCIPAL FINDINGS: Forty Chinese indigenous populations containing 2078 goats were sampled from different geographic regions of China. Moderate genetic diversity at the population level (H(S) of 0.644) and high population diversity at the species level (H(T) value of 0.737) were estimated. Significant moderate population differentiation was detected (F(ST) value of 0.129). Significant excess homozygosity (F(IS) of 0.105) and recent population bottlenecks were detected in thirty-six populations. Neighbour-joining tree, principal components analysis and Bayesian clusters all revealed that Chinese goat populations could be subdivided into at least four genetic clusters: Southwest China, South China, Northwest China and East China. It was observed that the genetic diversity of Northern China goats was highest among these clusters. The results here suggested that the goat populations in Southwest China might be the earliest domestic goats in China. CONCLUSIONS/SIGNIFICANCE: Our results suggested that the current genetic structure of Chinese goats were resulted from the special geographical structure, especially in the Western China, and the Western goat populations had been separated by the geographic structure (Hengduan Mountains and Qinling Mountains-Huaihe River Line) into two clusters: the Southwest and Northwest. It also indicated that the current genetic structure was caused by the geographical origin mainly, in close accordance with the human's migration history throughout China. This study provides a fundamental genetic profile for the conservation of these populations and better to understand the domestication process and origin of Chinese goats.

[Genetic diversity in goat breeds based on microsatellite analysis].

Fluorescence PCR was applied to investigate the genetic diversities of 9 indigenous Chinese goat breeds and 1 exotic breed with 10 microsatellite DNA markers recommended by the Food and Agriculture Organization of the United Nations and the International Livestock Research Institute of Animal Genetics, which provide data for the preservation and utilization of indigenous goat breeds genetic resource. We found that the 7 breeds were high polymorphic while 3 breeds were moderate polymorphic. We also detected 119 alleles, and the effective allele number ranged from 1.4641 to 9.2911. The average heterozygosity of loci and breeds respectively varied from 0.2618 to 0.7672 and from 0.5196 to 0.7024. As well as SRCRSP23 site and Hexi cashmere goat had the highest average heterozygosity. Then we analyzed the phylogenetic trees (NJ and UPGMA), and found both of them were generally in accordance with their original breeding history and localities.

[Individual identification and paternity testing of bulls using microsatellite].

The polymorphism distributions of 14 microsatellite loci were detected using the Bovine Paternity PCR Typing Kit (including 11 X-STR) and 3 selected Y-STR microsatellite DNA markers. The genetic diversity were evaluated, and the feasibility of the application to individual identification and paternity testing were discussed. The results showed that all the 14 microsatellite loci had genetic polymorphisms in bulls, and the polymorphism information content (PIC) in loci MCM158 was the biggest (0.888), while the ETH10 was the lowest (0.482). Power of discrimination (DP) value of the 14 STR loci ranged from 0.715 to 0.968. The Cumulate DP (CDP) was 99.99%, and the Cumulate PE (CPE) also reached 99.99%. These results indicate that the 14 microsatellites can be applied to the individual identification.

[Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.

[Genetic diversity and origin of mitochondrial DNA D-loop region for some domestic goat breeds of China].

The complete sequences of mitochondrial DNA D-loop of 140 individuals in 10 Chinese goat (Capra hiruc) breeds were analyzed by DNA sequencing technology. The results showed that the length of mtDNA D-loop in Chinese goats was 1 211-1 213 bp. There were 84 different haplotypes and 171 polymorphic sites. The mean nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.02063+/+0.00225 and 0.988+/-0.003, respectively, and the average number of nucleotide differences (k) was 24.896. The results showed an abundant genetic diversity in domestic goats of China. The NJ tree indicated that there were two main branches in Chinese domestic goats, thereinto, one branch was clustered with Capra aegagrus, and Capra falconeri was clustered alone, which indicated that Capra aegagrus had more contribution to Chinese domestic goat breeds.

[Establishment of fibroblast cell line and its biological characteristics in Matou goat].

Taking Matou goat ear margin as the study material, we succeeded in established a fibroblast cell line by the method of explant culture directly. Observations on morphology, dynamic growth, determination of viability, analysis of karyotype, test of microorganism and other characteristics were detected. Results showed: Population Doubling Time (PDT) of cells was approximately 36 h; Cell viability was 96.7% after thawing; The status of cell After passage was constant; Analysis of chromosomal karyotyps indicated that diploid (2n=60) account for 98% in the cell line. Every index in the cell line met all the standard quality controls of ATCC in USA. The established of Matou goat ear fibroblast cell line has not only important genetic resources preserved at the cell level, but also valuable material for genome, postgenome and somatic cell nuclear transfer research.

[Influence of infusion of Duroc blood on characteristics of chromosomes in Gansu black pig].

The variation of chromosomes of Gansu Black pig was caused by infusion of Duroc blood. The chromosome length ratios of No.1,6,7,13 chromosomes in the line I of Gansu Black pig were higher than those in the line II. It showed that the homogeneity in line I was higher than in line II. The number of Ag-staining nucleolus organizers (Ag-NOR) was between 1-4 in the line I and II, the average No. of Ag-NOR was different obviously between the line I and the line II. This indicated that the activity of translation in rRNA in the line I was different in the line II.