The microRNA miR-14 Regulates Egg-Laying by Targeting EcR in Honeybees (Apis mellifera).

Honeybees (Apis mellifera) are important pollinators and are commonly used for honey production. The oviposition behavior in honeybees is complex and errors in oviposition could affect the development of the bee colony. Recent studies reported that RNA-RNA cross-talk played a critical role in several biological processes, including reproduction. Ecdysone receptor (EcR) and miR-14 were previously reported to play important roles in egg-laying. Moreover, EcR was predicted to be the target gene of miR-14 and may form miR-14-EcR cross-talk. In this study, knocking down and overexpression of miR-14 and EcR in queen model were implemented. The effect of RNA expression of miR-14 and EcR on the number of eggs laid by honeybee queens were analyzed. Further, luciferase assay was used to confirm the target relation between miR-14 and 3'UTR of EcR. The results showed that the expression of miR-14 and EcR was associated with the number of eggs laid by queens. In specific, inhibition of miR-14 expression enhanced the number of eggs laid, while overexpression of EcR enhanced the number of eggs laid. Lastly, we determined that miR-14 directly targets the mRNA of EcR. These findings suggest that the cross-talk of miR-14-EcR plays an important role in the number of eggs laid by honeybee queens.

Introgression of Chinese haplotypes contributed to the improvement of Danish Duroc pigs.

The distribution of Asian ancestry in the genome of Danish Duroc pigs was investigated using whole-genome sequencing data from European wild boars, Danish Duroc, Chinese Meishan and Bamaxiang pigs. Asian haplotypes deriving from Meishan and Bamaxiang occur widely across the genome. Signatures of selection on Asian haplotypes are common in the genome, but few of these haplotypes have been fixed. By defining 50-kb windows with more than 50% Chinese ancestry, which did not exhibit extreme genetic differentiation between Meishan and Bamaxiang as candidate regions, the enrichment of quantitative trait loci in candidate regions supports that Asian haplotypes under selection play an important role in contributing genetic variation underlying production, reproduction, meat and carcass, and exterior traits. Gene annotation of regions with the highest proportion of Chinese ancestry revealed genes of biological interest, such as NR6A1. Further haplotype clustering analysis suggested that a haplotype of Chinese origin around the NR6A1 gene was introduced to Europe and then underwent a selective sweep in European pigs. Besides, functional genes in candidate regions, such as AHR and PGRMC2, associated with fertility, and SAL1, associated with meat quality, were identified. Our results demonstrate the contribution of Asian haplotypes to the genomes of European pigs. Findings herein facilitate further genomic studies such as genomewide association study and genomic prediction by providing ancestry information of variants.

Genome-wide detection of selection signatures in Chinese indigenous Laiwu pigs revealed candidate genes regulating fat deposition in muscle.

BACKGROUND: Currently, genome-wide scans for positive selection signatures in commercial breed have been investigated. However, few studies have focused on selection footprints of indigenous breeds. Laiwu pig is an invaluable Chinese indigenous pig breed with extremely high proportion of intramuscular fat (IMF), and an excellent model to detect footprint as the result of natural and artificial selection for fat deposition in muscle. RESULT: In this study, based on GeneSeek Genomic profiler Porcine HD data, three complementary methods, FST, iHS (integrated haplotype homozygosity score) and CLR (composite likelihood ratio), were implemented to detect selection signatures in the whole genome of Laiwu pigs. Totally, 175 candidate selected regions were obtained by at least two of the three methods, which covered 43.75 Mb genomic regions and corresponded to 1.79% of the genome sequence. Gene annotation of the selected regions revealed a list of functionally important genes for feed intake and fat deposition, reproduction, and immune response. Especially, in accordance to the phenotypic features of Laiwu pigs, among the candidate genes, we identified several genes, NPY1R, NPY5R, PIK3R1 and JAKMIP1, involved in the actions of two sets of neurons, which are central regulators in maintaining the balance between food intake and energy expenditure. CONCLUSIONS: Our results identified a number of regions showing signatures of selection, as well as a list of functionally candidate genes with potential effect on phenotypic traits, especially fat deposition in muscle. Our findings provide insights into the mechanisms of artificial selection of fat deposition and further facilitate follow-up functional studies.

Population admixture in Chinese and European Sus scrofa.

Relationships between different populations were investigated using Porcine 60 K data from 1,135 domestic pigs and wild boars across Europe and China. The results indicate that most European breeds have been introgressed with Chinese ancestry, but the extent of introgression varies considerably among breeds. Moreover, the main source of this introgression is pigs from South China, closely related to Bamaxiang and Dongshan pigs. Contributions from East and Central Chinese pig breeds are also detectable. Phylogeny reconstruction places European wild boars among European domestic breeds. Coalescent simulations indicate that this may be the result of gene flow from European wild boars to European domestic pigs. These results will facilitate further genomic studies such as genome-wide association studies, selection signature detection and genomic prediction.

Performance Gains in Genome-Wide Association Studies for Longitudinal Traits via Modeling Time-varied effects.

Complex traits with multiple phenotypic values changing over time are called longitudinal traits. In traditional genome-wide association studies (GWAS) for longitudinal traits, a combined/averaged estimated breeding value (EBV) or deregressed proof (DRP) instead of multiple phenotypic measurements per se for each individual was frequently treated as response variable in statistical model. This can result in power losses or even inflate false positive rates (FPRs) in the detection due to failure of exploring time-dependent relationship among measurements. Aiming at overcoming such limitation, we developed two random regression-based models for functional GWAS on longitudinal traits, which could directly use original time-dependent records as response variable and fit the time-varied Quantitative Trait Nucleotide (QTN) effect. Simulation studies showed that our methods could control the FPRs and increase statistical powers in detecting QTN in comparison with traditional methods where EBVs, DRPs or estimated residuals were considered as response variables. Besides, our proposed models also achieved reliable powers in gene detection when implementing into two real datasets, a Chinese Holstein Cattle data and the Genetic Analysis Workshop 18 data. Our study herein offers an optimal way to enhance the power of gene detection and further understand genetic control of developmental processes for complex longitudinal traits.

Characterization of genome-wide segmental duplications reveals a common genomic feature of association with immunity among domestic animals.

BACKGROUND: Segmental duplications (SDs) commonly exist in plant and animal genomes, playing crucial roles in genomic rearrangement, gene innovation and the formation of copy number variants. However, they have received little attention in most livestock species. RESULTS: Aiming at characterizing SDs across the genomes of diverse livestock species, we mapped genome-wide SDs of horse, rabbit, goat, sheep and chicken, and also enhanced the existing SD maps of cattle and pig genomes based on the most updated genome assemblies. We adopted two different detection strategies, whole genome analysis comparison and whole genome shotgun sequence detection, to pursue more convincing findings. Accordingly we identified SDs for each species with the length of from 21.7 Mb to 164.1 Mb, and 807 to 4,560 genes were harboured within the SD regions across different species. More interestingly, many of these SD-related genes were involved in the process of immunity and response to external stimuli. We also found the existence of 59 common genes within SD regions in all studied species except goat. These common genes mainly consisted of both UDP glucuronosyltransferase and Interferon alpha families, implying the connection between SDs and the evolution of these gene families. CONCLUSIONS: Our findings provide insights into livestock genome evolution and offer rich genomic sources for livestock genomic research.

Identification of selective sweeps reveals divergent selection between Chinese Holstein and Simmental cattle populations.

BACKGROUND: The identification of signals left by recent positive selection provides a feasible approach for targeting genomic variants that underlie complex traits and fitness. A better understanding of the selection mechanisms that occurred during the evolution of species can also be gained. In this study, we simultaneously detected the genome-wide footprints of recent positive selection that occurred within and between Chinese Holstein and Simmental populations, which have been subjected to artificial selection for distinct purposes. We conducted analyses using various complementary approaches, including LRH, XP-EHH and FST, based on the Illumina 770K high-density single nucleotide polymorphism (SNP) array, to enable more comprehensive detection. RESULTS: We successfully constructed profiles of selective signals in both cattle populations. To further annotate these regions, we identified a set of novel functional genes related to growth, reproduction, immune response and milk production. There were no overlapping candidate windows between the two breeds. Finally, we investigated the distribution of SNPs that had low FST values across five distinct functional regions in the genome. In the low-minor allele frequency bin, we found a higher proportion of low-FST SNPs in the exons of the bovine genome, which indicates strong purifying selection of the exons. CONCLUSIONS: The selection signatures identified in these two populations demonstrated positive selection pressure on a set of important genes with potential functions that are involved in many biological processes. We also demonstrated that in the bovine genome, exons were under strong purifying selection. Our findings provide insight into the mechanisms of artificial selection and will facilitate follow-up functional studies of potential candidate genes that are related to various economically important traits in cattle.

Expression of genes involved in progesterone receptor paracrine signaling and their effect on litter size in pigs.

BACKGROUND: Embryonic mortality during the period of implantation strongly affects litter size in pigs. Progesterone receptor (PGR) paracrine signaling has been recognized to play a significant role in embryonic implantation. IHH, NR2F2, BMP2, FKBP4 and HAND2 were proved to involve in PGR paracrine signaling. The objective of this study was to evaluate the expression of IHH, NR2F2, BMP2, FKBP4 and HAND2 in endometrium of pregnant sows and to further investigate these genes' effect on litter size in pigs. Real-time PCR, western blot and immunostaining were used to study target genes/proteins expression in endometrium in pigs. RFLP-PCR was used to detect single nucleotide polymorphisms (SNPs) of target genes. RESULTS: The results showed that the mRNA and protein expression levels of IHH, NR2F2 and BMP2 were up-regulated during implantation period (P < 0.05 or P < 0.01). All target proteins were mainly observed in luminal epithelium and glandular epithelium. Interestingly, the staining of NR2F2 and HAND2 was also strong in stroma. SNPs detection revealed that there was a -204C > A mutation in promoter region of NR2F2 gene. Three genotypes were found in Large White, Landrace and Duroc sows. A total of 1847 litter records from 625 sows genotyped at NR2F2 gene were used to analyze the total number born (TNB) and number born alive (NBA). The study of the effect on litter size suggested that sows with genotype CC tend to have higher litter size. CONCLUSIONS: These results showed the expression patterns of genes/proteins involved in PGR paracrine signaling over implantation time. And the candidate gene for litter size was identified from genes involved in this signaling. This study could be a resource for further studies to identify the roles of these genes for embryonic implantation in pigs.

Differential gene expression in uterine endometrium during implantation in pigs.

Embryonic mortality during the implantation period strongly affects litter size in pigs. To analyze the differentially expressed genes (DEGs) in the endometrium during implantation and further to identify candidate genes for litter size, tissues of endometrial attachment sites and intersites were collected from nine pregnant sows on Days 13, 18, and 24 of pregnancy. Endometrium tissue was also collected from another three nonpregnant sows. Samples were hybridized to the porcine Agilent GeneChip microarray. The analysis of gene expression patterns over the implantation period revealed 858 DEGs at endometrial attachment sites. Comparisons of the gene files of attachment sites and intersites revealed 12, 51, and 89 DEGs on Days 13, 18, and 24 of pregnancy, respectively. Annotated function was used to identify overrepresented genetic processes, and several biological processes were considered as the most enriched. Genes related to vascular development, proteolysis, RNA metabolism and translation, protein modification, immune response, and hormone-related are discussed in detail. Then we combined microarray technology and linkage analysis to select powerful candidate genes for quantitative trait loci affecting pig litter size. Eighty-seven DEGs were located in quantitative trait loci related to litter size, that is, total number born and number born alive. Those candidate genes were thought to affect litter size by influencing embryonic implantation. Furthermore, single nucleotide polymorphism of VEGFA was shown to be associated with litter size in pigs. This study identified candidate genes for litter size that were related to embryonic implantation and could be a resource for target studies of genetic markers for litter size in pigs.

[Analysis of association between CRS-PCR polymorphisms of integrin ß1 gene and litter size in pigs].

Single nucleotide polymorphisms of exon 5 T32207C and exon 7 A35230G of integrin ß1 gene were detected in Landrace, Large White and Duroc by CRS-RFLP. Association between the polymorphism and litter size was analyzed by the method of least square means. At 32207 polymorphic loci, there was no significant difference on TNB and NBA between the genotypes in Landrace, Large White, and Duroc. At 35230 polymorphic loci, there was significant difference (P<0.05) or greatly significant difference (P<0.01) on TNB and NBA between genotypes in the first, second, and all parities in Large White and Landrace. The effects of GG and AG genotypes were different from that of AA genotypes with the order of GG, AG>AA. These results suggested that the effect of G allele of integrin ß1 gene on litter size is significant in Large White and Landrace.

Molecular cloning, tissue expression and protein structure prediction of the porcine 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR) gene.

3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR) is the rate-limiting enzyme for cholesterol synthesis. Its activity is regulated via a negative feedback mechanism through sterols and non-sterol metabolites derived from mevalonate, the product of the reaction catalyzed by reductase. Here, we cloned a full-length transcript of porcine HMGR by RT-PCR and RACE. The porcine HMGR cDNA (2864 bp,GenBank accession no. DQ432054) contains a 2658 bp open reading frame and shares 91% identify with those of human and cattle, and 85%, 85% and 84% identify with the HMGR coding sequences of Norway rat, golden hamster, and house mouse, respectively. The deduced porcine HMGR protein consists of 885 amino acids with a calculated molecular mass of 97.15 kDa(GenBank accession no. ABD96089). The amino acid sequence similarities correspond to 95%, 95%, 92%, 92% and 92% when compared with human, cattle, Norway rat, golden hamster and house mouse sequences, respectively. The structure and function of HMGR deduced protein product were predicted by bioinformatic approaches. HMGR-specific transcripts were found in 15 different tissues from pig by RT-PCR and Real-time PCR. The relative expression level of HMGR was high in liver, heart, kidney, bladder and subcutaneous fat, medium in lung, uterus and large intestine, and low in cerebrum, spleen, spinal cord, stomach, ovary, longissimus muscle, and small intestine. The SNPs analysis of HMGR showed that there were five SNPS and three of them are synonymous mutations and the other two are missense mutations. Taken together, our data may lay a ground for further investigation of HMGR's functions and regulatory mechanisms in swine.

Association of EphA4 polymorphism with swine reproductive traits and mRNA expression of EphA4 during embryo implantation.

The swine erythropoietin-producing hepatocellular A4 (EphA4) gene, which was detected in the endometrium during embryo implantation in pigs, was one of the potential candidate genes for reproductive traits. In the study, two single nucleotide polymorphism (SNP) loci (EphA4_1 and EphA4_2) in exon 3 of EphA4 gene were analyzed to determine whether EphA4 influenced total number born (TNB) and number born alive (NBA). Association of two diallelic polymorphisms with reproductive traits was assessed in Landrace, Yorkshire and Duroc populations with 2,014 litter records of 765 sows. The results showed that G allele at EphA4_1 locus or C allele at EphA4_2 locus seemed to have advantageous effects on litter size. And the combined analyzed results demonstrated that genotype AGTC, AGCC and GGCC are better than genotype AATT, AATC and AGTT for TNB and NBA in either single parity or all parities. The results in this study demonstrated that EphA4 gene was significantly associated with litter size in pigs. In addition, a high mRNA expression of EphA4 was found in small intestine, large intestine, stomach and endometrium, and the expression decreased during implantation in pigs. Further studies were needed to confirm these preliminary researches.

Expression of Eph A molecules during swine embryo implantation.

Eph-Ephrin system can induce repulsive forces in cell migration and adhesion during embryonic development in various mammals. In this study, the attachment sites of swine endometrium during pregnancy were used and the physiological role of this system in the step of mammalian embryo implantation was estimated to investigate the involvement of the Eph-Ephrin system in swine embryo implantation. Real-time quantitative PCR indicated that mRNA expression of Eph A1 on endometrium increased extremely significantly around the implantation period (P < 0.01), while expression of Eph A2 and A4 decreased significantly during this period (P < 0.05). Immunostaining showed that protein expression of Eph A1, A2 and A4 in the endometrial stroma underlying the luminal epithelium was higher during mid-implantation compared with early or post-implantation. Western blotting examination demonstrated that protein expression of Eph A1, A2 and A4 at the attachment sites of swine endometrium increased from pregnancy day 13 to 18 (P < 0.01), and then decreased from pregnancy day 18 to 24 (P < 0.01). These findings suggest that the Eph-Ephrin A system might play an important role in regulating the swine contact between blastocysts and endometrium during embryo implantation.

Expression of tissue inhibitor of metalloproteinases-3 messenger RNA and protein in porcine endometrium during implantation.

Recent evidence points to a stromal decidualization-like response in the pregnant porcine uterus. The objective of this study was to evaluate expression of tissue inhibitors of metalloproteinase-3 (TIMP-3), a sensitive indicator of endometrial stromal decidualization, in endometrium of pregnant sows and to further investigate this phenomenon. Real-time PCR, Western blot and immunostaining analysis were used to study TIMP-3 expression between/at attachment sites of endometrium of Days 13, 18 and 24 pregnant sows. The results indicate that TIMP-3 protein expression was lowest by Day 13 compared with Day 18 (P<0.01) and 24 (P<0.01), and the expression was higher at attachment sites than between attachment sites on Day 13 (P<0.01) and 18 (P<0.01). TIMP-3 intensive immunostaining was observed in stroma of endometrium on Days 13, 18 and 24, and the staining at attachment sites was stronger than between attachment sites. Collectively, these results suggest the crucial role of TIMP-3 in successful implantation and embryo survival and indicate the endometrial stromal decidualization-like in pigs.

Temporal and spatial expression of Muc1 during implantation in sows.

Recent evidence points to an important role for Muc1 in embryo implantation. In this study, Real-time PCR and immunohistochemistry were used to study mRNA and protein levels at, and between, the attachment sites of the endometrium of Day 13, 18 and 24 pregnant sows. The results indicate that Muc1 mRNA expression was higher between attachment sites than at attachment sites during implantation and this effect was significant on Day 13 (P < 0.01) and 24 (P < 0.01). Intense Muc1 immunostaining was observed in luminal epithelium and stroma and the staining between attachment sites was stronger than at attachment sites on Days 13 and 18. Collectively, these results suggest the crucial role of Muc1 in successful implantation and embryo survival.