Transcriptome-wide m6A methylation profiling of Wuhua yellow-feathered chicken ovary revealed regulatory pathways underlying sexual maturation and low egg-laying performance.

RNA N6-melthyladenosine (m6A) can play an important role in regulation of various biological processes. Chicken ovary development is closely related to egg laying performance, which is a process primarily controlled by complex gene regulations. In this study, transcriptome-wide m6A methylation of the Wuhua yellow-feathered chicken ovaries before and after sexual maturation was profiled to identify the potential molecular mechanisms underlying chicken ovary development. The results indicated that m6A levels of mRNAs were altered dramatically during sexual maturity. A total of 1,476 differential m6A peaks were found between these two stages with 662 significantly upregulated methylation peaks and 814 downregulated methylation peaks after sexual maturation. A positive correlation was observed between the m6A peaks and gene expression levels, indicating that m6A may play an important role in regulation of chicken ovary development. Functional enrichment analysis indicated that apoptosis related pathways could be the key molecular regulatory pathway underlying the poor reproductive performance of Wuhua yellow-feathered chicken. Overall, the various pathways and corresponding candidate genes identified here could be useful to facilitate molecular design breeding for improving egg production performance in Chinese local chicken breed, and it might also contribute to the genetic resource protection of valuable avian species.

Resequencing and Signatures of Selective Scans Point to Candidate Genetic Variants for Hair Length Traits in Long-Haired and Normal-Haired Tianzhu White Yak.

Tianzhu white yak is a rare local yak breed with a pure white coat in China. In recent years, breeders have discovered long-haired individuals characterized by long hair on the forehead in the Tianzhu white yak, and the length and density of the hair on these two parts of the body are higher than that of the normal Tianzhu white yak. To elucidate the genetic mechanism of hair length in Tianzhu white yak, we re-sequence the whole genome of long-haired Tianzhu White yak (LTWY) (n = 10) and normal Tianzhu White yak (NTWY) (n = 10). Then, fixation index (F ST), θπ ratio, cross-population composite likelihood ratio (XP-CLR), integrated haplotype score (iHS), cross-population extended haplotype homozygosity (XP-EHH), and one composite method, the de-correlated composite of multiple signals (DCMS) were performed to discover the loci and genes related to long-haired traits. Based on five single methods, we found two hotspots of 0.2 and 1.1 MB in length on chromosome 6, annotating two (FGF5, CFAP299) and four genes (ATP8A1, SLC30A9, SHISA3, TMEM33), respectively. Function enrichment analysis of genes in two hotspots revealed Ras signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, and Rap1 signaling pathway were involved in the process of hair length differences. Besides, the DCMS method further found that four genes (ACOXL, PDPK1, MAGEL2, CDH1) were associated with hair follicle development. Henceforth, our work provides novel genetic insights into the mechanisms of hair growth in the LTWY.

Accuracies of Genomic Prediction for Growth Traits at Weaning and Yearling Ages in Yak.

Genomic selection is a promising breeding strategy that has been used in considerable numbers of breeding projects due to its highly accurate results. Yak are rare mammals that are remarkable because of their ability to survive in the extreme and harsh conditions predominantly at the so-called "roof of the world"-the Qinghai-Tibetan Plateau. In the current study, we conducted an exploration of the feasibility of genomic evaluation and compared the predictive accuracy of early growth traits with five different approaches. In total, four growth traits were measured in 354 yaks, including body weight, withers height, body length, and chest girth in two early stages of development (weaning and yearling). Genotyping was implemented using the Illumina BovineHD BeadChip. The predictive accuracy was calculated through five-fold cross-validation in five classical statistical methods including genomic best linear unbiased prediction (GBLUP) and four Bayesian methods. Body weights at 30 months in the same yak population were also measured to evaluate the prediction at 6 months. The results indicated that the predictive accuracy for the early growth traits of yak ranged from 0.147 to 0.391. Similar performance was found for the GBLUP and Bayesian methods for most growth traits. Among the Bayesian methods, Bayes B outperformed Bayes A in the majority of traits. The average correlation coefficient between the prediction at 6 months using different methods and observations at 30 months was 0.4. These results indicate that genomic prediction is feasible for early growth traits in yak. Considering that genomic selection is necessary in yak breeding projects, the present study provides promising reference for future applications.

Genome-wide detection and sequence conservation analysis of long non-coding RNA during hair follicle cycle of yak.

BACKGROUND: Long non-coding RNA (lncRNA) as an important regulator has been demonstrated playing an indispensable role in the biological process of hair follicles (HFs) growth. However, their function and expression profile in the HFs cycle of yak are yet unknown. Only a few functional lncRNAs have been identified, partly due to the low sequence conservation and lack of identified conserved properties in lncRNAs. Here, lncRNA-seq was employed to detect the expression profile of lncRNAs during the HFs cycle of yak, and the sequence conservation of two datasets between yak and cashmere goat during the HFs cycle was analyzed. RESULTS: A total of 2884 lncRNAs were identified in 5 phases (Jan., Mar., Jun., Aug., and Oct.) during the HFs cycle of yak. Then, differential expression analysis between 3 phases (Jan., Mar., and Oct.) was performed, revealing that 198 differentially expressed lncRNAs (DELs) were obtained in the Oct.-vs-Jan. group, 280 DELs were obtained in the Jan.-vs-Mar. group, and 340 DELs were obtained in the Mar.-vs-Oct. group. Subsequently, the nearest genes of lncRNAs were searched as the potential target genes and used to explore the function of DELs by GO and KEGG enrichment analysis. Several critical pathways involved in HFs development such as Wnt signaling pathway, VEGF signaling pathway, and signaling pathways regulating pluripotency of stem cells, were enriched. To further screen key lncRNAs influencing the HFs cycle, 24 DELs with differ degree of sequence conservation were obtained via a comparative analysis of partial DELs with previously published lncRNA-seq data of cashmere goat in the HFs cycle using NCBI BLAST-2.9.0+, and 3 DELs of them were randomly selected for further detailed analysis of the sequence conservation properties. CONCLUSIONS: This study revealed the expression pattern and potential function of lncRNAs during HFs cycle of yak, which would expand the knowledge about the role of lncRNAs in the HFs cycle. The findings related to sequence conservation properties of lncRNAs in the HFs cycle between the two species may provide valuable insights into the study of lncRNA functionality and mechanism.

Transcriptome and DNA Methylation Analyses of the Molecular Mechanisms Underlying with Longissimus dorsi Muscles at Different Stages of Development in the Polled Yak.

DNA methylation modifications are implicated in many biological processes. As the most common epigenetic mechanism DNA methylation also affects muscle growth and development. The majority of previous studies have focused on different varieties of yak, but little is known about the epigenetic regulation mechanisms in different age groups of animals. The development of muscles in the different stages of yak growth remains unclear. In this study, we selected the longissimus dorsi muscle tissue at three different growth stages of the yak, namely, 90-day-old fetuses (group E), six months old (group M), and three years old (group A). Using RNA-Seq transcriptome sequencing and methyl-RAD whole-genome methylation sequencing technology, changes in gene expression levels and DNA methylation status throughout the genome were investigated during the stages of yak development. Each group was represented by three biological replicates. The intersections of expression patterns of 7694 differentially expressed genes (DEGs) were identified (padj < 0.01, |log2FC| > 1.2) at each of the three developmental periods. Time-series expression profile clustering analysis indicated that the DEGs were significantly arranged into eight clusters which could be divided into two classes (padj < 0.05), class I profiles that were downregulated and class II profiles that were upregulated. Based on this cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs from class I profiles were significantly (padj < 0.05) enriched in 21 pathways, the most enriched pathway being the Axon guidance signaling pathway. DEGs from the class II profile were significantly enriched in 58 pathways, the pathway most strongly enriched being Metabolic pathway. After establishing the methylation profiles of the whole genomes, and using two groups of comparisons, the three combinations of groups (M-vs.-E, M-vs.-A, A-vs.-E) were found to have 1344, 822, and 420 genes, respectively, that were differentially methylated at CCGG sites and 2282, 3056, and 537 genes, respectively, at CCWGG sites. The two sets of data were integrated and the negative correlations between DEGs and differentially methylated promoters (DMPs) analyzed, which confirmed that TMEM8C, IGF2, CACNA1S and MUSTN1 were methylated in the promoter region and that expression of the modified genes was negatively correlated. Interestingly, these four genes, from what was mentioned above, perform vital roles in yak muscle growth and represent a reference for future genomic and epigenomic studies in muscle development, in addition to enabling marker-assisted selection of growth traits.

A Study of Genomic Prediction of 12 Important Traits in the Domesticated Yak (Bos grunniens).

The aim of this study was to explore the possibility of applying GP to important economic traits in the domesticated yak, thus providing theoretical support for its molecular breeding. A reference population was constructed consisting of 354 polled yaks, measuring four growth traits and eight hematological traits related to resistance to disease (involved in immune response and phagocytosis). The Illumina bovine HD 770k chip was used to obtain SNP information of all the individuals. With these genotypes and phenotypes, GBLUP, Bayes B and Bayes Cπ methods were used to predict genomic estimated breeding values (GEBV) and assess prediction capability. The correlation coefficient of the association of GEBV with estimated breeding value (EBV) was used as PA for each trait. The prediction accuracy varied from 0.043 to 0.281 for different traits. Each trait displayed similar PAs when using the three methods. Lymphocyte counts (LYM) exhibited the highest predictive accuracy (0.319) during all GP, while chest girth (CG) provided the lowest predictive accuracy (0.043). Our results showed moderate PA in most traits such as body length (0.212) and hematocrit (0.23). Those traits with lower PA could be improved by using SNP chips designed specifically for yak, a better optimized reference group structure, and more efficient statistical algorithms and tools.

Copy Number Variation of the CADM2 Gene and Its Association with Growth Traits in Yak.

Copy number variation (CNV) is currently accepted as a common source of genetic variation. It is reported that CNVs may influence the resistance to disease and complex economic traits, such as residual feed intake, muscle formation, and fat deposition in livestock. Cell adhesion molecule 2 (CADM2) is expressed widely in the brain and adipose tissue and can regulate body weight through the central nervous system. Growth traits are important economic traits for animal selection. In this study, we aimed to explore the effect of CADM2 gene copy number variants on yak growth traits. Here, two CNVs in the CADM2 gene were investigated using the quantitative polymerase chain reaction (qPCR), and the association of the CNVs with growth traits in yak was analyzed using statistical methods by SPSS software. Differences were considered significant if the p value was < 0.05. Statistical analysis indicated significant association of CADM2-CNV2 with the body weight of the Chinese Ashidan yak. A significant effect of CNV2 (p < 0.05) was found on body weight at 6 months. In CNV2, the gain-type copy number variation exhibited greater performance than the other variants, with greater body weight observed at 6 months (p < 0.05). To the best of our knowledge, this is the first attempt to investigate the function of CADM2-CNVs and their association with growth traits in animals. This may be a useful candidate marker in marker-assisted selection of yaks.

Analysis of Hematological Traits in Polled Yak by Genome-Wide Association Studies Using Individual SNPs and Haplotypes.

Yak (Bos grunniens) is an important domestic animal living in high-altitude plateaus. Due to inadequate disease prevention, each year, the yak industry suffers significant economic losses. The identification of causal genes that affect blood- and immunity-related cells could provide preliminary reference guidelines for the prevention of diseases in the population of yaks. The genome-wide association studies (GWASs) utilizing a single-marker or haplotype method were employed to analyze 15 hematological traits in the genome of 315 unrelated yaks. Single-marker GWASs identified a total of 43 significant SNPs, including 35 suggestive and eight genome-wide significant SNPs, associated with nine traits. Haplotype analysis detected nine significant haplotype blocks, including two genome-wide and seven suggestive blocks, associated with seven traits. The study provides data on the genetic variability of hematological traits in the yak. Five essential genes (GPLD1, EDNRA,APOB, HIST1H1E, and HIST1H2BI) were identified, which affect the HCT, HGB, RBC, PDW, PLT, and RDWSD traits and can serve as candidate genes for regulating hematological traits. The results provide a valuable reference to be used in the analysis of blood properties and immune diseases in the yak.

Genome-wide detection of copy number variations in polled yak using the Illumina BovineHD BeadChip.

BACKGROUND: Copy number variations (CNVs), which are genetic variations present throughout mammalian genomes, are a vital source of phenotypic variation that can lead to the development of unique traits. In this study we used the Illunima BovineHD BeadChip to conduct genome-wide detection of CNVs in 215 polled yaks. RESULTS: A total of 1066 CNV regions (CNVRs) were detected with a total length of 181.6 Mb, comprising ~ 7.2% of the bovine autosomal genome. The size of these CNVRs ranged from 5.53 kb to 1148.45 kb, with an average size of 170.31 kb. Eight out of nine randomly chosen CNVRs were successfully validated by qPCR. A functional enrichment analysis of the CNVR-associated genes indicated their relationship to a number of molecular adaptations that enable yaks to thrive at high altitudes. One third of the detected CNVRs were mapped to QTLs associated with six classes of economically important traits, indicating that these CNVRs may play an important role in variations of these traits. CONCLUSIONS: Our genome-wide yak CNV map may thus provide valuable insights into both the molecular mechanisms of high altitude adaptation and the potential genomic basis of economically important traits in yak.

Characterization of the complete mitochondrial genome sequence of Jialuo sheep (Ovis aries).

Jialuo sheep is one of the important production materials of herdsmen in the pastoral areas of northwestern Sichuan in China. We reported the complete mitogenome of Jialuo sheep for the first time. It is 16,617 bp in length, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (D-loop). In the evolutionary relationship, the mitogenome of Jialuo sheep is closer to Texel sheep. This report will help the further studies of sheep species classification and resource protection.

Characterization of the complete mitochondrial genome of the Bazhou yak (Bos Grunniens).

Yak is an ancient breed and Bazhou Yak is also one of China's unique yak breed resources. In the present study, its complete mitochondrial genome was assembled from Illumina sequencing data and we identified the complete mitochondrial genome of the Bazhou yak (Bos grunniens). The complete mitochondrial DNA is a circular molecule with 16,325 bp length consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a non-coding control region (D-loop). The overall nucleotide composition is A (33.69%), T (27.30%), C (25.79%), and G (13.22%), respectively. The content of C + G is 39.01%. Phylogenetic analysis of the mitochondrial genomes of 15 related species by MEGA7.0 showed that the genetic relationship of Bazhou yak is closer to Datong yak and polled yak.

Genomic Prediction for 25 Agronomic and Quality Traits in Alfalfa (Medicago sativa).

Agronomic and quality traits in alfalfa are very important to forage industry. Genomic prediction (GP) based on genotyping-by-sequencing (GBS) data could shorten the breeding cycles and accelerate the genetic gains of these complex traits, if they display moderate to high prediction accuracies. The aim of this study was to investigate the predictive potentials of these traits in alfalfa. A total of 322 genotypes from 75 alfalfa accessions were used for GP of the agronomic and quality traits, which were related to yield and nutrition value, respectively, using BayesA, BayesB, and BayesCπ methods. Ten-fold cross validation was used to evaluate the accuracy of GP represented by the correlation between genomic estimated breeding value (GEBV) and estimated breeding value (EBV). The accuracies ranged from 0.0021 to 0.6485 for different traits. For each trait, three GP methods displayed similar prediction accuracies. Among 15 quality traits, mineral element Ca had a moderate and the highest prediction accuracy (0.34). NDF digestibility after 48 h (NDFD 48 h) and 30 h (NDFD 30 h) and mineral element Mg had prediction accuracies varying from 0.20 to 0.25. Other traits, for example, fat and crude protein, showed low prediction accuracies (0.05 to 0.19). Among 10 agronomic traits, however, some displayed relatively high prediction accuracies. Plant height (PH) in fall (FH) had the highest prediction accuracy (0.65), followed by flowering date (FD) and plant regrowth (PR) with accuracies at 0.52 and 0.51, respectively. Leaf to stem ratio (LS), plant branch (PB), and biomass yield (BY) reached to moderate prediction accuracies ranging from 0.25 to 0.32. Our results revealed that a few agronomic traits, such as FH, FD, and PR, had relatively high prediction accuracies, therefore it is feasible to apply genomic selection (GS) for these traits in alfalfa breeding programs. Because of the limitations of population size and density of SNP markers, several traits displayed low accuracies which could be improved by a bigger reference population, higher density of SNP markers, and more powerful statistic tools.

MicroRNA-200a regulates adipocyte differentiation in the domestic yak Bos grunniens.

The domestic yak (Bos grunniens) is a culturally important animal that lives at high altitude and is farmed by Tibetan herders for its meat, milk, and other animal by-products. Within the animal, adipose tissue is an important store and source of energy and is used to maintain adequate body temperature during the extended cold seasons. Exploring the biomolecular role of microRNAs (miRNAs) in the regulation of growth, development, and metabolism of yak adipocytes may provide valuable insights into the physiology of adipogenesis in the yak. This study investigated whether and how miR-200a (a miRNA recently reported to promote adipogenesis in ST2 bone marrow stromal cells) regulates adipocyte differentiation in the yak. Expression levels of miR-200a gradually increased during day 0 to day 8 of adipocyte differentiation, and transfection of adipocytes with miR-200a enhanced lipid accumulation and triglyceride content compared to control (un-transfected) adipocytes. We additionally verified (using qRT-PCR analysis) that miR-200a increased the expression of adipocyte-specific genes involved in lipogenic transcription (PPARγ, ELVOL, and C/EBPα), fatty acid synthesis (ACC, ACS, SCD, and FAS), and fatty acid transport (DGAT, LPL, and FABP4). We also found that transfection of adipocytes with miR-200a resulted in suppression of the levels of noncanonical Wnt signaling transcription factors (Wnt5a, TAK1, and NLK). These results indicate that miRNA-200a plays an important role in promoting yak adipocyte differentiation that may operate via the suppression of noncanonical Wnt signaling.

Identification of genetic loci associated with crude protein and mineral concentrations in alfalfa (Medicago sativa) using association mapping.

BACKGROUND: Alfalfa (Medicago sativa) is one of the most important legume forage species in China and many other countries of the world. It provides a quality source of proteins and minerals to animals. Genetic underpinnings for these important traits, however, are elusive. An alfalfa (M. sativa) association mapping study for six traits, namely crude protein (CP), rumen undegraded protein (RUP), and four mineral elements (Ca, K, Mg and P), was conducted in three consecutive years using a large collection encompassing 336 genotypes genotyped with 85 simple sequence repeat (SSR) markers. RESULTS: All the traits were significantly influenced by genotype, environment, and genotype × environment interaction. Eight-five significant associations (P < 0.005) were identified. Among these, five associations with Ca were repeatedly observed and six co-localized associations were identified. CONCLUSIONS: The identified marker alleles significantly associated with the traits provided important information for understanding genetic controls of alfalfa quality. The markers could be used in assisting selection for the individual traits in breeding populations for developing new alfalfa cultivars.