Haifei Wang

Genome-wide detection of copy number variations using high-density SNP genotyping platforms in Holsteins

BackgroundCopy number variations (CNVs) are widespread in the human or animal genome and are a significant source of genetic variation, which has been demonstrated to play an important role in phenotypic diversity. Advances in technology have allowed for identification of a large number of CNVs in cattle. Comprehensive explore novel CNVs in the bovine genome would provide valuable information for functional analyses of genome structural variation and facilitating follow-up association studies between complex traits and genetic variants.ResultsIn this study, we performed a genome-wide CNV detection based on high-density SNP genotyping data of 96 Chinese Holstein cattle. A total of 367 CNV regions (CNVRs) across the genome were identified, which cover 42.74Mb of the cattle genome and correspond to 1.61% of the genome sequence. The length of the CNVRs on autosomes range from 10.76 to 2,806.42 Kb with an average of 96.23 Kb. 218 out of these CNVRs contain 610 annotated genes, which possess a wide spectrum of molecular functions. To confirm these findings, quantitative PCR (qPCR) was performed for 17 CNVRs and 13(76.5%) of them were successfully validated.ConclusionsOur study demonstrates the high density SNP array can significantly improve the accuracy and sensitivity of CNV calling. Integration of different platforms can enhance the detection of genomic structure variants. Our results provide a significant replenishment for the high resolution map of copy number variation in the bovine genome and valuable information for investigation of genomic structural variation underlying traits of interest in cattle.

Identification of Genome-Wide Copy Number Variations among Diverse Pig Breeds Using SNP Genotyping Arrays

Copy number variations (CNVs) are important forms of genetic variation complementary to SNPs, and can be considered as promising markers for some phenotypic and economically important traits or diseases susceptibility in domestic animals. In the present study, we performed a genome-wide CNV identification in 14 individuals selected from diverse populations, including six types of Chinese indigenous breeds, one Asian wild boar population, as well as three modern commercial foreign breeds. We identified 63 CNVRs in total, which covered 9.98 Mb of polymorphic sequence and corresponded to 0.36% of the genome sequence. The length of these CNVRs ranged from 3.20 to 827.21 kb, with an average of 158.37 kb and a median of 97.85 kb. Functional annotation revealed these identified CNVR have important molecular function, and may play an important role in exploring the genetic basis of phenotypic variability and disease susceptibility among pigs. Additionally, to confirm these potential CNVRs, we performed qPCR for 12 randomly selected CNVRs and 8 of them (66.67%) were confirmed successfully. CNVs detected in diverse populations herein are essential complementary to the CNV map in the pig genome, which provide an important resource for studies of genomic variation and the association between various economically important traits and CNVs.

Enhancing genome-wide copy number variation identification by high density array CGH using diverse resources of pig breeds.

Copy number variations (CNVs) are important forms of genomic variation, and have attracted extensive attentions in humans as well as domestic animals. In the study, using a custom-designed 2.1 M array comparative genomic hybridization (aCGH), genome-wide CNVs were identified among 12 individuals from diverse pig breeds, including one Asian wild population, six Chinese indigenous breeds and two modern commercial breeds (Yorkshire and Landrace), with one individual of the other modern commercial breed, Duroc, as the reference. A total of 1,344 CNV regions (CNVRs) were identified, covering 47.79 Mb (∼1.70%) of the pig genome. The length of these CNVRs ranged from 3.37 Kb to 1,319.0 Kb with a mean of 35.56 Kb and a median of 11.11 Kb. Compared with similar studies reported, most of the CNVRs (74.18%) were firstly identified in present study. In order to confirm these CNVRs, 21 CNVRs were randomly chosen to be validated by quantitative real time PCR (qPCR) and a high rate (85.71%) of confirmation was obtained. Functional annotation of CNVRs suggested that the identified CNVRs have important function, and may play an important role in phenotypic and production traits difference among various breeds. Our results are essential complementary to the CNV map in the pig genome, which will provide abundant genetic markers to investigate association studies between various phenotypes and CNVs in pigs.

Targeted resequencing of GWAS loci reveals novel genetic variants for milk production traits

BackgroundGenome wide association study (GWAS) has been proven to be a powerful tool for detecting genomic variants associated with complex traits. However, the specific genes and causal variants underlying these traits remain unclear.ResultsHere, we used target-enrichment strategy coupled with next generation sequencing technique to study target regions which were found to be associated with milk production traits in dairy cattle in our previous GWAS. Among the large amount of novel variants detected by targeted resequencing, we selected 200 SNPs for further association study in a population consisting of 2634 cows. Sixty six SNPs distributed in 53 genes were identified to be associated significantly with on milk production traits. Of the 53 genes, 26 were consistent with our previous GWAS results. We further chose 20 significant genes to analyze their mRNA expression in different tissues of lactating cows, of which 15 were specificly highly expressed in mammary gland.ConclusionsOur study illustrates the potential for identifying causal mutations for milk production traits using target-enrichment resequencing and extends the results of GWAS by discovering new and potentially functional mutations.

Global copy number analyses by next generation sequencing provide insight into pig genome variation

BackgroundCopy number variations (CNVs) confer significant effects on genetic innovation and phenotypic variation. Previous CNV studies in swine seldom focused on in-depth characterization of global CNVs.ResultsUsing whole-genome assembly comparison (WGAC) and whole-genome shotgun sequence detection (WSSD) approaches by next generation sequencing (NGS), we probed formation signatures of both segmental duplications (SDs) and individualized CNVs in an integrated fashion, building the finest resolution CNV and SD maps of pigs so far. We obtained copy number estimates of all protein-coding genes with copy number variation carried by individuals, and further confirmed two genes with high copy numbers in Meishan pigs through an enlarged population. We determined genome-wide CNV hotspots, which were significantly enriched in SD regions, suggesting evolution of CNV hotspots may be affected by ancestral SDs. Through systematically enrichment analyses based on simulations and bioinformatics analyses, we revealed CNV-related genes undergo a different selective constraint from those CNV-unrelated regions, and CNVs may be associated with or affect pig health and production performance under recent selection.ConclusionsOur studies lay out one way for characterization of CNVs in the pig genome, provide insight into the pig genome variation and prompt CNV mechanisms studies when using pigs as biomedical models for human diseases.

Structural variant detection by large-scale sequencing reveals new evolutionary evidence on breed divergence between Chinese and European pigs

In this study, we performed a genome-wide SV detection among the genomes of thirteen pigs from diverse Chinese and European originated breeds by next genetation sequencing, and constrcuted a single-nucleotide resolution map involving 56,930 putative SVs. We firstly identified a SV hotspot spanning 35 Mb region on the X chromosome specifically in the genomes of Chinese originated individuals. Further scrutinizing this region by large-scale sequencing data of extra 111 individuals, we obtained the confirmatory evidence on our initial finding. Moreover, thirty five SV-related genes within the hotspot region, being of importance for reproduction ability, rendered significant different evolution rates between Chinese and European originated breeds. The SV hotspot identified herein offers a novel evidence for assessing phylogenetic relationships, as well as likely explains the genetic difference of corresponding phenotypes and features, among Chinese and European pig breeds. Furthermore, we employed various SVs to infer genetic structure of individuls surveyed. We found SVs can clearly detect the difference of genetic background among individuals. This clues us that genome-wide SVs can capture majority of geneic variation and be applied into cladistic analyses. Characterizing whole genome SVs demonstrated that SVs are significantly enriched/depleted with various genomic features.

Differential expression of genes in milk of dairy cattle during lactation.

Summary The milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA‐seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305‐day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested.

A post-GWAS replication study confirming the PTK2 gene associated with milk production traits in Chinese Holstein.

Our initial genome-wide association study (GWAS) demonstrated that two SNPs (ARS-BFGL-NGS-33248, UA-IFASA-9288) within the protein tyrosine kinase 2 (PTK2) gene were significantly associated with milk production traits in Chinese Holstein dairy cattle. To further validate if the statistical evidence provided in GWAS were true-positive findings, a replication study was performed herein through genotype-phenotype associations. The two tested SNPs were found to show significant associations with milk production traits, which confirmed the associations observed in the original study. Specifically, SNPs lying in the PTK2 gene were also detected by sequencing 14 unrelated sires in Chinese Holsteins and a total of thirty-three novel SNPs were identified. Thirteen out of these identified SNPs were genotyped and tested for association with milk production traits in an independent resource population. After Bonferroni correction for multiple testing, twelve SNPs were statistically significant for more than two milk production traits. Analyses of pairwise D’ measures of linkage disequilibrium (LD) between all SNPs were also explored. Two haplotype blocks were inferred and the association study at haplotype level revealed similar effects on milk production traits. In addition, the RNA expression analyses revealed that a non-synonymous coding SNP (g.4061098T>G) was involved in the regulation of gene expression. Thus the findings presented here provide strong evidence for associations of PTK2 variants with dairy production traits and may be applied in Chinese Holstein breeding program.

Associations between variants of the HAL gene and milk production traits in Chinese Holstein cows

BackgroundThe histidine ammonia-lyse gene (HAL) encodes the histidine ammonia-lyase, which catalyzes the first reaction of histidine catabolism. In our previous genome-wide association study in Chinese Holstein cows to identify genetic variants affecting milk production traits, a SNP (rs41647754) located 357 bp upstream of HAL, was found to be significantly associated with milk yield and milk protein yield. In addition, the HAL gene resides within the reported QTLs for milk production traits. The aims of this study were to identify genetic variants in HAL and to test the association between these variants and milk production traits.ResultsFifteen SNPs were identified within the regions under study of the HAL gene, including three coding mutations, seven intronic mutations, one promoter region mutation, and four 3′UTR mutations. Nine of these identified SNPs were chosen for subsequent genotyping and association analyses. Our results showed that five SNP markers (ss974768522, ss974768525, ss974768531, ss974768533 and ss974768534) were significantly associated with one or more milk production traits. Haplotype analysis showed that two haplotype blocks were significantly associated with milk yield and milk protein yield, providing additional support for the association between HAL variants and milk production traits in dairy cows (P < 0.05).ConclusionOur study shows evidence of significant associations between SNPs within the HAL gene and milk production traits in Chinese Holstein cows, indicating the potential role of HAL variants in these traits. These identified SNPs may serve as genetic markers used in genomic selection schemes to accelerate the genetic gains of milk production traits in dairy cattle.

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs

MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.