Wenping Hu

Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization

Abstract Background Animal domestication has been extensively studied, but the process of feralization remains poorly understood. Results Here, we performed whole-genome sequencing of 99 sheep and identified a primary genetic divergence between 2 heterogeneous populations in the Tibetan Plateau, including 1 semi-feral lineage. Selective sweep and candidate gene analysis revealed local adaptations of these sheep associated with sensory perception, muscle strength, eating habit, mating process, and aggressive behavior. In particular, a horn-related gene, RXFP2, showed signs of rapid evolution specifically in the semi-feral breeds. A unique haplotype and repressed horn-related tissue expression of RXFP2 were correlated with higher horn length, as well as spiral and horizontally extended horn shape. Conclusions Semi-feralization has an extensive impact on diverse phenotypic traits of sheep. By acquiring features like those of their wild ancestors, semi-feral sheep were able to regain fitness while in frequent contact with wild surroundings and rare human interventions. This study provides a new insight into the evolution of domestic animals when human interventions are no longer dominant.

Single Nucleotide Polymorphisms in the HIRA Gene Affect Litter Size in Small Tail Han Sheep

Simple Summary Litter size is one of the most important reproductive traits in sheep. Two single nucleotide polymorphisms (SNPs), g.71874104G>A and g.71833755T>C, in the Histone Cell Cycle Regulator (HIRA) gene, were identified by whole-genome sequencing (WGS) and may be correlated with litter size in sheep. The two SNPs were genotyped and expression patterns of HIRA was determined in sheep breeds with different fecundity and in groups of Small Tail Han sheep producing large or small litters. Association analysis indicated that both SNPs were significantly correlated with litter size in Small Tail Han sheep. Furthermore, high levels of HIRA expression may have a negative effect on litter size in Small Tail Han sheep. Abstract Maintenance of appropriate levels of fecundity is critical for efficient sheep production. Opportunities to increase sheep litter size include identifying single gene mutations with major effects on ovulation rate and litter size. Whole-genome sequencing (WGS) data of 89 Chinese domestic sheep from nine different geographical locations and ten Australian sheep were analyzed to detect new polymorphisms affecting litter size. Comparative genomic analysis of sheep with contrasting litter size detected a novel set of candidate genes. Two SNPs, g.71874104G>A and g.71833755T>C, were genotyped in 760 Small Tail Han sheep and analyzed for association with litter size. The two SNPs were significantly associated with litter size, being in strong linkage disequilibrium in the region 71.80–71.87 Mb. This haplotype block contains one gene that may affect litter size, Histone Cell Cycle Regulator (HIRA). HIRA mRNA levels in sheep with different lambing ability were significantly higher in ovaries of Small Tail Han sheep (high fecundity) than in Sunite sheep (low fecundity). Moreover, the expression levels of HIRA in eight tissues of uniparous Small Tail Han sheep were significantly higher than in multiparous Small Tail Han sheep (p < 0.05). HIRA SNPs significantly affect litter size in sheep and are useful as genetic markers for litter size.

Subtraction suppressive hybridisation analysis of differentially expressed genes associated with puberty in the goat hypothalamus

The cost of developing replacement nanny goats could be reduced by decreasing the age at puberty because this way nanny goats could be brought into production at an earlier age. The aim of the present study was to screen genes related to puberty to investigate the molecular mechanisms of puberty. Subtracted cDNA libraries were constructed for hypothalami from juvenile (Group A), pubertal (Group B) and age-matched control pubertal (Group E) Jining grey (JG) and Liaoning cashmere (LC) goats using suppression subtractive hybridisation (SSH). Differentially expressed genes were analysed by bioinformatics methods. There were 203 expressed sequence tags (ESTs) in the subtracted cDNA libraries that were differentially expressed between JG and LC goats at the juvenile stage, 226 that were differentially expressed at puberty and 183 that were differentially expressed in the age-matched control group. The differentially expressed ESTs in each subtracted cDNA library were classified as known gene, known EST and unknown EST according to sequence homology in the GenBank non-redundant (NR) and EST database. According to gene function analysis in the COG (Cluster of Orthologous Groups) database, the known genes were grouped into 10 subdivisions in Group A, into seven subdivisions in Group E and into nine subdivisions in Group B under three categories: cellular processes and signalling, information storage and processing, and metabolism. Pathway analysis in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database of known genes revealed that the three pathways that most differentially expressed genes were involved in were metabolic pathways, Parkinsons disease and oxidative phosphorylation. Protein interaction analysis of the high homology genes revealed the most dominant network to be structure of ribosome/protein translation, oxidative phosphorylation and carbohydrate metabolism. The results reveal that the onset of puberty is a complex event involving multiple genes in multiple biological processes. The differentially expressed genes include genes related to both neuroendocrine and energy metabolism.

Metabolic Effects of FecB Gene on Follicular Fluid and Ovarian Vein Serum in Sheep (Ovis aries)

The FecB gene has been discovered as an important gene in sheep for its high relationship with the ovulation rate, but its regulatory mechanism remains unknown. In the present study, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were adopted to detect the metabolic effects of FecB gene in follicular fluid (FF) and ovarian vein serum (OVS) in Small Tail Han (STH) sheep. ANOVA and random forest statistical methods were employed for the identification of important metabolic pathways and biomarkers. Changes in amino acid metabolism, redox environment, and energy metabolism were observed in FF from the three FecB genotype STH ewes. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) showed that metabolic effects of FecB gene are more pronounced in FF than in OVS. Therefore, the difference of the metabolic profile in FF is also affected by the FecB genotypes. In Spearman correlation analysis, key metabolites (e.g., glucose 6-phosphate, glucose 1-phosphate, aspartate, asparagine, glutathione oxidized (GSSG), cysteine-glutathione disulfide, γ-glutamylglutamine, and 2-hydrosybutyrate) in ovine FF samples showed a significant correlation with the ovulation rate. Our findings will help to explain the metabolic mechanism of high prolificacy ewes and benefit fertility identification.

Expression and Functional Analysis of the BCL2-Associated Agonist of Cell Death (BAD) Gene in the Sheep Ovary During the Reproductive Cycle

Most ewes in China are seasonally polyestrous with normal ovulatory cycles, which is controlled by photoperiod (length of the daily light phase). These ewes are estrous in the short-day season and anestrus in the long-day season and cannot mate during anestrus. Thus seasonal breeding limits both diversification and intensification of production. If sheep can estrus all round year, it can be mated twice per year, which can greatly improve the economic benefits. To change seasonal estrus at the genetic level and cultivating new sheep breeds, it is important to understand the molecular mechanisms of seasonal breeding trait in sheep. The BCL2-associated agonist of cell death (BAD) gene being a regulator of cellular apoptosis was identified by our previous RNA-Seq, which is associated with follicular development in mammalian ovaries. However, the mechanism how BAD can regulate estrus in sheep was poorly understood. In this study, we characterized ovine BAD, including full-length mRNA cloning and protein sequence prediction, as well as BAD expression profile in Small-tailed Han (STH) sheep. The highest expression levels of BAD were observed in sheep hypothalamus, lung, and pituitary, while the lowest expression was in liver. Functional analysis of BAD was performed in primary granulosa cells of sheep. The concentration of P4 was significantly increased after RNAi interference of BAD, while P4 level was shown to be opposite after BAD overexpression in vitro. It has been found that BAD can reduce progesterone levels by promoting ovarian GC apoptosis, which might be involved in regulating the estrus cycle in sheep.

Molecular Cloning of the B4GALNT2 Gene and Its Single Nucleotide Polymorphisms Association with Litter Size in Small Tail Han Sheep

Simple Summary In French Lacaune sheep, the B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential gene for a FecL (mutation), which regulates the ovine ovulation rate. Three specific mutation sites linked with the FecL mutation have not been previously found in 11 sheep breeds. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 were found to have had a significant effect on the litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). B4GALNT2, which is mainly expressed in ovine ovary, also plays an important role in sheep reproduction. Furthermore, we discovered two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region in ovine granule cells in vitro. Abstract A new fecundity gene named the FecL (mutation), which regulates the ovulation rate, was discovered in French Lacaune sheep. The B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential FecL mutation gene. This study explores whether the effect of the FecL mutation exists in other sheep breeds, and the features of the B4GALNT2 gene in terms of the molecular structure and its expression profile. Using Sanger sequencing, we found that high and low fecundity breeds from among 11 measured sheep breeds all had no variation in the three specific mutation sites, which were linked with the FecL mutation. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 had a significant effect on litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). Two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region were discovered in ovine granule cells in vitro, through the RACE (Rapid amplification of cDNA ends) method. Except for in the kidney and oviduct, no significant difference in expression levels had been found between STH sheep and Tan sheep breeds. The B4GALNT2 gene, as a candidate for FecL, may have a relationship with the differences in litter size in STH sheep. B4GALNT2 is mainly expressed in the ovine ovary, which also suggests that B4GALNT2 plays an important role in sheep reproduction.

Expression Analysis of the Prolific Candidate Genes, BMPR1B, BMP15, and GDF9 in Small Tail Han Ewes with Three Fecundity (FecB Gene) Genotypes

Simple Summary As important prolific candidate genes, BMPR1B, BMP15, and GDF9 may affect the lambing performance of sheep. Therefore, regarding the three FecB genotypes of Small Tail Han (STH) sheep (FecB BB, FecB B+, and FecB ++), this study explored the gene expression characteristics of different tissues using reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR). The results showed that BMPR1B, BMP15, and GDF9 expression differed between the selected tissues, with all being highly expressed in the ovaries. Further analysis indicated that there was no significant difference in BMPR1B expression among the three FecB genotypes, but both GDF9 and BMP15 had the highest expression in FecB B+. As for other non-ovarian tissues, expression also varied. This study is relevant to understanding the high prolificacy of the STH breed. Abstract The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.

Selection signature in domesticated animals

Domesticated animals play an important role in the life of humanity. All these domesticated animals undergo same process, first domesticated from wild animals, then after long time natural and artificial selection, formed various breeds that adapted to the local environment and human needs. In this process, domestication, natural and artificial selection will leave the selection signal in the genome. The research on these selection signals can find functional genes directly, is one of the most important strategies in screening functional genes. The current studies of selection signal have been performed in pigs, chickens, cattle, sheep, goats, dogs and other domestic animals, and found a great deal of functional genes. This paper provided an overview of the types and the detected methods of selection signal, and outlined researches of selection signal in domestic animals, and discussed the key issues in selection signal analysis and its prospects.