Exploring genotype-phenotype relationships of the CRABP2 gene on growth traits in beef cattle.

Cellular retinoic acid binding protein 2 (CRABP2) is essential to myoblast differentiation. However, there was no report about the function of CRABP2 gene in cattle. This study explored the association of CRABP2 gene polymorphisms with growth traits in cattle breeds by several methods, such as DNA sequencing, PCR, PCR-RFLP and forced PCR-RFLP. Two sequence variants were determined. There were 621 individuals in six cattle breeds from China for the experiment, and three breeds were used to test validation of polymorphisms and extent of linkage disequilibrium (LD). The results showed that both SNPs (SNP1, g.2458 G > T, SNP2, g.3878 G > A) were in intron1. Two SNPs were in low linkage disequilibrium. Association analysis suggested that SNP1 had the significant difference on growth traits with body height, height at hip cross and body slanting length (P < .05), while SNP2 showed a significant difference in growth traits with body height, height at hip cross and body slanting length(P < .05). The results of this investigation displayed that the CRABP2 gene is an available candidate gene and may be used for breed selection and conservation.

Integrative analysis of APOL3 gene CNV for adult cattle stature.

Copy number variations (CNVs) have been identified as another important structural variation of genome. In recent years, a large amount of CNVRs have been identified in humans and animals. However, association and dosage effects studies of CNVs are very limited. Apolipoprotein L3 (APOL3) gene plays a central role in modulating gene transcription and is located within a CNVR that encompasses quantitative trait locis (QTLs) for economic traits like meat quality. Herein, we analyzed the CNV polymorphism of APOL3 in 421 individuals from five distinct cattle breeds, and then correlated their genotypes with growth traits. Association analysis revealed that the APOL3 CNV was significantly associated with hip height and cannon circumference of Xianan (XN) cattle (P < .01), and visibly associated with body slanting length and hucklebone width of Pinan (PN) cattle (P < .05). Overall, the data provide evidence for the functional role of APOL3 CNV and a basis for future applications in cattle breeding.

Copy number variation (CNV) in the IGF1R gene across four cattle breeds and its association with economic traits.

The insulin-like growth factor 1 receptor (IGF1R) plays a vital role in immunomodulation and muscle and bone growth. The copy number variation (CNV) is believed to the reason for many complex phenotypic variations. In this paper, we statistically analyzed the copy number and the expression profiling in different tissue types of the IGF1R gene using the 422 samples from four Chinese beef cattle breeds, and the mRNA of IGF1R was widely expressed in nine tissue types of adult cattle (heart, liver, kidney, muscle, fat, stomach, spleen, lung and testis). Results of CNV and growth traits indicated that the IGF1R CNV was significantly associated with body weight and body height of Jinnan (JN) cattle and was significantly associated with body height and hucklebone width of Qinchuan (QC) cattle, making IGF1R CNV a promising molecular marker to improve meat production in beef cattle breeding. Bioinformatics predictions show that the CNV region is highly similar to the human genome, and there are a large number of transcription factors, DNase I hypersensitive sites, and high levels of histone acetylation, suggesting that this region may play a role in transcriptional regulation, providing directions for further study of the role of bovine CNV and economic traits.

Genetic Variant of MYLK4 Gene and its Association with Growth Traits in Chinese Cattle.

As a member of MYLK family, MYLK4 gene may play a vital role in muscle development. In this study, one novel single-nucleotide polymorphism (SNP) was identified the bovine MYLK4 by sequencing pooled DNA samples (pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the intron 10 region within the bovine MYLK4 gene in 559 individuals representing five main cattle breeds from China (Nanyang, NY; Qinchuan; Jiaxian, JX; Pinan cattle; and Caidamu cattle, CDM). Genotype AA and allele A were predominant in the QC, PN, and XN populations. Association analysis with growth traits in the QC breed showed that the animals with genotype GG had significantly greater chest breadth and hip width (P < 0.05). Meanwhile, the genotype GG was strongly associated with withers height and body length than those with genotype AA (P < 0.01 or P < 0.05) at 12 months in the NY breed. These statistical results exhibited that the MYLK4 gene might be a potential candidate gene to improve cattle's growth traits, and the SNP could be used as molecular markers in early marker-assisted selection (MAS) in beef cattle breeding program.

Novel copy number variation of the KLF3 gene is associated with growth traits in beef cattle.

Copy number variation (CNV) related to complex traits, such as disease and quantitative phenotype, is considered an important and wealthy source of genetic and phenotypic diversity. It suggests that the copy number variation of function gene maybe leads to the phenotypic changes. Kupple like factor 3 (KLF3) gene is a vital transcription factor associated with the growth and development of muscle and adipose tissue. It has been mapped in a CNV region by animal genome re-sequencing. In this study, we detected the distribution diversity of KLF3 gene copy numbers in six Chinese cattle breeds (QC, NY, XN, PN, QDM and JX) and associated the phenotypic traits with it. Then, we analyzed the KLF3 gene transcription expression level in different tissues of Jiaxian (JX) cattle. Furthermore, we detected mRNA expression level of muscle and fat tissues of Jiaxian cattle (JX), Angus × Jiaxian (AJ). The results showed that the copy number in CNV loss was more frequent in QC than others. And we revealed a positive effect of KLF3 CNV on growth traits, such as body mass and heart girth (P < 0.05). In a word, we ascertained the significance between CNVs of KLF3 gene and growth traits in different cattle breeds, and our data indicates that the CNVs of KLF3 gene may as a marker for the future molecular breeding of Chinese beef cattle.

A novel SNP of PLAG1 gene and its association with growth traits in Chinese cattle.

Genetic polymorphism has great influences on the improvement of cattle traits. The polymorphism of the same gene family will greatly change the growth traits of cattle, such as the pleomorphic adenoma gene (PLAG) family. Many studies have shown that the PLAG family proteins are the transcription regulators of nuclear protein, which mainly regulates the expression of many important genes in the body. In cattle, single nucleotide polymorphisms (SNPs) within or near the PLAG1 gene is associated with economic traits of height in cattle. Here we investigate a SNPs of bovine PLAG1 in 646 cattle from five breeds. We found three different genotypes by using cleaved amplification polymorphism sequence-tagged sites (CAPs), includes some significant differences in body height, chest circumference and other shapes (P < 0.05), also we found that the TT genotype had no advantage in body shape. These results indicate that the selection of PLAG1 gene could be used to ensure the breeding direction for growth traits of the beef cattle.

DNA methylation status of CRABP2 promoter down-regulates its expression.

As an important epigenetic modification DNA methylation is catalyzed by DNA methylation transferases (DNMTs) and occurs mainly in CpG islands. DNA methylation plays an important role in regulates gene expression, cell differentiation, genetic imprinting and tumor therapy. Retinoic acid-binding proteins (RAC) is vital for the absorption, transport, metabolism and maintenance of homeostasis of retinoic acid, which in turn regulates the differentiation and proliferation of cells by regulating the transcription of many target genes, therefore, these proteins influence differentiation and proliferation of adipocytes and muscle fibroblasts. Thus, cellular retinoic acid binding protein 2 (CRABP2) may be a candidate gene which affects beef quality, yield and fat deposition. The aim of this study was to evaluate the expression and the methylation pattern on the differentially methylated region (DMR) of the promoter of CRABP2. The DNA methylation pattern was tested by bisulfite sequencing polymerase chain reaction (BSP), the quantitative real-time PCR (qPCR) was used to analysis the expression of CRABP2 gene. The results showed that the DNA methylation level was higher in purebred cattle breed than that in hybrid cattle breeds which was negative correlation with the expression of the gen. These results indicate that the methylation status of the CRABP2 DMR can regulate mRNA expression. What's more, there are different methylation and expression patterns in different breeds and tissues which may influence the phenotype, and the results may be a useful parameter to investigate the function of CRABP2 in muscle and fat developmental in Chinese cattle.

Detection of 19-bp deletion within PLAG1 gene and its effect on growth traits in cattle.

Polymorphic adenoma gene 1 (PLAG1) is a member of the pleomorphic adenoma gene family. PLAG family of proteins as a nuclear transcription factor mainly play a role in regulating a variety of important genes in the body. The aim of this study was to examine the association of the PLAG1 polymorphism with growth traits in 566 cattle. A novel 19-bp indel mutation was identified in the PLAG1 by sequencing pooled DNA samples (Pool-Seq) and agarose gel electrophoresis methods. The PCR products of PLAG1 exhibited 3 genotypes and 2 alleles: 142 bp (denoted as W) and 123 bp (denoted as D). Genotype WW and allele W were predominant in the studied populations. In addition, the 19-bp indel was significantly associated with the growth traits in cattle breeds, with the hip width and rump length of Pinan cattle (P < 0.05), heart girth and cannon bone circumference of Xianan cattle (P < 0.01 or P < 0.05), as well as the heart girth, hip width, hucklebone width, rump length, height at sacrum and chest depth of the Jiaxian cattle (P < 0.05). Our results indicate that the Indel marker of PLAG1 gene can be used as candidate molecular markers for the breeding in cattle.

Associations of GBP2 gene copy number variations with growth traits and transcriptional expression in Chinese cattle.

Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P > .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P < .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle.

Correlation between ZBED6 Gene Upstream CpG Island methylation and mRNA expression in cattle.

DNA methylation is essential for the regulation of gene expression and important roles in muscle development. To assess the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in ZBED6, we simultaneously examined DNA methylation and expression in six tissues from two different developmental stages (fetal bovine and adult bovine). The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The result of quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution and is highly expressed in adult bovine (P < 0.05 or P < 0.01). The DNA methylation level was significantly different in liver, lung and spleen between the two cattle groups (P < 0.05 or P < 0.01). The adult bovine group exhibited a significantly higher mRNA level and lower DNA methylation level than the fetal bovine group in liver, lung, and spleen. No significant association was detected between DNA methylation level and muscle, heart, and kidney at two different stages. In this study, the statistical analyses indicated that DNA methylation patterns are associated with mRNA level in some tissues, these results may be a useful parameter to investigate muscle developmental in cattle and as a model for studies in other species, potentially contributing to an improvement of growth performance selection in beef cattle breeding program.

Effect of Genetic Variations within the I-mfa Gene on the Growth Traits of Chinese Cattle.

I-mfa (inhibitor of the MyoD family a) is a transcription modulator that binds to MyoD family members and inhibits their transcriptional activities. It is highly expressed in the sclerotome and plays an important role in the patterning of the somite early in development. In this study, the polymorphisms of the bovine I-mfa gene were detected by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and DNA pool sequencing methods in 541 individuals from three Chinese cattle breeds. The results showed that P3 locus had two novel complete linked single nucleotide polymorphisms (NC_007324.4:g.12284A>G and g.12331T>C), resulting in a missense mutation p.S(AGC)113G(GGC) and a synonymous mutation p.H(CAT)128H(CAC), respectively. P4 locus had a novel SNP (NC_007324.4: g.16432C>A), which resulted in a nonsense mutation p.C(TGC)241X(TGA). The statistical analyses indicated that the three SNPs, are associated with the phenotypic traits in Luxi (LX), Qinchuan (QC), and Jiaxian (JX) cattle population (P < 0.05 or P < 0.01). The mutant-type variants were superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to wild-type homozygote. Our results provide evidence that polymorphisms in the I-mfa gene are associated with growth traits and may be used for marker-assisted selection in beef cattle breeding program.

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL.

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.

Linkage disequilibrium and haplotype distribution of the bovine LHX4 gene in relation to growth.

LIM homeobox transcription factor 4 (LHX4) is a LIM homeodomain transcription factor involved in pituitary gland and nervous system development. The aim of this study was to examine the association of the LHX4 polymorphisms with growth traits in beef cattle breed. A total of 7 single nucleotide polymorphyisms (SNPs) have been identified in the coding region and noncoding region of the bovine LHX4 by sequencing pooled DNA samples (Pool-Seq) and PCR-single strand conformation polymorphism (PCR-SSCP) methods. The linkage disequilibrium was assessed in 871 individuals representing four main cattle breeds from China. The SNPs 2-5 and 7-8 were found to be in complete linkage disequilibrium, respectively. The result of haplotype analysis of 13 SNPs showed that 31 haplotypes were found in four Chinese cattle breeds, and 20 genotypes were only found in Nanyang cattle. The statistical analyses indicated that the SNP1-5, and 6 are associated with the body weight at 18, and 6 months of age in Nanyang cattle population (P<0.05), but no significant associations between their twenty combined genotypes. Our results provide evidence that some polymorphisms in LHX4 are associated with growth traits at certain ages, and may be used as candidates for marker-assisted selection and management in cattle.

Genetic variants in BMP8B gene are associated with growth traits in Chinese native cattle.

As a signaling molecule, bone morphogenetic protein 8B (BMP8B) plays an essential role in bone metabolism and is able to regulate thermogenesis and energy balance, which suggests that BMP8B gene may be a new candidate for growth traits. Here, to characterize the effects of BMP8B gene on growth traits, we first used three Chinese indigenous cattle breeds (n=845) to detect single nucleotide polymorphisms (SNPs). Five novel SNPs of BMP8B gene (g.-242C>T, g.2164C>T, g.2639T>C, g.2900C>G and g.10817C>T) were identified by DNA pool sequencing and forced PCR-RFLP. And then we associated the five SNPs with four growth traits (body weight, body length, heart girth, and hucklebone width). Results from association analysis showed that the SNPs 1, 2, and 3 affected growth trait(s) markedly (P<0.05). Further, 6 combined haplotypes were constructed to guarantee the reliability of analysis results. There were also significant differences in body length, heart girth and body weight between the 6 combined haplotypes (P<0.05), but not in hucklebone width (P>0.05). Collectively, our results suggest a modulatory role of BMP8B gene in cattle growth and development, and 3 SNPs could be used as molecular markers in early marker assisted selection (MAS) in beef cattle breeding program.