Integrated Small RNA Sequencing, Transcriptome and GWAS Data Reveal microRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle

Milk protein is one of the most important economic traits in the dairy industry. Yet, the regulatory network of miRNAs for the synthesis of milk protein in mammary is poorly understood. Samples from 12 Chinese Holstein cows with three high ( ≥ 3.5%) and three low ( ≤ 3.0%) phenotypic values for milk protein percentage in lactation and non-lactation were examined through deep small RNA sequencing. We characterized 388 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. We analyzed for enrichments of GWAS signals in miRNAs and their correlated targets. Our results demonstrated that genomic regions harboring DE miRNA genes in lactation were significantly enriched with GWAS signals for milk protein percentage traits and that enrichments within DE miRNA targets were significantly higher than in random gene sets for the majority of milk production traits. This integrated study on the transcriptome and posttranscriptional regulatory profiles between significantly differential phenotypes of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

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Integrated Small RNA Sequencing, Transcriptome, and GWAS Data Reveal miRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle

10.21203/rs.3.rs-143526/v1 ◽

2021 ◽

Author(s):

Wentao Cai ◽

Cong Li ◽

Junya Li ◽

Jiuzhou Song ◽

Shengli Zhang

Keyword(s):

Protein Synthesis ◽

Rna Sequencing ◽

Small Rna ◽

Protein Concentration ◽

Milk Protein ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Production Traits ◽

Economic Traits ◽

Chinese Holstein

Abstract Background: Milk protein is one of the most important economic traits in the milk industry. Our previous study has revealed some functional genes responsible for milk protein synthesis in mammals. Yet, the miRNA-mediated gene regulatory network for the synthesis of milk protein in mammary is poorly understood. Results: 12 samples from Chinese Holstein Cows with three too high and three low phenotypic values for milk protein percentage in lactation and non-lactating were examined through deep small RNA sequencing. By bioinformatics analysis, we characterized 387 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. Using genome-wide association signal (GWAS) enrichment analysis among five milk production traits, we found the differentially expressed targets were significantly related to milk protein traits.Conclusions: This integrated study on the transcriptional and post-transcriptional regulatory profiles between significantly differential phenotype of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

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Integrated Small RNA Sequencing, Transcriptome, and GWAS Data Reveal miRNA Regulation in Response to Milk Protein Traits in Chinese Holstein Cattle

10.21203/rs.3.rs-142254/v1 ◽

2021 ◽

Author(s):

Wentao Cai ◽

Cong Li ◽

Junya Li ◽

Jiuzhou Song ◽

Shengli Zhang

Keyword(s):

Protein Synthesis ◽

Rna Sequencing ◽

Small Rna ◽

Protein Concentration ◽

Milk Protein ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Production Traits ◽

Economic Traits ◽

Chinese Holstein

Abstract BackgroundMilk protein is one of the most important economic traits in the milk industry. Our previous study has revealed some functional genes responsible for milk protein synthesis in mammals. Yet, the miRNA-mediated gene regulatory network for the synthesis of milk protein in mammary is poorly understood. Results12 samples from Chinese Holstein Cows with three too high and three low phenotypic values for milk protein percentage in lactation and non-lactating were examined through deep small RNA sequencing. By bioinformatics analysis, we characterized 387 known and 212 novel miRNAs in the mammary gland. Differentially expressed analysis detected 28 miRNAs in lactation and 52 miRNAs in the non-lactating period with a highly significant correlation with milk protein concentration. Target prediction and correlation analysis identified some key miRNAs and their targets potentially involved in the synthesis of milk protein. Using genome-wide association signal (GWAS) enrichment analysis among five milk production traits, we found the differentially expressed targets were significantly related to milk protein traits.ConclusionsThis integrated study on the transcriptional and post-transcriptional regulatory profiles between significantly differential phenotype of milk protein concentration provides new insights into the mechanism of milk protein synthesis, which should reveal the regulatory mechanisms of milk secretion.

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Association of UDP-galactose-4-epimerase with milk protein concentration in the Chinese Holstein population

Asian-Australasian Journal of Animal Sciences ◽

10.5713/ajas.19.0549 ◽

2020 ◽

Vol 33 (11) ◽

pp. 1725-1731

Author(s):

Cong Li ◽

Wentao Cai ◽

Shuli Liu ◽

Chenghao Zhou ◽

Mingyue Cao ◽

...

Keyword(s):

Protein Concentration ◽

Milk Protein ◽

Association Studies ◽

Regulatory Region ◽

Protein Yield ◽

Strong Linkage Disequilibrium ◽

Phenotypic Variance ◽

Production Traits ◽

Fat Percentage ◽

Chinese Holstein

Objective: An initial RNA-Sequencing study revealed that UDP-galactose-4-epimerase (GALE) was one of the most promising candidates for milk protein concentration in Chinese Holstein cattle. This enzyme catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. To further validate the genetic effect of GALE on milk protein traits, genetic variations were identified, and genotypes-phenotypes associations were performed.Methods: The entire coding region and the 5’-regulatory region (5’-UTR) of GALE were re-sequenced using pooled DNA of 17 unrelated sires. Association studies for five milk production traits were performed using a mixed linear animal model with a population encompassing 1,027 Chinese Holstein cows.Results: A total of three variants in GALE were identified, including two novel variants (g.2114 A>G and g.2037 G>A) in the 5’-UTR and one previously reported variant (g.3836 G>C) in an intron. All three single nucleotide polymorphisms (SNPs) were associated with milk yield (p<0.0001), fat yield (p = 0.0006 to <0.0001), protein yield (p = 0.0232 to <0.0001) and protein percentage (p<0.0001), while no significant associations were detected between the SNPs and fat percentage. A strong linkage disequilibrium (D’ = 0.96 to 1.00) was observed among all three SNPs, and a 5 Kb haplotype block involving three main haplotypes with GAG, AGC, and AGG was formed. The results of haplotype association analyses were consistent with the results of single locus association analysis (p<0.0001). The phenotypic variance ratio above 3.00% was observed for milk protein yield that was explained by SNP-g.3836G >C.Conclusion: Overall, our findings provided new insights into the polymorphic variations in bovine GALE gene and their associations with milk protein concentration. The data indicate their potential uses for marker-assisted breeding or genetic selection schemes.

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Genetic Analyses Confirm SNPs in HSPA8 and ERBB2 are Associated with Milk Protein Concentration in Chinese Holstein Cattle

Genes ◽

10.3390/genes10020104 ◽

2019 ◽

Vol 10 (2) ◽

pp. 104 ◽

Cited By ~ 1

Author(s):

Cong Li ◽

Miao Wang ◽

Wentao Cai ◽

Shuli Liu ◽

Chenghao Zhou ◽

...

Keyword(s):

Dairy Cattle ◽

Milk Production ◽

Protein Concentration ◽

Milk Protein ◽

Genetic Effects ◽

Production Traits ◽

Rna Seq ◽

Milk Production Traits ◽

Chinese Holstein ◽

Tyrosine Kinase 2

Heat shock 70 kDa protein 8 (HSPA8) and erb-b2 receptor tyrosine kinase 2 (ERBB2) were the promising candidates for milk protein concentration in dairy cattle revealed through previous RNA sequencing (RNA-Seq) study. The objective of this post-RNA-Seq study was to confirm genetic effects of HSPA8 and ERBB2 on milk protein concentration in a large Chinese Holstein population and to evaluate the genetic effects of both genes on other milk production traits. There were 2 singlenucleotide polymorphisms (SNPs) identified for HSPA8 and 11 SNPs for ERBB2 by sequencing 17 unrelated Chinese Holstein sires. The SNP-rs136632043 in HSPA8 had significant associations with all five milk production traits (p = 0.0086 to p < 0.0001), whereas SNP-rs132976221 was remarkably associated with three yield traits (p < 0.0001). Nine (ss1996900615, rs109017161, rs109122971, ss1996900614, rs110133654, rs109941438, rs110552983, rs133031530, and rs109763505) of 11 SNPs in ERBB2 were significantly associated with milk protein percentage (p = 0.0177 to p < 0.0001). A 12 Kb haplotype block was formed in ERBB2 and haplotype associations revealed similar effects on milk protein traits. Our findings confirmed the significant genetic effects of HSPA8 and ERBB2 on milk protein concentration and other milk production traits and SNP phenotypic variances above 1% may serve as genetic markers in dairy cattle breeding programs.

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Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

Frontiers in Genetics ◽

10.3389/fgene.2020.548268 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xiaogang Cui ◽

Shengli Zhang ◽

Qin Zhang ◽

Xiangyu Guo ◽

Changxin Wu ◽

...

Keyword(s):

Mammary Gland ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Small Rna ◽

Milk Protein ◽

Milk Composition ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

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Faculty Opinions recommendation of Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1161246.621733 ◽

2009 ◽

Author(s):

Alain Gojon

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Chain Reaction ◽

Polymerase Chain

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Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

International Journal of Toxicology ◽

10.1177/1091581820961515 ◽

2020 ◽

pp. 109158182096151

Author(s):

Jennifer C. Shing ◽

Kai Schaefer ◽

Shaun E. Grosskurth ◽

Andy H. Vo ◽

Tatiana Sharapova ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Exploratory Study ◽

Quantitative Polymerase Chain Reaction ◽

Small Rna Sequencing ◽

Circulating Microrna ◽

Testicular Toxicity ◽

Potential Candidate ◽

Drug Induced ◽

Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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