Lattice Oxygen Activation through Deep Oxidation of Co4N by Jahn-Teller-active Dopants for Improved Electrocatalytic Oxygen Evolution.

Triggering the lattice oxygen oxidation mechanism is crucial for improving oxygen evolution reaction (OER) performance, because it could bypass the scaling relation limitation associated with the conventional adsorbate evolution mechanism through the directly formation of oxygen-oxygen bond. High-valence transition metal sites are favorable for activating the lattice oxygen, but the deep oxidation of pre-catalysts suffers from a high thermodynamic barrier. Here, taking advantage of the Jahn-Teller (J-T) distortion induced structural instability, we incorporate high-spin Mn3+ (t2g3eg1) dopant into Co4N. Mn dopants enable a surface structural transformation from Co4N to CoOOH, and finally to CoO2, as observed by various in-situ spectroscopic investigations. Furthermore, the reconstructed surface on Mn doped Co4N triggers the lattice oxygen activation, as evidenced experimentally by pH-dependent OER, tetramethylammonium cation adsorption and on-line electrochemical mass spectrometry measurements of 18O-labelled catalysts. In general, this work not only offers the introducing J-T effect approach to regulate the structural transition, but also provides an understanding about the influence of catalyst's electronic configuration on determining the reaction route, which may inspire the design of more efficient catalysts with activated lattice oxygen.

Entropy engineering of La-based perovskite for simultaneous photocatalytic CO2 reduction and biomass oxidation.

Herein, the high-entropy perovskite, i.e. La(FeCoNiCrMn)O3, was prepared for simultaneous CO2 reduction and biomass upgrading. Based on the synergistic effect between the elements in the high-entropy material, an excellent CO evolution rate of 131.8 µmol g-1 h-1 and a xylonic acid yield of 63.9% were gained.

Identifying Candidate Genes for Litter Size and Three Morphological Traits in Youzhou Dark Goats Based on Genome-Wide SNP Markers.

This study aimed to reveal the potential genetic basis for litter size, coat colour, black middorsal stripe and skin colour by combining genome-wide association analysis (GWAS) and selection signature analysis and ROH detection within the Youzhou dark (YZD) goat population (n = 206) using the Illumina GoatSNP54 BeadChip. In the GWAS, we identified one SNP (snp54094-scaffold824-899720) on chromosome 11 for litter size, two SNPs on chromosome 26 (snp11508-scaffold142-1990450, SORCS3) and chromosome 12 (snp55048-scaffold842-324525, LOC102187779) for coat colour and one SNP on chromosome 18 (snp56013-scaffold873-22716, TCF25) for the black middorsal stripe. In contrast, no SNPs were identified for skin colour. In selection signature analysis, 295 significant iHS genomic regions with a mean |iHS| score > 2.66, containing selection signatures encompassing 232 candidate genes were detected. In particular, 43 GO terms and one KEGG pathway were significantly enriched in the selected genes, which may contribute to the excellent environmental adaptability and characteristic trait formation during the domestication of YZD goats. In ROH detection, we identified 4446 ROH segments and 282 consensus ROH regions, among which nine common genes overlapped with those detected using the iHS method. Some known candidate genes for economic traits such as reproduction (TSHR, ANGPT4, CENPF, PIBF1, DACH1, DIS3, CHST1, COL4A1, PRKD1 and DNMT3B) and development and growth (TNPO2, IFT80, UCP2, UCP3, GHRHR, SIM1, CCM2L, CTNNA3 and CTNNA1) were revealed by iHS and ROH detection. Overall, this study is limited by the small population size, which affects the results of GWAS to a certain extent. Nevertheless, our findings could provide the first overview of the genetic mechanism underlying these important traits and provide novel insights into the future conservation and utilisation of Chinese goat germplasm resources.

Genome-wide association analysis of nine reproduction and morphological traits in three goat breeds from Southern China.

OBJECTIVE: This study aimed to investigate the significant single nucleotide polymorphisms (SNPs) and genes associated with nine reproduction and morphological traits in three breed populations of Chinese goats. METHODS: The genome-wide association of nine reproduction and morphological traits (litter size, nipple number, wattle, skin color, coat color, black dorsal line, beard, beard length, and hind leg hair) were analyzed in three Chinese native goat breeds (n = 336) using an Illumina Goat SNP50 Beadchip. RESULTS: A total of 17 genome-wide or chromosome-wide significant SNPs associated with one reproduction trait (litter size) and six morphological traits (wattle, coat color, black dorsal line, beard, beard length, and hind leg hair) were identified in three Chinese native goat breeds, and the candidate genes were annotated. The significant SNPs and corresponding putative candidate genes for each trait are as follows: two SNPs located on chromosomes 6 (CSN3) and 24 (TCF4) for litter size trait; two SNPs located on chromosome 9 (KATNA1) and 1 (UBASH3A) for wattle trait; three SNPs located on chromosome 26 (SORCS3), 24 (DYM), and 20 (PDE4D) for coat color trait; two SNPs located on chromosome 18 (TCF25) and 15 (CLMP) for black dorsal line trait; four SNPs located on chromosome 8, 2 (PAX3), 5 (PIK3C2G), and 28 (PLA2G12B and OIT3) for beard trait; one SNP located on chromosome 18 (KCNG4) for beard length trait; three SNPs located on chromosome 17 (GLRB and GRIA2), 28 (PGBD5), and 4 for hind leg hair trait. In contrast, there were no SNPs identified for nipple number and skin color. CONCLUSION: The significant SNPs or genes identified in this study provided novel insights into the genetic mechanism underlying important reproduction and morphological traits of three local goat breeds in Southern China as well as further potential applications for breeding goats.

Genome-Wide Selective Analysis of Boer Goat to Investigate the Dynamic Heredity Evolution under Different Stages.

Boer goats, as kemp in meat-type goats, are selected and bred from African indigenous goats under a long period of artificial selection. Their advantages in multiple economic traits, particularly their plump growth, have attracted worldwide attention. The current study displayed the genome-wide selection signature analyses of South African indigenous goat (AF), African Boer (BH), and Australian Boer (AS) to investigate the hereditary basis of artificial selection in different stages. Four methods (principal component analysis, nucleotide diversity, linkage disequilibrium decay, and neighbor-joining tree) implied the genomic diversity changes with different artificial selection intensities in Boer goats. In addition, the θπ, FST, and XP-CLR methods were used to search for the candidate signatures of positive selection in Boer goats. Consequently, 339 (BH vs. AF) and 295 (AS vs. BH) candidate genes were obtained from SNP data. Especially, 10 genes (e.g., BMPR1B, DNER, ITGAL, and KIT) under selection in both groups were identified. Functional annotation analysis revealed that these genes are potentially responsible for reproduction, metabolism, growth, and development. This study used genome-wide sequencing data to identify inheritance by artificial selection. The results of the current study are valuable for future molecular-assisted breeding and genetic improvement of goats.

Genetic basis investigation of wattle phenotype in goat using genome-wide sequence data.

In domestic goats, wattles often appear in even numbers, mostly on the neck and a few under the ear. Goat wattle is composed of ectopic cartilage tissue covered by skin and was reported as a dominant inheritance. Thirty-eight goats from two Southwest Chinese breeds were studied to elucidate the genetic basis of wattle phenotype in goat. Their genomes were sequenced for wide-genome selective sweep analysis (WGSA) and a genome-wide association study (GWAS). The WGSA results revealed 500 candidate genes identified by fixation index and π ratio and 261 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched with 195 genes and 38 significantly enriched KEGG items. In particular, three chondrogenesis-related pathways (Wnt, Hippo and MAPK signaling pathways) were found. Among the 500 genes, 474 were enriched to 2855 Gene Ontology items, and four (BMP2, BMP4, RARA and MSX1) were annotated in the regulation and development of chondrogenesis. Four chondrogenesis-related genes (GREM1, NEDD4, ATG7 and ITGA1) were identified from 519 single-nucleotide polymorphisms (SNPs) with a GWAS above the threshold. Six and 11 SNPs on chromosome 10 are located on GREM1 and NEDD4 respectively, and the highest numbers of SNPs on chromosomes 20 and 22 are located on ITGA1 and ATG7 respectively. All of these genes are related to cartilage development. This study identified a series of genes related to chondroplasia by GWAS and WGSA and presented the possibility that wattle inheritance may be influenced by multiple genes. This work provides a new theoretical understanding of the hereditary basis of wattle phenotype.

MicroRNA-127-3p regulates myoblast proliferation by targeting Sept7.

OBJECTIVE: MicroRNAs (miRNAs) are highly conserved, endogenous small RNAs that regulate gene expression at the post-transcriptional level. miR-127 plays an essential role in myogenic differentiation in vivo and in vitro. However, it is not clear whether miR-127-3p affects myogenic cell proliferation. METHODS: The detailed function of miR-127-3p in proliferative C2C12 cell lines and further identified its regulatory mechanism by qRT-PCR, western blot, flow cytometry analysis and luciferase reporter assay. RESULTS: Overexpression of miR-127-3p significantly inhibited proliferation of C2C12 cells and vice versa. Sept7 was a target gene of miR-127-3p using dual-luciferase reporter assay, qRT-PCR, and western blotting. The RNA interference analysis, in which Sept7 was downregulated, showed that Sept7 significantly promoted the proliferation of C2C12 cells. Besides, the expression level of Sept7 was detected analysis in muscle cells and tissues. CONCLUSIONS: These findings reveal that miR-127-3p regulates myoblast proliferation by targeting Sept7.

The microRNA-127-3p directly targeting Vamp2 in C2C12 myoblasts.

MicroRNAs (miRNAs) have been reported that can regulate skeletal muscle growth and development. Previously, we demonstrated that miR-127-3p were differently expressed in skeletal muscle and muscle cells. However, the molecular mechanism of miR-127-3p regulation of skeletal myogenesis are not well elucidated. In this study, we transfected miR-127-3p into C2C12 cells, and found miR-127-3p induces myogenesis by targeting Vamp2. Moreover, the regulatory mechanism of Vamp2 in myoblasts proliferation and differentiation was further confirmed. In conclusion, our data providedevidences that miR-127-3p reciprocally regulated myoblasts proliferation and differentiation through directly targeting Vamp2.

Genetic diversity of the Chinese goat in the littoral zone of the Yangtze River as assessed by microsatellite and mtDNA.

The objective of this study was to assess the genetic diversity and population structure of goats in the Yangtze River region using microsatellite and mtDNA to better understand the current status of those goat genetic diversity and the effects of natural landscape in fashion of domestic animal genetic diversity. The genetic variability of 16 goat populations in the littoral zone of the Yangtze River was estimated using 21 autosomal microsatellites, which revealed high diversity and genetic population clustering with a dispersed geographical distribution. A phylogenetic analysis of the mitochondrial D-loop region (482 bp) was conducted in 494 goats from the Yangtze River region. In total, 117 SNPs were reconstructed, and 173 haplotypes were identified, 94.5% of which belonged to lineages A and B. Lineages C, D, and G had lower frequencies (5.2%), and lineage F haplotypes were undetected. Several high-frequency haplotypes were shared by different ecogeographically distributed populations, and the close phylogenetic relationships among certain low-frequency haplotypes indicated the historical exchange of genetic material among these populations. In particular, the lineage G haplotype suggests that some west Asian goat genetic material may have been transferred to China via Muslim migration.

Comparative transcriptome and histological analyses provide insights into the prenatal skin pigmentation in goat (Capra hircus).

The Youzhou dark goat is a natural mutant with dark skin over the whole body including the visible mucous membranes. In the present study, we characterized 100-day-old fetal skin at the histomorphological and transcriptomic levels in dark-skinned (Youzhou dark goat) and white-skinned (Yudong white goat) goats with deep RNA sequencing, quantitative PCR, and histological methods. Histological analysis indicated that there were marked differences in both melanin distribution and epidermal ultrastructure between the hyperpigmented and normal skin in two breeds of goat. Subsequent analyses suggested that a presumed structure variation (duplication or insertion) in ASIP might be responsible for its lower expression in the hyperpigmented skin (Youzhou dark goat) by determining the distribution of melanocytes across the body at early development stage. Analyses for genes with differential expression between the dark-skinned and white-skinned goats indicated the network composed of ASIP-MC1R, ECM-receptor interaction, and MAPK signaling might play crucial roles in the determination of skin pigmentation in fetal goats. Moreover, we also identified 1,616 novel transcripts in goat skin by RNA sequencing, which may represent two distinct groups of transcript based on their characteristics. Our findings contribute to the understanding of the characteristics of global gene expression in early-stage skin pigmentation and development and describe an animal model for human diseases associated with pigmentation.

Dynamical Expression of MicroRNA-127-3p in Proliferating and Differentiating C2C12 Cells.

MicroRNAs (miRNAs) are highly conserved, short non-coding RNAs that regulate gene expression at the posttranscriptional level. Although many miRNAs are identified in muscles and muscle cells, their individual roles are still not fully understood. In the present study, we investigated a muscle highly-expressed miRNA, miR-127-3p, in C2C12 myoblasts and tissues of goats with different muscle phenotypes (Boer vs Wushan black goats). Our results demonstrated that i) miR-127-3p was extensively expressed in tissues of goats; ii) miR-127-3p was higher expressed in muscle, spleen, heart, and skin in the muscular goats (Boer goats) than the control (Wushan black goats). Then we further characterized the dynamical expression of miR-127-3p, MyoD, MyoG, Myf5, Mef2c, and Myosin in the proliferating and differentiating C2C12 myoblasts at day of 0, 1, 3, 5, and 7 in culture mediums. Especially, we found that miR-127-3p was significantly higher expressed in the proliferating than differentiating cells. Our findings suggest that miR-127-3p probably plays roles in the proliferation and differentiation of myoblasts, which further underlies regulation of muscle phenotype in goats.

Genome-wide analysis of long non-coding RNAs at early stage of skin pigmentation in goats (Capra hircus).

BACKGROUND: Long noncoding RNAs (lncRNAs) play roles in almost all biological processes; however, their function and profile in skin development and pigmentation is less understood. In addition, because lncRNAs are species-specific, their function in goats has not been established. RESULT: We systematically identified lncRNAs in 100-day-old fetal skin by deep RNA-sequencing using the Youzhou dark goat (dark skin) and Yudong white goat (white skin) as a model of skin pigmentation. A total of 841,895,634 clean reads were obtained from six libraries (samples). We identified 1336 specific lncRNAs in fetal skin that belonged to three subtypes, including 999 intergenic lncRNAs (lincRNAs), 218 anti-sense lncRNAs, and 119 intronic lncRNAs. Our results demonstrated significant differences in gene architecture and expression among the three lncRNA subtypes, particularly in terms of density and position bias of transpose elements near the transcription start site. We also investigated the impact of lncRNAs on its target genes in cis and trans, indicating that these lncRNAs have a strict tissue specificity and functional conservation during skin development and pigmentation. CONCLUSION: The present study provides a resource for lncRNA studies in diseases involved in pigmentation and skin development. It expands our knowledge about lncRNA biology as well as contributes to the annotation of the goat genome.

Signals of Ezh2, Src, and Akt Involve in myostatin-Pax7 pathways regulating the myogenic fate determination during the sheep myoblast proliferation and differentiation.

Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis.

Co-expression analysis of fetal weight-related genes in ovine skeletal muscle during mid and late fetal development stages.

BACKGROUND: Muscle development and lipid metabolism play important roles during fetal development stages. The commercial Texel sheep are more muscular than the indigenous Ujumqin sheep. RESULTS: We performed serial transcriptomics assays and systems biology analyses to investigate the dynamics of gene expression changes associated with fetal longissimus muscles during different fetal stages in two sheep breeds. Totally, we identified 1472 differentially expressed genes during various fetal stages using time-series expression analysis. A systems biology approach, weighted gene co-expression network analysis (WGCNA), was used to detect modules of correlated genes among these 1472 genes. Dramatically different gene modules were identified in four merged datasets, corresponding to the mid fetal stage in Texel and Ujumqin sheep, the late fetal stage in Texel and Ujumqin sheep, respectively. We further detected gene modules significantly correlated with fetal weight, and constructed networks and pathways using genes with high significances. In these gene modules, we identified genes like TADA3, LMNB1, TGF-ß3, EEF1A2, FGFR1, MYOZ1, and FBP2 correlated with fetal weight. CONCLUSION: Our study revealed the complex network characteristics involved in muscle development and lipid metabolism during fetal development stages. Diverse patterns of the network connections observed between breeds and fetal stages could involve some hub genes, which play central roles in fetal development, correlating with fetal weight. Our findings could provide potential valuable biomarkers for selection of body weight-related traits in sheep and other livestock.

Identification of the crucial molecular events during the large-scale myoblast fusion in sheep.

It is well known that in sheep most myofibers are formed before birth; however, the crucial myogenic stage and the cellular and molecular mechanisms underpinning phenotypic variation of fetal muscle development remain to be ascertained. We used histological, microarray, and quantitative real-time PCR (qPCR) methods to examine the developmental characteristics of fetal muscle at 70, 85, 100, 120, and 135 days of gestation in sheep. We show that day 100 is an important checkpoint for change in muscle transcriptome and histomorphology in fetal sheep and that the period of 85-100 days is the vital developmental stage for large-scale myoblast fusion. Furthermore, we identified the cis-regulatory motifs for E2F1 or MEF2A in a list of decreasingly or increasingly expressed genes between 85 and 100 days, respectively. Further analysis demonstrated that the mRNA and phosphorylated protein levels of E2F1 and MEF2A significantly declined with myogenic progression in vivo and in vitro. qRT-PCR analysis indicated that PI3K and FST, as targets of E2F1, may be involved in myoblast differentiation and fusion and that downregulation of MEF2A contributes to transition of myofiber types by differential regulation of the target genes involved at the stage of 85-100 days. We clarify for the first time the timing of myofiber proliferation and development during gestation in sheep, which would be beneficial to meat sheep production. Our findings present a repertoire of gene expression in muscle during large-scale myoblast fusion at transcriptome-wide level, which contributes to elucidate the regulatory network of myogenic differentiation.

Genome-wide association studies for growth and meat production traits in sheep.

BACKGROUND: Growth and meat production traits are significant economic traits in sheep. The aim of the study is to identify candidate genes affecting growth and meat production traits at genome level with high throughput single nucleotide polymorphisms (SNP) genotyping technologies. METHODOLOGY AND RESULTS: Using Illumina OvineSNP50 BeadChip, we performed a GWA study in 329 purebred sheep for 11 growth and meat production traits (birth weight, weaning weight, 6-month weight, eye muscle area, fat thickness, pre-weaning gain, post-weaning gain, daily weight gain, height at withers, chest girth, and shin circumference). After quality control, 319 sheep and 48,198 SNPs were analyzed by TASSEL program in a mixed linear model (MLM). 36 significant SNPs were identified for 7 traits, and 10 of them reached genome-wise significance level for post-weaning gain. Gene annotation was implemented with the latest sheep genome Ovis_aries_v3.1 (released October 2012). More than one-third SNPs (14 out of 36) were located within ovine genes, others were located close to ovine genes (878bp-398,165bp apart). The strongest new finding is 5 genes were thought to be the most crucial candidate genes associated with post-weaning gain: s58995.1 was located within the ovine genes MEF2B and RFXANK, OAR3_84073899.1, OAR3_115712045.1 and OAR9_91721507.1 were located within CAMKMT, TRHDE, and RIPK2 respectively. GRM1, POL, MBD5, UBR2, RPL7 and SMC2 were thought to be the important candidate genes affecting post-weaning gain too. Additionally, 25 genes at chromosome-wise significance level were also forecasted to be the promising genes that influencing sheep growth and meat production traits. CONCLUSIONS: The results will contribute to the similar studies and facilitate the potential utilization of genes involved in growth and meat production traits in sheep in future.

Analysis of copy number variations in the sheep genome using 50K SNP BeadChip array.

BACKGROUND: In recent years, genome-wide association studies have successfully uncovered single-nucleotide polymorphisms (SNPs) associated with complex traits such as diseases and quantitative phenotypes. These variations account for a small proportion of heritability. With the development of high throughput techniques, abundant submicroscopic structural variations have been found in organisms, of which the main variations are copy number variations (CNVs). Therefore, CNVs are increasingly recognized as an important and abundant source of genetic variation and phenotypic diversity. RESULTS: Analyses of CNVs in the genomes of three sheep breeds were performed using the Ovine SNP50 BeadChip array. A total of 238 CNV regions (CNVRs) were identified, including 219 losses, 13 gains, and six with both events (losses and gains), which cover 60.35 Mb of the sheep genomic sequence and correspond to 2.27% of the autosomal genome sequence. The length of the CNVRs on autosomes range from 13.66 kb to 1.30 Mb with a mean size of 253.57 kb, and 75 CNVRs events had a frequency > 3%. Among these CNVRs, 47 CNVRs identified by the PennCNV overlapped with the CNVpartition. Functional analysis indicated that most genes in the CNVRs were significantly enriched for involvement in the environmental response. Furthermore, 10 CNVRs were selected for validation and 6 CNVRs were further experimentally confirmed by qPCR. In addition, there were 57 CNVRs overlapped in our new dataset and other published ruminant CNV studies. CONCLUSIONS: In this study, we firstly constructed a sheep CNV map based on the Ovine SNP50 array. Our results demonstrated the differences of two detection tools and integration of multiple algorithms can enhance the detection of sheep genomic structure variations. Furthermore, our findings would be of help for understanding the sheep genome and provide preliminary foundation for carrying out the CNVs association studies with economically important phenotypes of sheep in the future.

Identification and characterization of the miRNA transcriptome of Ovis aries.

The discovery and identification of Ovis aries (sheep) miRNAs will further promote the study of miRNA functions and gene regulatory mechanisms. To explore the microRNAome (miRNAome) of sheep in depth, samples were collected that included eight developmental stages: the longissimus dorsi muscles of Texel fetuses at 70, 85, 100, 120, and 135 days, and the longissimus dorsi muscles of Ujumqin fetuses at 70, 85, 100, 120, and 135 d, and lambs at 0 (birth), 35, and 70 d. These samples covered all of the representative periods of Ovis aries growth and development throughout gestation (about 150 d) and 70 d after birth. Texel and Ujumqin libraries were separately subjected to Solexa deep sequencing; 35,700,772 raw reads were obtained overall. We used ACGT101-miR v4.2 to analyze the sequence data. Following meticulous comparisons with mammalian mature miRNAs, precursor hairpins (pre-miRNAs), and the latest sheep genome, we substantially extended the Ovis aries miRNAome. The list of pre-miRNAs was extended to 2,319, expressing 2,914 mature miRNAs. Among those, 1,879 were genome mapped to unique miRNAs, representing 2,436 genome locations, and 1,754 pre-miRNAs were mapped to chromosomes. Furthermore, the Ovis aries miRNAome was processed using an elaborate bioinformatic analysis that examined multiple end sequence variation in miRNAs, precursors, chromosomal localizations, species-specific expressions, and conservative properties. Taken together, this study provides the most comprehensive and accurate exploration of the sheep miRNAome, and draws conclusions about numerous characteristics of Ovis aries miRNAs, including miRNAs and isomiRs.

[Animal gene pyramiding in cross populations].

Gene pyramiding aims at producing individuals with one superior economic trait according to the optimal breeding scheme involving selection of favorable target alleles or linked markers after crossing basal populations and pyramiding them into a single individual. In consideration of animal traditional cross program along with the features of animal segregating population, four types of cross programs and two types of selection strategies for gene pyramiding are performed from practice perspective of view, two population cross for pyramiding two genes (denoted II), three populations cascading cross for pyramiding three genes (denoted III), four population symmetrical (denoted IV-S) and cascading cross for pyramiding four genes (denoted IV-C), and various schemes (denoted cross program-A-E) were designed for each cross program with different levels of initial favorable allele frequencies, basal population sizes, and trait heritabilities. The process of gene pyramiding for various schemes were simulated and compared based on the population hamming distance, average superior genotype frequencies, and average phenotypic values. By simulation, the results showed that larger base population size and higher initial favorite allele frequency resulted in higher efficiency of gene pyramiding. The order of parent crossing was shown to be the most important factor in cascading cross, but had no significant influence on the symmetric cross. The results also showed that genotypic selection strategy was superior to phenotypic selection in accelerating gene pyramiding. The method and corresponding software would be used to compare different cross schemes and selection strategies. Moreover, our study would help to build the optimal gene pyramiding simulation platform.

Transcript characteristic of myostatin in sheep fibroblasts.

Myostatin, a secreted growth factor highly expressed in skeletal muscle, negatively regulates skeletal muscle growth and differentiation. Recently, myostatin is emerged as a potential target for anti-atrophy and anti-fibrotic therapies. Therefore, to investigate the regulation of myostatin in sheep adult fibroblasts, we used the RNA interference mediated by lentiviral vector to gene silence myostatin. Simultaneously, we also had constructed the sheep myostatin overexpression vector to further explore the function of myostatin in fibroblasts. The results here demonstrated that the lentiviral vector could significantly reduce myostatin gene both at mRNA and protein level by 71% and 67%, respectively (P < 0.01). Inhibition of myostatin also resulted in a remarkable increase of activin receptor 2B (ACV2B), p21, PPARγ, leptin, C/EBPß, and MEF2A expression, and a decrease of Akt1, CDK2, MEF2C, and Myf5 expression. Ectopic myostatin mRNA and protein were also present in the fibroblasts transfection. Furthermore, we observed that overexpression of myostatin contributed to an increase of Akt1, CDK2, Myf5 and PPARγ, and a decrease of p21, C/EBPα and leptin at the transcript level. These results suggested that myostatin positively regulated Akt1, CDK2, Myf5, leptin, and C/EBPα, but negatively regulated p21 mRNA expression in adult fibroblasts, and it also expanded our understanding of the regulation mechanism of myostatin. Moreover, the lentiviral system inactivated myostatin gene in fibroblasts would be used to generate transgenic sheep and to ameliorate muscle fibrosis and atrophy by gene therapy in the future.