Genetic variation in the OPN gene affects milk composition in Chinese Holstein cows.

The aim of this study was to investigate the association between genotypes and haplotypes of OPN, and milk composition in dairy cows. A total of 317 Chinese Holstein cows were genotyped via DNA sequencing in this study. Three single nucleotide polymorphisms (SNPs), g.2916G > A, g.58675C > T and g.58899C > A, and eight haplotypes were identified. Of the eight possible haplotypes, four haplotypes i.e., Hap2 (ACC; 55.30%), Hap6 (GCC, 15.6%), Hap1 (ACA, 13.6%) and Hap4 (ATC, 5.70%), were considered to be major with a cumulative estimated frequency of >90%. Single markers (g.2916G > A and g.58899C > A) and Haplotype Hap6/4 were found to be associated with an increase in butter-fat percentage (p < 0.05). Taken together, our results provided evidence that polymorphisms in OPN are associated with milk composition, and could potentially be used for marker-assisted selection in Chinese Holstein cows.

Variations in the insulin receptor substrate 1 (IRS1) and its association with growth traits in Chinese black Tibetan sheep (Ovis aries).

This paper presented the results on the study of insulin receptor substrate 1 (IRS1) polymorphisms in Chinese black Tibetan sheep. Via DNA direct sequencing, four variations within 3' untranslated region (UTR) of IRS1, including g.9382T > G, g.9569T > G, g.9572C > T and g.9695A > C were detected in the black Tibetan sheep population. Based on the χ2 test, those four loci deviated from Hardy-Weinberg equilibrium (p < 0.05). In g.9569T > G locus, genotype of GG possessed advantage on body weight (p < 0.05). In g.9572C > T locus, individuals with genotype of TT homozygous mutation decreased significantly on body weight, withers height, body length and chest circumference (p < 0.05 or p < 0.01). In g.9695A > C locus, the body weight and chest circumference were also higher in AA carriers than in CC carriers (p < 0.05). Our results provided evidence that polymorphisms in IRS1 were associated with growth efficiency traits by quantitative genetic analysis, and may be used for marker-assisted selection in Chinese indigenous sheep.

Expression of the bovine KLF6 gene polymorphisms and their association with carcass and body measures in Qinchuan cattle (Bos Taurus).

Kruppel-like factor 6 (KLF6) genes plays a significant role in the regulation of cell differentiation, proliferation and muscle development. The aim of this study is to investigate the genetic variation and the haplotype combination of the KLF6 gene in Qinchuan cattle and verify its contribution to bovine carcass traits and body measurements. The data were analyzed by real-time quantitative PCR (qPCR) to detect the expression profile of the KLF6 gene in the various tissues of Qinchuan cattle. PCR amplicons sequencing explored three novel SNPs at loci 3332C > G; 3413C > T and 3521G > A in the 2nd exon region of the KLF6 gene. The expression of KLF6 in the liver, kidney and lung was greater than that of other tissues. Allelic and genotypic frequencies of these SNPs were found to be in Hardy Weinberg equilibrium (P < 0.05). In SNP1, genotype CC, in SNP2, genotype CT and in SNP3 genotype GG were associated (P < 0.05) with larger body and carcass measurements. Association analysis results indicated that individuals with the Hap1/4 diplotype had a longer body and rump, were taller at the withers, and were wider at the hip than the other combinations. In terms of ultrasound carcass measures, Hap1/4 was associated with a larger muscle area and more intramuscular fat than other combinations. The bioinformatics study of the KLF6 protein showed a high degree of conservation in different mammalian species. The above results suggest that the KLF6 gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection of Qinchuan cattle.

Association of hormone-sensitive lipase (HSL) gene polymorphisms with the intramuscular fat content in two Chinese beef cattle breeds.

Hormone-sensitive lipase (HSL) was considered as an essential enzyme in glucolipid metabolism. It has been proposed to be a lead candidate gene for genetic markers of lipid deposition in livestock. The aim of this study was to identify sequence variants (SVs) of the bovine HSL gene and evaluate the relations to intramuscular fat in two indigenous Chinese beef cattle breeds. Expression analysis by quantitative real-time polymerase chain reactions (qPCR) indicated that expression levels of bovine HSL gene were highest in the perirenal fat and heart within two different age stage (adult and calf), respectively. Five SVs were identified by direct DNA sequencing, which included four missense mutations (g.16563C>T, g.16734G>A, g.16896A>G, g.17388G>T) in exon 8 and a synonymous mutation (g.17402C>T) in exon 9. Population genetic analysis showed that except for g.16563C>T and g.17402C>T, all the other detected SVs strongly affected the bovine intramuscular fat content (P < 0.01 or P < 0.05). The individuals with Hap5/5 diplotypes (CC-GG-GG-GG-CC) was highly significantly associated with intramuscular fat content than the other diplotypes (P < 0.01). The above results suggested that the HSL gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection in Chinese cattle breeds.

Analysis of the oxidized low density lipoprotein receptor 1 gene as a potential marker for carcass quality traits in Qinchuan cattle.

OBJECTIVE: The oxidized low density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low-density lipoprotein and adipocyte proliferation in mammals. For this reason, we aimed at investigating the association of OLR1 gene polymorphisms with carcass quality traits in Chinese Qinchuan cattle. METHODS: The single nucleotide polymorphism (SNP) was identified in the 3' untranslated region of bovine OLR1 gene by DNA sequencing. In addition, the haplotype frequency and linkage disequilibrium estimates of three SNPs were evaluated in 520 individuals. RESULTS: Results indicated that the studied three SNPs were within the range of moderate genetic diversity (0.25< polymorphism information content<0.5). Haplotype analysis of three SNPs showed that ten different haplotypes were identified, but only five haplotypes were listed as those with a frequency of <0.05 were excluded. The Hap3 (-G1T2C3-) had the highest haplotype frequency (42.10%). Linkage disequilibrium analysis showed that the three SNPs had a low linkage (r2<0.001). The T10588C and C10647T were significantly associated with backfat thickness and intramuscular fat content in Qinchuan cattle. CONCLUSION: Based on our results, we believe that the OLR1 gene could be a strong candidate gene for influencing carcass quality traits in Qinchuan cattle.

Genetic variants in the SIRT6 transcriptional regulatory region affect gene activity and carcass quality traits in indigenous Chinese beef cattle (Bos taurus).

BACKGROUND: The aim of this study was to analyze potential influences of polymorphisms within the regulatory region of the bovine SIRT6 gene on carcass quality traits. Expression analyses suggested that SIRT6 gene is predominately expressed in kidney, compared with other tissues. In 535 indigenous Chinese beef cattle, two novel single nucleotide polymorphisms (SNPs) were identified within the promoter region of the SIRT6 gene. RESULTS: Association analysis indicated that G allele of the c.-1100 A > G had a positive effect on fat deposition, and the Hap4/4 diplotype had more favourable results than other dipoltypes with respect to the evaluation of carcass quality traits. Furthermore, promoter activity associated with the Hap3 haplotype was measured at higher levels than the Hap1 haplotype, which would be in agreement with the previously described association analysis. CONCLUSION: The SIRT6 promoter variants significantly affect transcriptional levels and subsequently significantly influence bovine intramscular fat content.

Expression of the SIRT2 gene and its relationship with body size traits in Qinchuan cattle (Bos taurus).

Silent information regulator 2 (SIRT2) is a member of the sirtuin family of class III NAD (nicotinamide adenine dinucleotide)-dependent protein deacetylases and may regulate senescence, metabolism and apoptosis. The aims of this study were to investigate whether the SIRT2 gene could be used as a candidate gene in the breeding of Qinchuan cattle. Real-time polymerase chain reaction (RT-PCR) results showed that among all types of tissue that were analyzed, the highest mRNA expression levels of the gene were found in subcutaneous fat. DNA sequencing of 468 individual Qinchuan cattle identified two novel, single nucleotide polymorphisms (g.19501 C > T and g.19518 C > T) in the 3' untranslated region (3'UTR) of the SIRT2 gene. The frequencies of SNP g.19501 C > T and g.19518 C > T were in Hardy-Weinberg disequilibrium in all the samples (chi-square test, χ2 < χ0.052). An association analysis showed that the two loci were significantly correlated with some body size traits and the H2H2 (-CT-CT-) diplotypes performed better than other combinations. These results indicated that the variations in the SIRT2 gene and their corresponding genotypes may be considered as molecular markers for economic traits in cattle breeding.

Molecular Characterization of Bovine SMO Gene and Effects of Its Genetic Variations on Body Size Traits in Qinchuan Cattle (Bos taurus).

Smoothened (Smo)-mediated Hedgehog (Hh) signaling pathway governs the patterning, morphogenesis and growth of many different regions within animal body plans. This study evaluated the effects of genetic variations of the bovine SMO gene on economically important body size traits in Chinese Qinchuan cattle. Altogether, eight single nucleotide polymorphisms (SNPs: 1-8) were identified and genotyped via direct sequencing covering most of the coding region and 3'UTR of the bovine SMO gene. Both the p.698Ser.>Ser. synonymous mutation resulted from SNP1 and the p.700Ser.>Pro. non-synonymous mutation caused by SNP2 mapped to the intracellular C-terminal tail of bovine Smo protein; the other six SNPs were non-coding variants located in the 3'UTR. The linkage disequilibrium was analyzed, and five haplotypes were discovered in 520 Qinchuan cattle. Association analyses showed that SNP2, SNP3/5, SNP4 and SNP6/7 were significantly associated with some body size traits (p < 0.05) except SNP1/8 (p > 0.05). Meanwhile, cattle with wild-type combined haplotype Hap1/Hap1 had significantly (p < 0.05) greater body length than those with Hap2/Hap2. Our results indicate that variations in the SMO gene could affect body size traits of Qinchuan cattle, and the wild-type haplotype Hap1 together with the wild-type alleles of these detected SNPs in the SMO gene could be used to breed cattle with superior body size traits. Therefore, our results could be helpful for marker-assisted selection in beef cattle breeding programs.

RNA-Seq revealed the effect of adding different proportions of wheat diet on fat metabolism of Tibetan lamb.

The objective of this study was to evaluate the effect of the level of wheat substitution for corn on the fat metabolism of Tibetan lamb. A total of 90 Tibetan lambs [body weight (BW) of 19.78 ± 2.45 kg] were arranged with three substitution levels of wheat: WC (100 % corn), WL (10 % wheat substitution for corn), and WH (15 % wheat substitution for corn) on a dry matter basis. After the experiment, cry section technology was used to scrutinize the subcutaneous adipose tissue morphology, and genes related to fat metabolism were excavated using high-throughput sequencing technology. According to the study results, fat diameter and fat biovolume of the WL and WH groups were less than the WC group. A total of 506 differentially expressed genes (DEGs) were identified by RNA sequencing (RNA-Seq) technology. Compared with the WC group, 66 DEGs were upregulated and 59 DEGs were downregulated in the WL group, and 179 DEGs were upregulated and 269 DEGs were downregulated in the WH group. The top 20 DEGs were analyzed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway, indicating significant differences in the fat metabolism pathway. Five DEGs were randomly screened for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) verification, and the results were consistent with the RNA-Seq results, which proved the accuracy of sequencing. In summary, with the increase in the proportion of supplemental wheat, the fat cells became smaller, and the genes related to fat decomposition were significantly upregulated.

Association between Single Nucleotide Polymorphisms in SIRT1 and SIRT2 Loci and Growth in Tibetan Sheep.

Silent information regulator 1 and 2 (SIRT1, 2) were NAD+-dependent histone or non-histone deacetylase, which emerged as key metabolic sensors in several tissues of mammals. In the present study, the search for polymorphisms within the ovine SIRT1 and SIRT2 loci as well as association analyses between SNPs and growth-related traits were performed in Tibetan sheep. To determine the expression pattern of SIRT1 and SIRT2 genes in Tibetan sheep, the quantitative real-time polymerase chain reaction (qPCR) analysis revealed that those two genes were widely expressed in diverse tissues. Expression of SIRT1 was less in abomasum of lamb, whereas it was greater in duodenum within adult stage. In the case of SIRT2, the greatest expression was observed in reticulum (lamb) and in muscle (adult), whereas the least expression was in liver for lamb and in kidney for adult animals. The association analysis demonstrated that g.3148 C > T polymorphism of SIRT1 affected heart girth (p = 0.002). The g.8074 T > A SNP of SIRT2 had a significant correlation with body weight (p = 0.011) and body length (p = 0.008). These findings suggested that the SIRT1 and SIRT2 polymorphism was involved in growth-related traits in Tibetan sheep, which may be considered to be genetic markers for improving the growth traits of Tibetan sheep.

Detection of polymorphisms in the bovine leptin receptor gene affects fat deposition in two Chinese beef cattle breeds.

Leptin receptor (LEPR) gene play a pivotal role in the regulation of fat deposition and energy homeostasis. This study investigated the presence and frequency of polymorphisms of bovine LEPR gene and determine whether the polymorphisms are associated with the fat deposition in two Chinese beef cattle breeds. Quantitative real-time polymerase chain reactions identified that the expression of LEPR gene was highest in the liver and subcutaneous fat. Four single nucleotide polymorphisms (SNPs) were identified including g.24169C > T, g.24256T > A, g.24267 G > C and g.24413T > A. A greater backfat thickness was associated with the AA genotype of g.24256T > A compared to the TT genotype. A greater intramuscular fat content was associated with the GG genotype of g.24267 G > C compared to the CC genotype. Both g.24169C > T and g.24413T > A were not correlated with fat deposition. These results indicated that the SNP g.24256T > A and g.24267 G > C of LEPR gene may be useful markers for genetic improvement of fat deposition in Chinese beef cattle breeds.

The genetic polymorphisms of melanocortin-4 receptor gene are associated with carcass quality traits in a Chinese indigenous beef cattle breed.

Melanocortin-4 receptor (MC4R) was considered as an essential modifiers in feelings intake, the regulation of metabolism and body weight. This study aimed at identifying polymorphisms in MC4R gene that might associate with carcass quality traits in Chinese indigenous beef cattle breed. qPCR analysis showed that the MC4R gene was widely expressed in various tissues, with predominantly expression levels in heart. Three single-nucleotide polymorphisms (SNPs) were identified, including a mutation (g.85A > G) in 5'untranslated regions (UTR) and two mutations (g.927C > T and g.1069C > G) in exon 1. Based on the χ2 test, both g.927C > T and g.1069C > G loci fitted with Hardy-Weinberg equilibrium (P > .05). Population genetic analysis showed that except for g.85A > G, the other detected SNPs strongly affected the bovine back fat thickness and intramuscular fat content (P < .05). The individuals with Hap1/4 diplotypes (ACC-ATG) were highly significantly associated with carcass quality traits than the other diplotypes (P < .01 or P < .05). Results indicated that the bovine MC4R gene polymorphisms were implicated as genetic markers of potential importance in marker-assisted selection (MAS) strategies to improve carcass quality in Chinese Qinchuan cattle.

Detection of polymorphisms in the promoter of bovine SIRT1 gene and their effects on intramuscular fat content in Chinese indigenous cattle.

Intramuscular fat content (IMF) is one of the most significant factors for meat quality affecting tenderness, flavor, and juiciness of meat. For this reason, we aimed to investigate the association of SIRT1 gene polymorphisms with intramuscular fat content in Chinese Qinchuan cattle (Bos taurus). Using DNA sequencing, three single nucleotide polymorphisms (SNPs) within the promoter regions of SIRT1 gene were identified in 535 Qinchuan cattle, and the five haplotypes representing five potential different compositions of polymorphic potential cis-acting elements. Results indicated that both c.-107 G>A and c.-274 A>G were significantly associated with intramuscular fat content in Qinchuan cattle, and Hap5/5 diplotype showed higher (P < 0.05) intramuscular fat content than other combinations (P < 0.05 or P < 0.01). In addition, the Hap5 haplotype had much lower (P < 0.05) transcriptional activity, consistent with the association analysis. Based on our results, the polymorphisms in transcription factor binding sites of SIRT1 gene promoter may affect the transcriptional activity of SIRT1 gene, and thus alter intramuscular fat content in beef cattle.

Polymorphisms in adrenergic receptor genes in Qinchuan cattle show associations with selected carcass traits.

The beta-adrenergic receptors coded by the ADRB1, ADRB2 and ADRB3 genes play important roles in mediating metabolic effects, especially lipolysis, insulin resistance and energy balance. This study investigated the expression levels of these three genes in different tissues of Qinchuan cattle by real-time polymerase chain reaction (RT-PCR). Expressed levels of RNA from the ADRB2 gene were generally much higher than for ADRB1 and ADRB3. ADRB1 and ADRB2 expression levels were highest in subcutaneous fat and lower in muscle, whereas ADRB3 expression was higher in muscle tissue. Eight single nucleotide polymorphisms (SNPs) were discovered in 503 Qinchuan cattle by DNA sequencing, containing three missense mutations (g.1148G>C in ADRB1, g.1293C>T and g.1311T>C in ADRB2), four synonymous mutations (g.1054T>C, g.1122C>T and g.1143G>T in ADRB1 and g.506A>G in ADRB3), as well as one mutation in 3'untranslated region (3'UTR) (g.2799G>A in ADRB3). Interestingly, five of them were located in regions predicted to contain multiple repeats of CG nucleotides (CpG islands). Association analysis showed relationships between most of those SNPs or combined haplotypes and carcass traits of Qinchuan cattle. This study association analysis suggests that polymorphisms in these genes might be useful for selection in beef cattle breeding.

NRF1 and ZSCAN10 bind to the promoter region of the SIX1 gene and their effects body measurements in Qinchuan cattle.

The SIX1 homeobox gene belongs to the six homeodomain family and is widely thought to play a principal role in mediating of skeletal muscle development. In the present study, we determined that the bovine SIX1 gene was highly expressed in the longissimus thoracis and physiologically immature individuals. DNA sequencing of 428 individual Qinchuan cattle identified nine single nucleotide polymorphisms (SNPs) in the promoter region of the SIX1 gene. Using a series of 5' deletion promoter plasmid luciferase reporter assays and 5'-rapid amplification of cDNA end analysis (RACE), two of these SNPs were found to be located in the proximal minimal promoter region -216/-28 relative to the transcriptional start site (TSS). Correlation analysis showed the combined haplotypes H1-H2 (-GG-GA-) was significantly greater in the body measurement traits (BMTs) than the others, which was consistent with the results showing that the transcriptional activity of Hap2 was higher than the others in Qinchuan cattle myoblast cells. Furthermore, the electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assay (ChIP) demonstrated that NRF1 and ZSCAN10 binding occurred in the promoter region of diplotypes H1-H2 to regulate SIX1 transcriptional activity. This information may be useful for molecular marker-assisted selection (MAS) in cattle breeding.

Characterization of the promoter region of the bovine SIX1 gene: Roles of MyoD, PAX7, CREB and MyoG.

The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5' regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5' flanking region of bovine SIX1. However, a CpG island was predicted in the region -235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5' flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region -689/-40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.

Altered milk yield and rumen microbial abundance in response to concentrate supplementation during the cold season in Tibetan sheep

BACKGROUND Weight loss and decline of milk yield in Tibetan sheep was a challenge for the dairy industry in Qinghai-Tibet Plateau, which were considered to be caused by underfeeding of the sheep during the harsh winter. The objective of this study was to assess the role of feed supplementation in the milk performance and rumen microbiome of ewes under forage-based diets. Based on parity, milking period, milk yield, and body weight, ten 1.5-yr-old ewes were allocated randomly into two groups. One group of ewes was fed no supplement Control group (CON) and the other group was fed with concentrate feed supplement (Treatment group, T). Individual milk yield was determined daily; both the milk composition and rumen bacterial characteristics were analyzed after the end of feeding trials. RESULTS Results showed that lactose in the milk of the CON group was significantly lower (P < 0.05) than that of the T group at days 30 and 60. Milk yield in the T group was greater than in the CON group at day 30 (P < 0.05). Additionally, the dominant ruminal bacteria (phyla Bacteroidetes, Firmicutes, and Verrucomicrobia) were shared by both groups through 16S rRNA gene pyrosequencing. Greater relative abundance of Bacteroidales RF16 group in family level, Victivallales in order level, Lentisphaeria in class level, and Lachnospiraceae bacterium in species level were observed in the T group than in the CON group (P < 0.05). CONCLUSIONS These results demonstrated that supplementation of concentrate in the cold season improved milk lactose yield and milk production, and the rumen microbial abundance of Tibetan sheep.