Mapping of quantitative trait loci for carcass traits in a Japanese Black (Wagyu) cattle population

To detect quantitative trait loci (QTL) that influence economically important traits in a purebred Japanese Black cattle population, we performed a preliminary genome-wide scan using 187 microsatellite markers across a paternal half-sib family composed of 258 offspring. We located six QTL at the 1% chromosome-wise level on bovine chromosomes (BTA) 4, 6, 13, 14 and 21. A second screen of these six QTL regions using 138 additional paternal offspring half-sib from the same sire, provided further support for five QTL: carcass weight on BTA14 (22-39 cM), one for rib thickness on BTA6 (27-58 cM) and three for beef marbling score (BMS) on BTA4 (59-67 cM), BTA6 (68-89 cM) and BTA21 (75-84 cM). The location of QTL for subcutaneous fat thickness on BTA13 was not supported by the second screen (P > 0.05). We determined that the combined contribution of the three QTLs for BMS was 10.1% of the total variance. The combined phenotypic average of these three Q was significantly different (P < 0.001) from those of other allele combinations. Analysis of additional half-sib families will be necessary to confirm these QTL.     

Contributions of FASN and SCD gene polymorphisms on fatty acid composition in muscle from Japanese Black cattle

The fatty acid synthase (FASN) and stearoyl‐CoA desaturase (delta‐9‐desaturase) (SCD) genes affect fatty acid composition. This study evaluated the contributions of polymorphisms of these genes on fatty acid composition in muscle in two different populations: 1189 and 1058 Japanese Black cattle from the Miyagi and the Yamagata populations respectively. We sampled intramuscular fat from the longissimus thoracis muscle in the Miyagi population and from the trapezius muscle in the Yamagata population. The collective contributions of FASN and SCD polymorphisms to total additive genetic variance for oleic acid were 13.46% in the Miyagi population and 16.29% in the Yamagata population and to phenotypic variance were 5.45% and 6.54% respectively. Although the individual effects of FASN and SCD polymorphisms on fatty acid composition were small, overall gene substitution may effectively improve fatty acid composition. In addition, we found that gene polymorphism contributions of fatty acids varied by population even in the same breed.     

Fine-mapping of a marbling trait to a 2.9-cM region on bovine chromosome 7 in Japanese Black cattle

To locate quantitative trait loci (QTL) for intramuscular fat deposition (marbling) in a local population of Japanese Black cattle, we performed a genome scan using a paternal half-sib family of Bull A. A marbling QTL was mapped in the region flanked by DIK0079 (20.7 cM) and TGLA303 (39.3 cM) on bovine chromosome (BTA) 7, affecting 5.0% of the total family variance. Haplotype analysis of the QTL region revealed that the marbling-increasing Q allele was transmitted from the dam. On the other hand, Bull B, a maternal half-sib of Bull A, did not receive the Q allele from its dam, based on the following findings: (i) a marbling QTL on BTA7 was not detected in the Bull B paternal half-sib family; (ii) recombination between DIK0079 (20.7 cM) and RM006 (25.4 cM) in the QTL region was observed in the maternal chromosome of Bull B; and (iii) the Q -harbouring steers from Bull A exhibited significantly higher marbling than the steers from Bull B and the remaining steers from Bull A. To precisely compare the maternal chromosomes of both bulls, we constructed a bacterial artificial chromosome contig covering the region between DIK0079 and RM006 and developed DNA markers. The recombination occurred between DIK8042 and DIK8044 , indicating that the marbling QTL was in a 2.9-cM region flanked by DIK0079 and DIK8044 .     

Whole‐genome association study for fatty acid composition of oleic acid in Japanese Black cattle

Fatty acid composition, especially oleic acid (C18:1), plays an important role in the eating quality of meat in Japanese Black cattle. Therefore, the objective of this study was to identify loci associated with C18:1 in the intramuscular fat of the trapezius muscles in Japanese Black cattle using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. We also evaluated the relationship between C18:1 and three fatty acid synthesis genes, fatty acid synthase (FASN), stearoyl‐CoA desaturase and sterol regulatory element‐binding protein‐1. In this experiment, we applied a mixed model and Genomic Control approach using selective genotyping to perform a genome‐wide association study. A total of 160 animals (80 animals with higher values and 80 animals with lower values), selected from 3356 animals based on corrected phenotype, were genotyped using the Illumina BovineSNP50 BeadChip and three fatty acid synthesis genes, and the quality of these SNPs was assessed. In this study, a total of 38 955 SNPs, which included SNPs in the three fatty acid synthesis genes, were used, and the estimated inflation factor was 1.06. In the studied population, a total of 32 SNPs, including the FASN gene, had significant effects, and in particular 30 SNPs of all significant SNPs were located between 49 and 55 Mbp on chromosome 19. This study is one of the first genome‐wide association studies for fatty acid composition in a cattle population using the recently released Illumina BovineSNP50 BeadChip.     

Mapping of a Quantitative Trait Locus for Beef Marbling on Bovine Chromosome 9 in Purebred Japanese Black Cattle

The goal of this study is to detect quantitative trait loci (QTL) for carcass traits applicable for a DNA-based breeding system in a Japanese Black cattle population. A purebred paternal half-sib family from a commercial line composed of 65 steers was initially analyzed using 188 informative microsatellites giving a 16-cM average interval covering 29 autosomes. A significant QTL for marbling was detected in the centromeric portion of bovine chromosome (BTA) 9. After additional marker genotyping across a larger sample size composed of 169 individuals, this locus was refined to a 20-cM confidence interval between microsatellites BM1227 (24 cM) and DIK2741 (50 cM) at a 1% chromosome-wise threshold. The allele substitution effect between Q and q for a beef marbling standard score (1 to 12 range) on BTA9 was 1.0 (5.7% of total phenotypic variance in QTL contribution in this family). This result provides a primary platform for a marker-assisted selection system of the beef marbling trait within the Japanese Black (Wagyu) cattle population.     

Factors influencing the priority of access to food and their effects on the carcass traits for Japanese Black (Wagyu) cattle

The factors influencing the priority of access to food and the effects of the priority of access to food on their carcass traits were analyzed for Japanese Black (Wagyu) cattle in a semi-intensive fattening production system. The records of 96 clinically healthy steers and heifers were analyzed. The calves at ∼3 to 4 months of age were allocated to pens with four animals per pen; all four animals in the same pen were of the same sex and of similar body size. The ranking of the animals’ priority of access to food (1^st, 2^nd, 3^rd and 4^th), which was determined by the farm manager, was used as an indicator of social dominance in the present study. Four models including sire line, maternal grandsire line and the difference in the animals’ birth dates as fixed effects were used to analyze factors influencing the priority of access to food. Ranking was represented by ordinal scores (highest=4, lowest=1) in Model 1, and the binary scores were assigned in Model 2 (highest=1; 2^nd, 3^rd and 4^th=0), Model 3 (1^st and 2^nd=1; 3^rd and 4^th=0) and Model 4 (1^st, 2^nd and 3^rd=1; lowest=0). The results showed that the difference in the animals’ birth dates had a significant effect on the establishment of the priority of access to food in Model 3 (P<0.05), suggesting that animals born earlier may become more dominant in the pen. The maternal grandsire line tended to affect the social rank score in Models 2 and 3 (P<0.10). Our results indicated that the maternal grandsire line may affect the temperament of calves through their mothers’ genetic performance and thereby more aggressive calves may be more dominant and have higher priority of access to food. On the other hand, there was a significant effect of the priority of access to food on beef marbling score (BMS; P<0.05), and the priority of access to food also tended to influence the carcass weight (P=0.09). The highest BMS was observed for animals with the first rank of the priority of access to food (P<0.05), and the higher-ranking animals had the tendency to be heavier carcass than the lower-ranking animals. Our findings emphasized the importance of information about the priority of access to food determined by farmers’ own observation on implementing best management practices in small-scaled semi-intensive beef cattle production systems.     

Indels within promoter and intron 1 of bovine prion protein gene modulate the gene expression levels in the medulla oblongata of two Japanese cattle breeds

Genetic differences which exist in the prion protein gene (PRNP) have been reported to influence susceptibility of humans, sheep and goats to prion diseases. In cattle, however, none of the known coding polymorphisms has a direct effect on bovine spongiform encephalopathy (BSE). It has been reported that 23‐bp insertion/deletion (indel) polymorphisms within the promoter region have a tentative association to BSE susceptibility in German cattle, and a lower number of 24‐bp repeat units in the open reading frame (ORF) was reported to reduce BSE susceptibility in transgenic mice. In this study, because of the hypothesis that bovine PRNP promoter polymorphisms cause changes in PRNP expression, we genotyped PRNP polymorphisms in the promoter and intron 1 using 218 genomic DNA samples from two Japanese cattle breeds. We also analysed the expression levels of prion in 40 animals by quantification of real‐time PCR using mRNAs extracted from the medulla oblongata to study the relationship between PRNP genotypes and PRNP expression. We found a significant correlation between promoter indel polymorphisms and PRNP‐mRNA expression (P_0.0413) and therefore hypothesize that differences in polymorphisms could be one of the causes of differences in PRNP expression levels. We also report a novel difference in PRNP expression (P < 0.0001) between Japanese Black and Japanese Brown cattle breeds. There was no significant difference based on age and sex of the animals.     

Linkage mapping of the locus responsible for forelimb-girdle muscular anomaly of Japanese black cattle on bovine chromosome 26

Forelimb-girdle muscular anomaly is an autosomal recessive disorder of Japanese black cattle characterized by tremor, astasia and abnormal shape of the shoulders. Pathological examination of affected animals reveals hypoplasia of forelimb-girdle muscles with reduced diameter of muscle fibres. To identify the gene responsible for this disorder, we performed linkage mapping of the disorder locus using an inbred pedigree including a great-grand sire, a grand sire, a sire and 26 affected calves obtained from a herd of Japanese black cattle. Two hundred and fifty-eight microsatellite markers distributed across the genome were genotyped across the pedigree. Four markers on the middle region of bovine chromosome 26 showed significant linkage with the disorder locus. Haplotype analysis using additional markers in this region refined the critical region of the disorder locus to a 3.5-Mb interval on BTA26 between BM4505 and MOK2602 . Comparative mapping data revealed several potential candidate genes for the disorder, including NRAP , PDZD8 and HSPA12A , which are associated with muscular function.     

荷斯坦牛BoLA-DRA基因SNPs筛选及生物信息学分析

为了探讨BoLA-DRA基因多态性,本研究利用DNA混合池扩增产物直接测序的方法对荷斯坦牛BoLA-DRA基因编码区SNPs进行筛选,并利用生物信息学软件预测该基因m RNA二级结构。结果表明:荷斯坦牛BoLA-DRA基因编码区共有4个SNPs (exon2-A82T,exon2-G116A,exon2-C197T,exon2-C233A)。生物信息学预测结果显示,exon2-A82T、exon2-G116A和exon2-C233A增大了m RNA二级结构的稳定性,而exon2-C197T降低了mRNA二级结构的稳定性。本研究结果可为荷斯坦牛抗病和抗逆性能研究积累更多的分子遗传学数据,并为经济性状相关基因筛选提供理论依据。     

细胞培养用牛血清污染大肠杆菌噬菌体的检测

用大肠杆菌噬菌体增殖法检测２１６批细胞培养用牛血清,结果１４２批（６５．７％）显示阳性结果。由此提示牛血清在采集制备过程中的污染严重,应引起重视。     

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.     

Genetic Variants in SDC3 Gene are Significantly Associated with Growth Traits in Two Chinese Beef Cattle Breeds

Identification of the genes and polymorphisms underlying quantitative traits, and understanding these genes and polymorphisms affect economic growth traits, are important for successful marker-assisted selection and more efficient management strategies in commercial cattle (Bos taurus) population. Syndecan-3 (SDC3), a member of the syndecan family of type I transmembrane heparan sulfate proteoglycans is a novel regulator of feeding behavior and body weight. The aim of this study is to examine the association of the SDC3 polymorphism with growth traits in Chinese Jiaxian and Qinchuan cattle breeds (). Four single nucleotide polymorphisms (SNPs: 1–4) were detected in 555 cows from three Chinese native cattle breeds by means of sequencing pooled DNA samples and polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) methods. We found one SNP (g.28362A > G) in intron and three SNPs (g.30742T > G, g.30821C > T and 33418 A > G) in exons. The statistical analyses indicated that these SNPs of SDC3 gene were associated with bovine body height, body length, chest circumference, and circumference of cannon bone (P < 0.05). The mutant-type variant was superior for growth traits; the heterozygote was associated with higher growth traits compared to wild-type homozygote. Our result confirms the polymorphisms in the SDC3 gene are associated with growth traits that may be used for marker-assisted selection in beef cattle breeding programs.     

Efficient Genetic Mapping of Single Nucleotide Polymorphisms Based Upon DNA Mismatch Digestion

A single-strand specific (sss) nuclease, found in extracts of celery juice, can be used to digest heteroduplex DNA and hence identify heterozygous single nucleotide polymorphism (SNP) sites in PCR products. Here we show this method can be used to map specific genes with relative simplicity and low cost. A particular nucleotide substitution does not need to be identified, and in fact, a priori knowledge of the presence of a SNP is not required, as the entire length of the PCR product is interrogated for the presence of SNPs. This characteristic enables application of this technique to genomes that are not well characterized with regard to SNP polymorphism, and for rapidly placing particular genes onto linkage maps. While this technique is best suited for mapping markers in a backcross configuration, we show that in an F₂ configuration, where alternative homozygotes cannot be discerned by this technique, data are still relatively informative about linkage.     

务川黑牛mtDNA遗传变异及起源进化分析

目的:测定务川黑牛mtDNA D-loop区全序列,以了解其遗传背景。方法:采用PCR直接测序方法测定务川黑牛mtD-NA D-loop区全序列。结果:32个个体D-loop区的全序列中,四种碱基A、T、C和G含量分别为:33.1%、28.4%、25.0%和13.5%,A+T平均含量61.5%。经过序列比对,共检测到57个变异位点,占所测mtDNA序列总长的6.26%,其中转换54个,颠换3个;产生17种单倍型,其中6种是普通牛单倍型,11种是瘤牛单倍型;平均核苷酸差异为22.623,单倍型多样度0.901±0.039,核苷酸多样度0.0249±0.00147。结论:务川黑牛这一品种遗传多样性非常丰富,据此构建的NJ进化树显示务川黑牛含有普通牛和瘤牛的血统。     

微卫星标记与奶牛数量性状QTL定位

本文对奶牛产奶性能（产奶量、乳脂率、乳脂量、蛋白率、蛋白量）、乳房炎及体细胞数、繁殖性能、生产寿命等数量性状QTL定位研究进展进行了综述。 Abstract:Mapping of quantitative trait loci for some important traits (milk yield,fat percentage,fat yield,protein percentage,protein yield,clinical mastitis and somatic cell count,reproductive performance,productive life,etc.) in dairy cattle was introduced in this review.     

Maintenance behavior of cattle in a young tree plantation in southern Kyushu,Japan

This 4-year study monitored maintenance behavior of beef cattle (Japanese Black) foraging in a young Chamaecyparis obtusa (an evergreen conifer) plantation in southwestern Japan. The animals spent 551 ± 8 min (mean ± SE) per day grazing, 436 ± 4 min ruminating, 313 ± 9 min resting, 44 ± 2 min moving, and 96 ± 4 min on other activities. Grazing activity was high during 0600–1800 h and low during 2200–0600 h. Ruminating activity was high during 2200–0600 h and low during 0600–1800 h. Resting activity was high during 2200–0600 h and low for the remainder of the day. Air temperature was not a dominant factor determining the daily duration of grazing, rumination, or resting. The activity of the animals was usually synchronized, with the degree of synchronization being highest in grazing followed by ruminating and resting. Daily liveweight change of the animals was positively related with daily ruminating time, i.e., animals gained more when they ruminated longer. The maintenance behavior of cattle in the young tree plantation, as compared with that on grasslands, is characterized by (1) high grazing activity around midday, (2) low ruminating activity in the daytime, and (3) midday adjustment of the daily total grazing time. Daily rumination time may be used as an indicator of animal performance on the bases of a herd (not individual animals) and a period of 2–15 weeks (not individual days).     

Mother–young distance in Japanese Black cattle at pasture

Distance between dam and offspring (1–121 days old) in a herd of Japanese Black cattle (Bos taurus) grazing a tropical grass (Paspalum notatum) pasture (1.5 ha) was investigated during 7-h grazing periods over grazing seasons from May (spring) to October (autumn). The mother–young distance was not constant throughout the grazing period, repeatedly increasing and decreasing. Although significant periodicity was always detected in the mother–young distance, there was no consistent dominant cycle, indicating the complexity of the within-day pattern of mother–young distance. The mean mother–young distance over the grazing period increased as a calf aged, reaching a plateau at an age of about 33 days. The mean distance of a calf from its mother was usually shorter than that from a non-mother cow, with the difference between the mean distances decreasing sharply until a calf became about 35 days old. The results and literature show that mutual independence of mother and young rapidly develops in the first 30–50 days after parturition. Electronic Publication     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.     

Properties of Particulate and Detergent-Solubilized Phospholipid N-Methyltransferase Activity from Calf Brain

Abstract: Calf brain membranes catalyze the enzymatic transfer of [CH₃-³H]methyl groups from S-adenosyl-l -[CH₃-³H]methionine into endogenous phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N,N-dimethylethanolamine (PDE), and phosphatidylcholine (PC). Phospholipid N-methylation can be stimulated by the addition of exogenous PME or PDE, added in aqueous dispersions with sodium taurocholate. When membranes are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the labeled phospholipid products. When exogenous PME is replaced by PDE, 91% of the label is incorporated into PC. Thus, under these in vitro conditions it is possible to assay PME- and PDE-N-methyitransferase activity separately. The calf brain phospholipid N-methyltransferase activity has also been solubilized by treating the membranes ultrasonically in the presence of Triton X-100 and 10 mM monothioglycerol. When the detergent extracts are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the enzymatically labeled products. In the presence of exogenous PDE, more than 97% of the label is incorporated into PC. Optimal conditions for the membrane-bound and detergent-solubilized PME- and PDE-N-methyltransferase activity have been established. These conditions have been used as a basis for testing the hypothesis that the conversion of PME to PC is catalyzed by a single enzyme in calf brain. In these studies, PME- and PDE-N-methyltransferase activities have been found to be similar, if not identical, with respect to: (1) extractability with Triton X-100; (2) pH optimum; (3) response to divalent cations; (4) apparent K_m, for S-adenosyl-l -methionine and K_I for S-adenosyl-l -homocysteine, (5) sensitivity to N-ethylmaleimide; and (6) thermal inactivation at 55°. Overall, these results are consistent with the conclusion that in calf brain, PME and PDE are methylated by the same enzyme or by two phospholipid N-methyltransferases having very similar properties.     

Profiles of growth hormone and insulin secretion, and glucose response to insulin in growing Japanese Black heifers (beef type): comparison with Holstein heifers (dairy type)

Nine Japanese Black and 10 Holstein heifers ranging from 1 week (wk) to 18 months (mo) old received a single bolus intravenous injection of GH-releasing factor (GRF, 0.25-microg/kg BW), glucose (112.5-mg/kg BW) or insulin (0.2-U/kg BW) at various stages through 18 mo of age. The GH secretory response to exogenous GRF in Japanese Black heifers was lower than that in Holstein heifers at all stages of growth. While insulin secretory function was not very different in both breeds from 1 to 12 mo of age, the insulin response was much higher in Japanese Black heifers than in Holstein heifers after sexual maturation. The degree of decrease in plasma glucose following insulin injection was similar in both breeds at each stage of growth. It is concluded that compared with Holstein heifers, Japanese Black heifers have lower GH and higher insulin secretory functions, and that the two breeds have similar glucose response to insulin.