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Litter size is one of the most important reproductive traits of sheep, which has pronounced effects on the profit of husbandry enterprises and enthusiasm of breeders. Despite the importance of litter size, the underlying genetic mechanisms have not been entirely elucidated. Therefore, based on a high-density SNP chip, genome-wide comparative analysis was performed between two groups with different fecundity to reveal candidate genes linked to litter size via detection of homozygosity and selection signatures in Luzhong mutton sheep. Consequently, nine promising genes were identified from six runs of homozygosity islands, and functionally linked to reproduction (ACTL7A, ACTL7B, and ELP1), embryonic development (KLF5 and PIBF1), and cell cycle (DACH1, BORA, DIS3, and MZT1). A total of 128 genes were observed under selection, of which HECW1 and HTR1E were related to total lambs born, GABRG3, LRP1B, and MACROD2 to teat number, and AGBL1 to reproductive seasonality. Additionally, the presence of inbreeding depression implies the urgency of reasonable mating system to increase litter size in the present herd. These findings provide a comprehensive insight to the genetic makeup of litter size, and also contribute to implementation of marker-assisted selection in sheep.

The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms (SNPs) of two genes were associated with multiple thoracic vertebrae traits in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen, lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal adipose tissues of two sheep breeds with different thoracic vertebral number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279C>T and SYNDIG1L gene g.82573325C>A in T14 and T13 were genotyped by the Sequenom MassARRAY® SNP assay, and association analysis was performed with the TVN. The results demonstrated that UNC13C gene was extensively expressed in 11 tissues. The expression of UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher than that of T13 groups (PA was significantly correlated with the TVN in both STH (P

Background Many recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays. Results A total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3’UTR at a unique binding site. Conclusions Our results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

The objective of this study was to assess the genetic diversity and phylogenetic relationship of nine sheep populations, including two famous high prolific populations and seven popular mutton populations raised in China. Overall, these sheep populations in this study exhibited a rich genetic diversity. Both the expected heterozygosity and Nei's unbiased gene diversity ranged from 0.64 to 0.75, with the lowest value found in Dorset sheep (DST) and the highest in Hu sheep (HUS) and Ba Han sheep (BAS). The polymorphic information content (PIC) varied between 0.59 in DST and 0.71 in HUS and BAS. Specifically, for individual breeds, the small-tail Han sheep (STH) and the four introduced populations did not display the expected diversity; therefore more attention should be paid to the maintenance of diversity during management of these populations. The results of un-weighted pair-group method (UPGMA) phylogenetic tree and structure analysis indicated that the nine investigated populations can be divided into two groups. Suffolk (SUF) and DST were clustered in one group, and the other group can be further divided into three clusters: German Mutton Merino (GMM)–BAS–Bamei Mutton sheep (BAM), HUS–STH and Du Han (DOS)–Dorper (DOP). This clustering result is consistent with sheep breeding history. TreeMix analysis also hinted at the possible gene flow from GMM to SUF. Together, an in-depth view of genetic diversity and genetic relationship will have important implications for breed-specific management.

The expression characteristics of the hypothalamic–pituitary–gonadal (HPG) axis-related candidate genes, DIO2, EYA3, KISS1 and GPR54, were analyzed in year-round estrous rams (small-tail Han sheep, STH) and seasonally estrous rams (Sunite sheep, SNT) using qPCR. The results were as follows: DIO2 was mainly expressed in pituitary, and KISS1 was specifically expressed in hypothalamus in the two groups. However, EYA3 and GPR54 were widely expressed in the cerebrum, cerebellum, hypothalamus, pituitary, testis, epididymis, vas deferens and adrenal gland tissues in both breeds, with significant differences in the cerebellum, hypothalamus, pituitary, testis and vas deferens tissues. We speculated that DIO2 and KISS1 may have positive roles in different regions in ram year-round estrus. Moreover, the expression patterns of EYA3 and GPR54 suggested that they may regulate the estrous mode of ram via testis and vas deferens. This is the first study to systematically analyze the expression patterns of HPG axis-related genes in rams.

Intense artificial selection has been imposed to Luzhong mutton sheep population in the past years. Improvements on growth and reproductive performance are two breeding goals in the present herd. Although some progresses were phenotypically observed possibly due to inbreeding induced by strong selection in terms of these traits, the genomic evaluation was poorly understood. Therefore, a high-density SNP array was used to characterize the pattern of runs of homozygosity (ROH), estimate inbreeding and inbreeding depressions on early growth performance and litter size based upon ROH, and scan positive selection signatures of recent population. Consequently, a low inbreeding level was observed which had negative effects on litter size, but not on early growth performance. And 160 genes were under selection, of which some were reported to be linked to several traits of sheep including body weight, litter size, carcass and meat quality, milk yield and composition, fiber quality and health, and the top genes were associated with growth (growth hormone [GH]- growth hormone receptor [GHR]- Insulin-like growth factor 1 [IGF1] axis) and litter size (bone morphogenic proteins [BMPs]-associated). The effectiveness of previous breeding measures was highlighted, but purging selection was proposed to alleviate the inbreeding depression on litter size, providing some genomic insights to breeding management of Luzhong mutton sheep.

Prolificacy of most local goat breeds in China is low. Jining Grey goat is one of the most prolific goat breeds in China, it is an important goat breed for the rural economy. ASMT (acetylserotonin O‐methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are essential for animal reproduction. Single nucleotide polymorphisms (SNPs) of ASMT and ADAMTS1 genes in the highly prolific breed (Jining Grey goats), medium prolific breed (Boer goats and Guizhou White goats) and low prolific breeds (Angora goats, Liaoning Cashmere goats and Inner Mongolia Cashmere goats) were detected by polymerase chain reaction‐restriction fragment length polymorphism and sequencing. Two SNPs (g.158122T>C, g.158700G>A) of ASMT gene and two SNPs (g.7979798A>G, g.7979477C>T) of ADAMTS1 gene were identified. For g.158122T>C of ASMT gene, further analysis revealed that genotype TC or CC had 0.66 (p 0.05) was found in litter size between TC and CC genotypes. The SNP (g.158122T>C) caused a p.Tyr298His change and this SNP mutation resulted in changes in protein binding sites and macromolecule‐binding sites. The improvement in reproductive performance may be due to changes in the structure of ASMT protein. For g.7979477C>T of ADAMTS1 gene, Jining Grey does with genotype CT or TT had 0.82 (p 0.05) was found in litter size between CT or TT genotypes. These results preliminarily indicated that C allele (g.158122T>C) of ASMT gene and T allele (g.7979477C>T) of ADAMTS1 gene are potential molecular markers which could improve litter size of Jining Grey goats and be used in goat breeding., ASMT (acetylserotonin O‐methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are very important for female and animal reproduction. One mutation (g.158122T>C) of ASMT gene and one mutation (g.7979477C>T) of ADAMTS1 gene were found in goats in this study, and the association between polymorphisms of ASMT or ADAMTS1 genes and prolificacy in Jining Grey goats were investigated. It suggested the ASMT gene with C allele and ADAMTS1 gene with T allele may increase the litter size in goats.

The 2019 new coronavirus epidemic potentially induced by wild animals has drawn tremendous attention. Wild animal meat contamination and adulteration have become increasingly serious, particularly for highly cooked wild animal meats that are difficult to be detected. In this study, a highly specific polyclonal antibody targeting the cooked rat proteins was developed. The corresponding sandwich ELISA (swELISA) was developed and found highly sensitive and specific for cooked rat meat, while there are no cross-reactions to the cooked chicken, pork and beef meats. The limit of detection (LOD) is determined to be as low as 0.01 ug/L based OD values. The coefficient variation (CV) is 5% and 8% for intra and inter assays, respectively. The recovery efficiencies are between 90% and 110%. The sandwich ELISA can detect both raw and cooked rat meat and is also suitable for Swab test of rat contamination. The results indicated a highly reliable and robust ELISA-based assay for cooked rat meat identification and contamination.

The Booroola fecundity gene (FecB) has a mutation that was found to increase the ovulation rate and litter size in Booroola Merino sheep. This mutation is also associated with the fecundity of small-tail han (STH) sheep, an important maternal breed used to produce hybrid offspring for mutton production in China. Previous research showed that the FecB gene affects reproduction in STH sheep, based on litter size records. However, the effects of this gene on estrus, ovulation, and endocrine characteristics in these sheep remain unclear. Here, we analyzed the traits mentioned earlier and compared them among the three FecB genotypes of STH ewes using estrus synchronization. Overall, 53 pluriparous ewes were selected from among 890 STH ewes and subjected to FecB genotyping for experiments to characterize estrous and ovulation rates. FecB heterozygous (+B) ewes presented an earlier onset of estrus (42.9 ± 2.2 h) and a shorter estrous cycle (17.2 ± 0.2 days) (P ≤ 0.05). The ovulation rates increased with the increasing copy number of the B allele (P ≤ 0.01). Ovulation time showed no significant differences among the three FecB genotypes. The serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, estrogen (E2), and progesterone (P4) were measured in 19 of the ewes. Serum concentrations of E2 and FSH dramatically varied around the time of behavioral estrus. In FecB mutant homozygous (BB) ewes, E2 concentration had two peaks, which were higher (P ≤ 0.05) than those of ++ genotypes. FSH concentration of BB ewes was higher (P ≤ 0.05) than that of the ++ ewes just after estrus. The expression of the estrogen receptor 1 (ESR1) gene in the +B genotype was higher than in the other genotypes. Based on the data for the reproductive performance of STH ewes with the three FecB genotypes, our study suggests that the development of follicles in ewes with the B allele is dependent on the response to FSH regulated by E2 in the early stage. +B ewes, exhibiting moderate ovulation and litter size and a shorter estrous cycle, can be highly recommended in sheep crossbreeding systems for commercial mutton production. Moreover, this study provides useful information to conserve better and use the genetic resources of STH sheep in China.

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Improvement of ewe reproduction is considerable by appropriately increasing litter size and sustaining non-seasonal breeding. However, their genetic makeups have not been entirely elucidated. Genome-wide analyses of 821 individuals were performed by combining three genomic approaches (genome-wide association study, XP-nSL, and runs of homozygosity). Consequently, 35 candidate genes including three domestication genes (TSHR, GTF2A1, and KITLG) were identified. Other than the FecB mutation at BMPR1B, we described a significant association of a missense mutation rs406686139 at seasonal lambing-associated TSHR gene with litter size. Some promising novel genes may be relevant for sheep reproduction by multitude biological processes, such as FETUB functioning in fertilization, HNRNPA1 in oogenesis, DCUN1D1 in spermatogenesis, and HRG in fertility outcome. The present study suggests that improvement of ewe reproduction is attributed to selective breeding, and casts light on the genetic basis and improvement of sheep reproduction.

The pituitary pars tuberalis (PT) is the regulating center of seasonal reproduction, which can sense the melatonin signal and eventually cause downstream changes of GnRH secretion through TSHβ. Recently, lncRNAs have been identified in animal reproductive-related tissues, and they play important roles in reproductive regulation. Therefore, in this study, we expect to identify photoperiod-induced lncRNAs and genes in pituitary PT of sheep by comparison of expression profiles between short photoperiod (SP) and long photoperiod (LP). Through RNA-Seq, a total of 55,472 lncRNAs were identified in pituitary PT of Sunite ewes. The number of differentially expressed (DE) genes and lncRNAs between SP and LP increased gradually with the extension of LP (from LP7 to LP42). The notable LP-induced candidate genes included EYA3, TSHB, SIX1, DCT, VMO1, AREG, SUV39H2, and EZH2, and SP-induced genes involved ENSOARG00000012585, CHGA, FOS, SOCS3, and TH. In enriched pathways for DE genes and lncRNA target genes between SP and LP, the reproduction- and circadian-related pathways were highlighted. In addition, the interactome analysis of lncRNAs and their targets implied that MSTRG.209166 and its trans-target TSHB, MSTRG.288068 and its cis-target SIX1, and ENSOARG00000026131 and its cis-target TH might participate in regulation of seasonal reproduction. Together, these results will help to determine important photoperiod-induced lncRNAs and genes and give us some new insights into the epigenetic regulation of seasonal reproduction in sheep.

Although it has been investigated for many years, the physiological processes regulating prolificacy in sheep remains unclear because of regulation by many genes. To better understand the effects of three single nucleotide polymorphisms (SNPs) comprising g.48462350CT in BMP2, g.58171856CG and g.58171886AC in BMP7, a population genetic analysis was conducted using data obtained from genotyping in 768 sheep from six breeds (three polytocous and three monotocous). The results indicate that all the sheep breeds were considered to conform to the Hardy-Weinberg equilibrium (P 0.05). The associations of these three SNPs with litter size in 384 Small Tail Han sheep were analyzed, therefore, and found to be correlated with fecundity as assessed by mean litter size (P 0.05). Bioinformatic analysis indicated there was a transmembrane domain change that occurred after a mutation in BMP2 at g.48462350CT, and changes involving transcription factors such as USF1, USF2 and INMS1 in the BMP7 promoter region might be involved in greater sheep prolificacy.

Small-tailed Han sheep, with different FecB genotypes, manifest distinct ovulation rates and fecundities, which are due to differences in reproductive hormones secreted by the hypothalamic–pituitary–ovarian axis. Nevertheless, the function of the hypothalamus against a FecB mutant background on increasing ovulation rate is rarely reported. Therefore, we determined the expression profiles of hypothalamus tissue collected from six wild-type (WW) and six FecB mutant homozygous (BB) ewes at the follicular and luteal phases by whole-transcriptome sequencing. We identified 53 differentially expressed mRNAs (DEGs) and 40 differentially expressed long non-coding RNAs (DELs) between the two estrus states. Functional annotation analysis revealed that one of the DEGs, PRL, was particularly enriched in the hypothalamic function, hormone-related, and reproductive pathways. The lncRNA–target gene interaction networks and KEGG analysis in combination suggest that the lncRNAs LINC-676 and WNT3-AS cis-acting on DRD2 and WNT9B in different phases may induce gonadotropin-releasing hormone (GnRH) secretion. Furthermore, there were differences of regulatory elements and WNT gene family members involved in the follicular–luteal transition in the reproductive process between wild-type (WNT7A) and FecB mutant sheep (WNT9B). We combined the DEG and DEL data sets screened from different estrus states and genotypes. The overlap of these two sets was identified to select the mRNAs and lncRNAs that have major effects on ovulation. Among the overlapping molecules, seven DEGs and four DELs were involved in the follicular–luteal transition regulated by FecB mutation. Functional annotation analysis showed that two DEGs (FKBP5 and KITLG) were enriched in melanogenesis, oxytocin, and GnRH secretion. LINC-219386 and IGF2-AS were highly expressed in the BB ewes compared with WW ewes, modulating their target genes (DMXL2 and IGF2) to produce more GnRH during follicular development, which explains why mutated ewes produced more mature follicles. These results from expression profiling of the hypothalamus with the FecB mutation at different estrus states provide new insights into how the hypothalamus regulates ovulation under the effect of the FecB mutation.

Qira black sheep is a famous indigenous sheep breed in China. The objectives of this study are to identify candidate genes related to body size, and to estimate the level of inbreeding depression on body size based on runs of homozygosity in Qira black sheep. Here, 188 adult Qira black sheep were genotyped with a high density (630 K) SNP chip and genome-wide association study for body weight and body size traits (including withers height, body slanting length, tail length, chest girth, chest width, and chest depth) were performed using an additive linear model. In consequence, 12 genome- and chromosome-wide significant SNPs and, accordingly, six candidate genes involved in muscle differentiation, metabolism and cell processes were identified. Of them, ZNF704 (zinc finger protein 704) was identified for body weight; AK2 (adenylate kinase 2) and PARK2 (parkin RBR E3 ubiquitin protein ligase) for tail length; MOCOS (molybdenum cofactor sulfurase) and ELP2 (elongator acetyltransferase complex subunit 2) for chest width; and MFAP1 (microfibril associated protein 1) for chest girth. Additionally, inbreeding depressions on body size were observed in the current herd. These results will provide insightful understandings into the genetic mechanisms of adult body size, and into the conservation and utilization of Qira black sheep.

Simple Summary Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. There is large variation in the reproductive abilities of Bamei mutton sheep. After recent artificial selection, the average lambing rate of the Bamei mutton nucleus group was over 150%. We used the FST (Fixation Index) and XP-EHH (The Cross-Population Extended Haplotype Homozygosity) statistical approach to detect the selective sweeps between high- and low-fecundity Bamei mutton sheep groups. JUN (JUN proto-oncogene, AP-1 transcription factor subunit), ITPR3 (inositol 1,4,5-trisphosphate receptor type 3, PLCB2 (phospholipase C beta 2), HERC5 (HECT and RLD domain containing E3 ubiquitin protein ligase 5), and KDM4B (lysine demethylase 4B) were detected that are potential responsible for litter size. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. Abstract Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection.

Ovine litter size (LS) is an important trait showing variability within breeds. It remains largely unknown whether inbreeding depression on LS exists based on genomic homozygous regions, and whether the homozygous regions resulted from inbreeding are significantly associated with LS in sheep. We here reanalyze a set of single nucleotide polymorphism (SNP) chip of six breeds to characterize the patterns of runs of homozygosity (ROH), to evaluate inbreeding levels and inbreeding depressions on LS, and to identify candidate homozygous regions responsible for LS. Consequently, unique ROH patterns were observed among six sheep populations. Inbreeding depression on LS was only found in Hu sheep, where a significant reduction of 0.016, 0.02, and 0.02 per 1% elevated inbreeding FROH4&ndash, 8, FROH&gt, 8 and the total inbreeding measure was observed, respectively. Nine significantly homozygous regions were found for LS in Hu sheep, where some promising genes for LS possibly via regulation of the development of oocytes (NGF, AKT1, and SYCP1), fertilization (SPAG17, MORC1, TDRD9, ZFYVE21, ADGRB3, and CKB), embryo implantation (PPP1R13B, INF2, and VANGL1) and development (DPPA2, DPPA4, CDCA4, CSDE1, and ADSSL1), and reproductive health (NRG3, BAG5, CKB, and XRCC3) were identified. These results from the present study would provide insights into the genetic management and complementary understandings of LS in sheep.

Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction, however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small-tailed Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as LGALS3 in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.

Follistatin‐like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA‐Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.

Simple Summary Cashmere goats are a unique husbandry resource in China. These goats are well known for producing the highest cashmere yield and best fiber quality in the world. Although cashmere is highly valued and also known as “fiber gem” and “soft gold”, few studies have examined the genetic basis of cashmere traits in cashmere goats. Here, we identified selection signals by comparing Fst and XP-EHH (the cross population extend haplotype homozygosity test) of a non-cashmere breed (Huanghuai goat) with those of two cashmere breeds (Inner Mongolia and Liaoning cashmere goats). Two genes (WNT10A and CSN3) were potentially associated with cashmere traits. This information may be valuable for studying the genetic uniqueness of cashmere goats and elucidating the mechanisms underlying cashmere traits in cashmere goats. Abstract Inner Mongolia and Liaoning cashmere goats in China are well-known for their cashmere quality and yield. Thus, they are great models for identifying genomic regions associated with cashmere traits. Herein, 53 Inner Mongolia cashmere goats, Liaoning cashmere goats and Huanghuai goats were genotyped, and 53,347 single-nucleotide polymorphisms (SNPs) were produced using the Illumina Caprine 50K SNP chip. Additionally, we identified some positively selected SNPs by analyzing Fst and XP-EHH. The top 5% of SNPs had selection signatures. After gene annotation, 222 and 173 candidate genes were identified in Inner Mongolia and Liaoning cashmere goats, respectively. Several genes were related to hair follicle development, such as TRPS1, WDR74, LRRC14, SPTLC3, IGF1R, PADI2, FOXP1, WNT10A and CSN3. Gene enrichment analysis of these cashmere trait-associated genes related 67 enriched signaling pathways that mainly participate in hair follicle development and stem cell pluripotency regulation. Furthermore, we identified 20 overlapping genes that were selected in both cashmere goat breeds. Among these overlapping genes, WNT10A and CSN3, which are associated with hair follicle development, are potentially involved in cashmere production. These findings may improve molecular breeding of cashmere goats in the future.

Tibetan goat is an ancient breed, which inhabits the adverse conditions of the plateaus in China. To investigate the role of selection in shaping its genomes, we genotyped Tibetan goats (Nagqu Prefecture, above 4500 m) and three lowland populations (Xinjiang goats, Taihang goats and Huanghuai goats). The result of PCA, neighbor-joining (N-J) tree and model-based clustering showed that the genetic structure between the Tibetan goat and the three lowland populations has significant difference. As demonstrated by the di statistic, we found that some genes were related to the high-altitude adaptation of Tibetan goats. Functional analysis revealed that these genes were enriched in the VEGF (vascular endothelial growth factor) signaling pathway and melanoma, suggesting that nine genes (FGF2, EGFR, AKT1, PTEN, MITF, ENPEP, SIRT6, KDR, and CDC42) might have important roles in the high-altitude adaptation of Nagqu Tibetan goats. We also found that the LEPR gene was under the strongest selection (di value = 16.70), and it could induce upregulation of the hypoxic ventilatory response. In addition, five genes (LEPR, LDB1, EGFR, NOX4 and FGF2) with high di values were analyzed using q-PCR. Among them, we found that LEPR, LDB1 and FGF2 exhibited higher expression in the lungs of the Tibetan goats, LEPR, EGFR and LDB1 exhibited higher expression in the hearts of the Huanghuai goat. Our results suggest that LEPR, LDB1, EGFR and FGF2 genes may be related to the high-altitude adaptation of the goats. These findings improve our understanding of the selection of the high-altitude adaptability of the Nagqu Tibetan goats and provide new theoretical knowledge for the conservation and utilization of germplasm resources.

The objective of this study was to assess the genetic diversity and population structure of nine sheep populations (four famous mutton breeds worldwide, three popular mutton populations and two most famous high prolific breeds in China) and predict the heterosis among these populations based on microsatellite markers. Results showed the relatively rich genetic diversity in Chinese sheep populations. The results of UPGMA phylogenetic tree and Structure analysis indicated that the nine investigated populations can be divided into two groups. Suffolk and Dorset were clustered in one group, and the other group can be further divided into three clusters: GMM-BAS-BAM, HUS-STH and DOS-DOP. This is consistent with sheep breeding history. TreeMix analysis also hinted the possible gene flow from GMM to SUF. Additionally, based on the calculation of genetic distance, the optimal cross combinations are recommended for two popular mutton sheep populations (BAM and BAS) in the southern Mongolian Plateau and the most famous high prolific breeds (Small Tail Han sheep and Hu sheep) in China, respectively. Together, these results bring us an in-depth view of genetic diversity within sheep population and the genetic relationship among populations.

The pituitary is a vital endocrine organ that regulates animal seasonal reproduction by controlling the synthesis and secretion of the hormone. The change of photoperiod is the key factor affecting the function of the pituitary in animals, but the mechanism is unclear. Here, we studied the transcriptomic variation in pars distalis (PD) of the pituitary between short photoperiod (SP) and long photoperiod (LP) using RNA sequencing based on the OVX+E2 sheep. 346 differentially expressed (DE) lncRNAs and 186 DE-mRNA were found in the PD. Moreover, function annotation analysis indicated that the reproductive hormones and photoperiod response-related pathways including aldosterone synthesis and secretion, insulin secretion, thyroid hormone synthesis, and circadian entrainment were enriched. The interaction analysis of mRNA-lncRNA suggested that MSTRG.240648, MSTRG.85500, MSTRG.32448, and MSTRG.304959 targeted CREB3L1 and DUSP6, which may be involved in the photoperiodic regulation of the PD. These findings provide resources for further study on the seasonal reproductive in ewes.

Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes, however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY&reg, SNP system at the loci g.60495375A &gt, G, g.60495363G &gt, A, and g.60499690C &gt, A in NLRP5, and g.59030623T &gt, C and g.59043397A &gt, C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy&ndash, Weinberg equilibrium (p &gt, 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G &gt, A in NLRP5 and g.59030623T &gt, C in NLRP9, were highly associated with litter size. The mutation in g.60495363G &gt, A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T &gt, C may enhance the combining capacity of NLRP9 with these proteins, consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals.

Photoperiod is one of the important factors leading to seasonal reproduction of sheep. However, the molecular mechanisms underlying the photoperiod regulation of seasonal reproduction remain poorly understood. In this study, we compared the expression profiles of mRNAs, lncRNAs, and circRNAs in uterine tissues from Sunite sheep during three different photoperiods, namely, the short photoperiod (SP), short transfer to long photoperiod (SLP), and long photoperiod (LP). The results showed that 298, 403, and 378 differentially expressed (DE) mRNAs, 171, 491, and 499 DE lncRNAs, and 124, 270, and 400 DE circRNAs were identified between SP and LP, between SP and SLP, and between LP and SLP, respectively. Furthermore, functional enrichment analysis showed that the differentially expressed RNAs were mainly involved in the GnRH signaling pathway, thyroid hormone synthesis, and thyroid hormone signaling pathway. In addition, co-expression networks of lncRNA&ndash, mRNA were constructed based on the correlation analysis between the differentially expressed RNAs. Our study provides new insights into the expression changes of RNAs in different photoperiods, which might contribute to understanding the molecular mechanisms of seasonal reproduction in sheep.

Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., POMC, GNRH1, PRL, GH, TRH, and TTR) and mRNA-miRNAs pairs (e.g., TRH co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and PRL regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropin-releasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release via direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy.

MicroRNA (miRNA) is a sort of endogenous ~20-25 nt non-coding RNAs, and it can regulate a variety of biological events. We found the miR-378 may involve in regulating the muscle development of sheep during our previous research. However, the molecular mechanism of miR-378 regulating myoblast proliferation is still unclear. In this research, we predicted that BMP2 (Bone morphogenetic protein 2) was the target gene of miR-378 and the BMP-Smad signal pathway that BMP2 participated in playing an important role in the muscle development. Therefore, we tried to determine whether miR-378 influence myoblast proliferation of sheep through the BMP-Smad signal pathway. The results indicated that inhibit BMP-Smad signal pathway by interfering Smad4 to promote proliferation of sheep myoblasts; promote BMP-Smad signal pathway by interfering Smad7 to inhibit proliferation of sheep myoblasts; over-expression miR-378 promotes BMP-Smad signal pathway and myoblast proliferation in sheep; interfering miR-378 inhibits BMP-Smad signal pathway and myoblast proliferation in sheep. However, when both of which functioned at the myoblast, miR-378 could not fully depend on BMP-Smad signal pathway to regulate myoblast proliferation. In sum, both miR-378 and BMP-Smad can influence the proliferation of myoblast, but miR-378 does not target the 3' UTR of sheep BMP2.

Inner Mongolia and Liaoning cashmere goats are two outstanding Chinese multipurpose breeds that adapt well to the semi-arid temperate grassland. These two breeds are characterized by their soft cashmere fibers, thus making them great models to identify genomic regions that are associated with cashmere fiber traits. Whole-genome sequencing of 70 cashmere goats produced more than 5.52 million single-nucleotide polymorphisms and 710,600 short insertions and deletions. Further analysis of these genetic variants showed some population-specific molecular markers for the two cashmere goat breeds that are otherwise phenotypically similar. By analyzing FST and θπ outlier values, we identified 135 genomic regions that were associated with cashmere fiber traits within the cashmere goat populations. These selected genomic regions contained genes, which are potential involved in the production of cashmere fiber, such as FGF5, SGK3, IGFBP7, OXTR, and ROCK1. Gene ontology enrichment analysis of identified short insertions and deletions also showed enrichment in keratinocyte differentiation and epidermal cell differentiation. These findings demonstrate that this genomic resource will facilitate the breeding of cashmere goat and other Capra species in future.

CircRNA plays important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports on circRNAs related to livestock reproduction. In this study, we identified circRNAs by deep sequencing and analyzed their expression in the uteri of polytocous and monotocous sheep (FecB++) during follicular and luteal phases. There were 147 and 364 circRNAs with differential expression in the follicular and luteal phases, respectively. GO and KEGG enrichment analysis was performed for the host genes of the circRNAs to predict the functions of differentially expressed circRNAs. These source genes were mainly involved in the estrogen signaling pathway, TGF&beta, signaling pathway, GnRH signaling pathway, oxytocin signaling pathway, pentose phosphate pathway, and starch and sucrose metabolism related to reproduction and energy metabolism. CircRNA expression patterns were validated by RT-qPCR. Our findings provide a solid foundation for the identification and characterization of key important circRNAs involved in reproduction.

Background Long non-coding RNAs (lncRNAs) regulate endometrial secretion and uterine volume. However, there is little research on the role of lncRNAs in the uterus of Small Tail Han sheep (FecB++). Herein, RNA-seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous and monotocous sheep (FecB++) in follicular and luteal phases. Methods To identify lncRNA and mRNA expressed in the uterus, the expression of lncRNA and mRNA in the uterus of Small Tail Han sheep (FecB++) from the polytocous group (n = 6) and the monotocous group (n = 6) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the two groups and two phases . Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to further analyses the function of related genes. Results In the follicular phase, 473 lncRNAs and 166 mRNAs were differentially expressed in polytocous and monotocous sheep; in the luteal phase, 967 lncRNAs and 505 mRNAs were differentially expressed in polytocous and monotocous sheep. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes are mainly involved in ovarian steroidogenesis, retinol metabolism, the oxytocin signaling pathway, steroid hormone biosynthesis, and the Foxo signaling pathway. Key lncRNAs may regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, UGT1A1, LHB, TGFB1, TAB1, and RHOA, which are targeted by MSTRG.134747, MSTRG.82376, MSTRG.134749, MSTRG.134751, and MSTRG.134746, may play key regulatory roles. These results offer insight into molecular mechanisms underlying sheep prolificacy.

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5'-3' exoribonuclease 2 (XRN2) in PF versus MF, and bcl-2-associated athanogene 4 (BAG4), insulin-like growth factor-1 receptor (IGF1R), hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin-releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.

Ovarian activity, which is mainly controlled by follicle-stimulating hormone and luteinizing hormone, is vital to successful reproduction and maintaining reproductive efficiency in livestock. To determine if the regulation of follicular-luteal transition occurs at the post-transcriptional level in hircine ovaries, the expression patterns of small RNAs in the ovarian tissues of Anhui white goats in the follicular and luteal phases were analyzed using Solexa sequencing. In total, 1039 miRNAs were co-expressed in the two libraries, and 278 and 469 miRNAs were specifically expressed in the hircine ovaries during the follicular and luteal phases, respectively. A total of 43 potential novel miRNAs were predicted in the two libraries. GO annotation and KEGG pathway analysis were applied to analyze the target genes of all miRNAs predicted in the two libraries. The highly and differentially expressed miRNAs included miR-26-5p, miR-145-5p, miR-145, miR-145a-5p, miR-125a-5p, miR-320d, and miR-320c, which may participate in follicular-luteal transition. Five co-expressed miRNAs, of which 2 were differentially expressed between the two libraries, were randomly selected to validate the expression pattern using RT-PCR, and the results were consistent with the Solexa sequencing data. Our present results help to clarify the roles of miRNAs in the regulation of follicular-luteal transition in goat ovaries, which may further enhance the reproductive efficiency of commercially important animals in the future.

DIO2 was studied as a candidate gene for reproduction in sheep. Three pairs of primers were designed to detect single nucleotide polymorphisms (SNPs) of exon 2 of DIO2 in nonseasonal and seasonal reproduction breeds. The polymorphisms were detected using SeqMan II and analyzed by PCR-RFLP. The sheep DIO2 gene was cloned and its mutations were detected in five sheep breeds whose reproductive seasonality and litter sizes were different. Association analyses were performed to reveal the relationships between SNPs and reproductive seasonality as well as litter size in sheep. The results showed that a 1041-bp DNA fragment of the sheep DIO2 gene was obtained and a part of the coding sequence of 591 bp encodes 196 amino acids, having high homology with that of other mammals. Moreover, three mutations (G318T, G482C, and C885T) were detected. The test of genotype distribution in five sheep breeds showed that there were significant differences between nonseasonal and seasonal breeds for 318 and 482 loci. However, the associations between the three polymorphisms and sheep seasonal reproduction were not very distinct. Additionally, there was no significant associations between polymorphisms and litter sizes in Small Tail Han sheep. These results may provide the basic theory for further study.

Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p &lt, 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (&minus, 1453 to &minus, 1854) of GDF9 promoter in ovary and heart was determined. In this region (&minus, 1987 to &minus, 1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary, with increased (p &lt, 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = &minus, 0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p &lt, 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

Background Sheep have developed the ability to store fat in their tails, which is a unique way of reserving energy to survive a harsh environment. However, the mechanism underlying this adaptive trait remains largely unsolved. Results In the present study, we provide evidence for the genetic determinants of fat tails, based on whole genome sequences of 89 individual sheep. A genome-wide scan of selective sweep identified several candidate loci including a region at chromosome 13, a haplotype of which underwent rapid evolution and spread through fat-tailed populations in China and the Middle East. Sequence analysis revealed an inter-genic origin of this locus, which later became a hotspot of ruminant-specific retro-transposon named BovB. Additionally, the candidate locus was validated based on a fat- and thin-tailed cross population. The expression of an upstream gene BMP2 was differentially regulated between fat-tailed and thin-tailed individuals in tail adipose and several other tissue types. Conclusions Our findings suggest the fixation of fat tails in domestic sheep is caused by a selective sweep near a retro-transposable hotspot at chromosome 13, the diversity of which specifically affects the expression of BMP2. The present study has shed light onto the understanding of fat metabolism. Electronic supplementary material The online version of this article (10.1186/s12864-019-5620-6) contains supplementary material, which is available to authorized users.

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.

The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic&ndash, pituitary&ndash, 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 &lt, 0.05, p &lt, 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.

Simple Summary LncRNAs may play important role in many biological processes. The aims of this research were to identify potential lncRNAs active in skeletal muscle of the Texel and Ujumqin sheep and investigate their functions. Overall, 2002 lncRNA transcripts were found, some of which may be related to muscle development. The findings obtained here should promote understanding of the regulatory functions of lncRNAs in ovine muscle development and potentially also in other mammals. Abstract Long noncoding RNAs (lncRNAs) are increasingly being recognized as key regulators in many cellular processes. However, few reports of them in livestock have been published. Here, we describe the identification and characterization of lncRNAs in ovine skeletal muscle. Eight libraries were constructed from the gastrocnemius muscle of fetal (days 85 and 120), newborn and adult Texel and Ujumqin sheep. The 2002 identified transcripts shared some characteristics, such as their number of exons, length and distribution. We also identified some coding genes near these lncRNA transcripts, which are particularly associated with transcriptional regulation- and development-related processes, suggesting that the lncRNAs are associated with muscle development. In addition, in pairwise comparisons between the libraries of the same stage in different breeds, a total of 967 transcripts were differentially expressed but just 15 differentially expressed lncRNAs were common to all stages. Among them, we found that TCONS_00013201 exhibited higher expression in Ujumqin samples, while TCONS_00006187 and TCONS_00083104 were higher in Texel samples. Moreover, TCONS_00044801, TCONS_00008482 and TCONS_00102859 were almost completely absent from Ujumqin samples. Our results suggest that differences in the expression of these lncRNAs may be associated with the muscular differences observed between Texel and Ujumqin sheep breeds.

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.

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.

Compared with the commercially available single nucleotide polymorphism (SNP) chip based on the Bead Chip technology, the solution hybrid selection (SHS)-based target enrichment SNP chip is not only design-flexible, but also cost-effective for genotype sequencing. In this study, we propose to design an animal SNP chip using the SHS-based target enrichment strategy for the first time. As an update to the international collaboration on goat research, a 66 K SNP chip for cashmere goat was created from the whole-genome sequencing data of 73 individuals. Verification of this 66 K SNP chip with the whole-genome sequencing data of 436 cashmere goats showed that the SNP call rates was between 95.3% and 99.8%. The average sequencing depth for target SNPs were 40X. The capture regions were shown to be 200 bp that flank target SNPs. This chip was further tested in a genome-wide association analysis of cashmere fineness (fiber diameter). Several top hit loci were found marginally associated with signaling pathways involved in hair growth. These results demonstrate that the 66 K SNP chip is a useful tool in the genomic analyses of cashmere goats. The successful chip design shows that the SHS-based target enrichment strategy could be applied to SNP chip design in other species.

The existence of major genes affecting fecundity in sheep flocks throughout the world has been demonstrated. Three major genes whose mutations can increase ovulation rate have been discovered, and all related to the transforming growth factor β (TGF-β) superfamily. The mutant FecB of bone morphogenetic protein receptor 1B (BMPR1B) has an additive effect on ovulation rate. Six mutations (FecXI, FecXH, FecXG, FecXB, FecXL, FecXR) of bone morphogenetic protein 15 (BMP15) related with fertility have been identified that share the same mechanism. All the mutants can increase ovulation rate in heterozygotes and cause complete sterility in homozygotes. Homozygous ewes with two new mutations (FecXGr, FecXO) of BMP15 had increased ovulation rate without causing sterility. There are five mutations in growth differentiation factor 9 (GDF9) associated with sheep prolificacy where FecGE and FecGF have additive an effect on ovulation rate and litter size. The newly identified β-1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) gene of FecL is proposed as a new mechanism of ovulation rate regulation in sheep. Woodlands is an X-linked maternally imprinted gene which increases ovulation rate. In addition, several putative major genes need to be verified. This review is focused on the identification of the mutations and mechanisms whereby the major genes affecting ovulation rate.

In our early work, one mutation (A898G, which caused Ser32Gly (S32G) change of mature peptide) was identified in the goat BMP15 gene and the allele G was associated with high litter size in Jining Grey goats (the goat breed with the highest fecundity in China). The aim of this research was to investigate the genetic structure of the prolific BMP15 gene in 18 goat breeds reared in China, including 12 native breeds, 3 introduced breeds, and 2 cultivated breeds. Genomic DNA of 1011 goats was screened for the S32G mutation. The results showed that this mutation existed in all 3 cultivated and 8 native goat breeds but in none of the 3 introduced goat breeds, which hinted that this mutation may originate from native goat breeds of China. Besides the Jining Grey goats, the BMP15 gene was also a potential prolificacy gene in Matou goats. Moreover, the structure prediction indicated that the S32G mutation might participate in the binding of BMP15 with receptors.

Simple Summary The hypothalamus has an important role in sheep reproduction. In this study, the key long noncoding RNAs (lncRNAs) associated with sheep fecundity were detected and characterized using the RNA Sequencing technique in sheep hypothalami. The results indicated that several key lncRNAs may affect crucial reproductive processes by differentially influencing the expression of their target genes in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and in polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML). These results provide an insight into the prolificacy mechanism in sheep without FecB mutation in terms of the hypothalamus. Abstract The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.