Genotype imputation in a tropical crossbred dairy cattle population

The objective of this study was to investigate different strategies for genotype imputation in a population of crossbred Girolando (Gyr × Holstein) dairy cattle. The data set consisted of 478 Girolando, 583 Gyr, and 1,198 Holstein sires genotyped at high density with the Illumina BovineHD (Illumina, San Diego, CA) panel, which includes ～777K markers. The accuracy of imputation from low (20K) and medium densities (50K and 70K) to the HD panel density and from low to 50K density were investigated. Seven scenarios using different reference populations (RPop) considering Girolando, Gyr, and Holstein breeds separately or combinations of animals of these breeds were tested for imputing genotypes of 166 randomly chosen Girolando animals. The population genotype imputation were performed using FImpute. Imputation accuracy was measured as the correlation between observed and imputed genotypes (CORR) and also as the proportion of genotypes that were imputed correctly (CR). This is the first paper on imputation accuracy in a Girolando population. The sample-specific imputation accuracies ranged from 0.38 to 0.97 (CORR) and from 0.49 to 0.96 (CR) imputing from low and medium densities to HD, and 0.41 to 0.95 (CORR) and from 0.50 to 0.94 (CR) for imputation from 20K to 50K. The CORR_anim exceeded 0.96 (for 50K and 70K panels) when only Girolando animals were included in RPop (S1). We found smaller CORR_anim when Gyr (S2) was used instead of Holstein (S3) as RPop. The same behavior was observed between S4 (Gyr + Girolando) and S5 (Holstein + Girolando) because the target animals were more related to the Holstein population than to the Gyr population. The highest imputation accuracies were observed for scenarios including Girolando animals in the reference population, whereas using only Gyr animals resulted in low imputation accuracies, suggesting that the haplotypes segregating in the Girolando population had a greater effect on accuracy than the purebred haplotypes. All chromosomes had similar imputation accuracies (CORR_snp) within each scenario. Crossbred animals (Girolando) must be included in the reference population to provide the best imputation accuracies.     

Proline and arginine accumulation in developing berries of Vitis vinifera L. in Australian vineyards: Influence of vine cultivar, berry maturity and tissue type

Using HPLC analysis, free amino acid profiles were obtained for ripe berries of six grapevine cultivars (Cabernet Sauvignon, Grenache, Muscat Gordo, Pinot Noir, Riesling and Sangiovese) grown under comparable conditions and harvested at similar maturities. Compositional differences were observed between cultivars, however proline and arginine were always the major amino acids. Mature berries of Cabernet Sauvignon contained a very high concentration of proline, but a much lower concentration of arginine. Those of other cultivars contained moderate levels of both arginine and proline.
Changes in free amino acid profiles during grape berry development were further investigated in four cultivars (Cabernet Sauvignon, Chardonnay, Gewurztraminer and Muscat Gordo). In all cases, most of the proline accumulation occurred late in ripening, around four weeks post-veraison. In contrast, arginine accumulation began before veraison and continued to full maturity, except for those cultivars in which a high concentration of proline accumulated, in which case the concentration of arginine reached a plateau relatively early in development. Accumulation of both proline and arginine appears to be develop-mentally regulated. These observations are discussed with reference to proline and arginine metabolism and possible links between them.
The distribution of free proline, arginine and other amino acids amongst pulp, skin and seed was examined using mature fruit of Cabernet Sauvignon and Riesling. Amino acid profiles were found to vary considerably between the berry components. The skin contained a greater ratio of arginine:proline compared with the pulp. This suggests that the yeast-assimilable nitrogen content of juices, and therefore their fermentability, could be enhanced in the presence of skins.     

Associations between casein haplotypes and milk production traits of Swiss Brown cattle

Effects of casein haplotypes and beta-lactoglobulin (LG) genotypes on milk protein fractions and on daughter yield deviations for milk performance traits were estimated from a daughter design. Offspring of seven Swiss Brown sires with the haplotypes B-A-B-A and B-A-B-B for alpha s1-, alpha s2-, beta-, and kappa-caseins were selected. The milk of daughter groups with paternal haplotype B-A-B-A was associated with lower casein content and higher whey protein content compared with B-A-B-B. Because of these contrary effects, the true protein content was not affected by the paternal haplotypes. The effects of maternal haplotypes were significant on true protein and casein content but not on whey protein content. The beta-LG genotypes had highly significant effects on casein and whey protein content. The effect of beta-LG BB was positive on casein and negative on whey protein content compared with beta-LG AA; the effect of beta-LG AB was intermediate. No significant effects of paternal haplotypes were found for daughter yield deviation on kilograms of milk, fat, and protein or percentages of fat and protein. The effects of the beta-LG genotypes were, independent of the parental haplotypes, close to significant on daughter yield deviation for percentage of protein. The beta-LG BB tended to be associated with a higher protein content compared with beta-LG AA. The effects for beta-LG genotypes showed additive gene effects. The analysis of paternal haplotypes within sires revealed a contrary effect of haplotypes for two of the seven sires for casein content. The paternal haplotypes within sire showed, although not significant, that haplotypes of the two sires had a contrary effect on daughter yield deviation for percentage of protein as well.     

Sequential hydrolysis of swine carcass samples and determination of amino acid concentrations using pre‐column derivatization with phenyl isothiocyanate

Comparing amino acid (AA) retention levels in pig carcass to true ileal digestible AA intake provides an estimate of the marginal efficiency of AA utilization. Accurate analysis of AA levels in the carcass samples is critical. However, the standard 24 h of hydrolysis does not always provide maximum AA values. A study was carried out to investigate the effect of hydrolysis time on AA measurements in pig carcass. Correction factors to standardize AA levels to 24 h of hydrolysis were also determined. Ground carcass samples were hydrolysed with 6 mol litre⁻¹ hydrochloric acid (HCl) in a 110 °C oven for nine different time periods. Pre‐column derivatization with phenyl isothiocyanate (PITC) was used to determine AA concentrations in all of the samples. Hydrolysis time significantly affected (P < 0.001) AA levels. The highest levels (P > 0.05) of valine, isoleucine, serine, glycine, threonine, alanine, arginine, proline, histidine and phenylalanine were not observed with 24 h hydrolysis. Therefore, correction factors and sequential hydrolysis curves are important for these amino acids. In conclusion, the effect of hydrolysis time should be considered in amino acids analysis. © 2000 Society of Chemical Industry     

Antioxidant Bioactive Compounds Changes in Fruit of Quince Genotypes Over Cold Storage

Quince fruit has many benefits to human health and is excellent source of bioactive compounds. The fruit of 15 quince genotypes stored at 2 °C for 5 mo to study fruit quality changes during cold storage. Fruit were sampled monthly and stored at 20 °C for 24 h. Fruit ascorbic acid (AA), total phenol (TP), and total flavonoid (TF) concentrations, total antioxidant activity (TAA), flesh browning (FB) incidence, polyphenol oxidase (PPO), peroxidase (POX), and superoxide dismutase (SOD) activities were measured during storage. A high variation in bioactive compounds was observed across genotypes. The range of 26.8 to 44.4 mg/100 g FW for AA, 86.7% to 98.2% for TAA, 157.7 to 380.7 mg GAE 100^?1 g FW for TP, and 5.3 to 10.7 mg/100 g FW for TF were observed across genotypes at harvest time. The overall AA, TAA, TP, TF, and SOD decreased while PPO and POX increased during storage. FB was first observed after 4 mo and increased thereafter while the FB index was different across genotypes. Higher bioactive content may prevent or reduce FB index so that a negative correlation was found between FB and AA, TAA, TP, TF, and SOD.     

Short communication: Polymorphism of casein cluster genes in Czech local goat breeds

The 4 casein loci were evaluated as haplotypes in 2 dairy goat breeds kept in the Czech Republic. Analysis of 41 White Shorthaired (WSH) trio families and 44 Brown Shorthaired (BSH) trio families revealed 14 and 20 haplotypes, respectively. Various genomic techniques were used to type the casein loci. Twenty-two different combinations of these alleles (casein haplotypes, in the order CSN1S1-CSN2-CSN1S2-CSN3) were found. Only 5 haplotypes in the WSH breed and 6 haplotypes in the BSH breed occurred at frequencies >0.05. For the WSH breed, the most common haplotype was FCFB (0.260), whereas for the BSH breed, the most common haplotype was FCFA (0.217). The information on the haplotype variability in both breeds could be used in breeding programs aimed at preserving biodiversity or selecting animals for specific protein production and cheesemaking.     

烟草叶绿体密码子的偏好性及聚类分析

综合运用多种软件对3种烟草属植物以及其他对照植物的6个叶绿体蛋白编码基因进行了分析.结果表明,普通烟草在密码子使用特性上与林生烟草最为接近,在所分析的59个密码子中,有39个密码子的RSCU( Relative Synonymous Codon Usage)值完全一样;8种参与分析的植物中共出现了30个具有偏好使用的密码子,其中7种双子叶植物在碱基组成上偏爱以C结尾的密码子,而水稻偏爱以T和C结尾的密码子;以RSCU值为基础的聚类结果显示出植物的密码子偏好性具有种族特征,表明基于叶绿体蛋白编码基因的密码子使用特性的聚类结果较适合用于系统发育分析的重要补充.     

Methods for extracting the taste compounds from water soluble extract of Jinhua ham

The present paper reports the determination of 22 amino acids and ammonium along the acetification process by high-performance liquid chromatography (HPLC), employing 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as precolumn derivatization reagent. The method was successfully validated obtaining adequate values to selectivity, response linearity, precision and accuracy, as well as low detection and quantification limits. Its utility for routine analysis of amino acids along acetification has been proved. Three red wine substrates were fermented employing two kinds of acetification methods (surfaced and submerged culture) and the requirements of amino acids and ammonium as nitrogen source by acetic acid bacteria were evaluated. The identification of the acetic acid bacteria at species level was also done. At the beginning, the principal amino acid, in terms of abundance, was proline followed by arginine. A different behavior between both the acetification methods was observed. First of all, the consumption of amino acids is much lower in submerged than in surface acetifications. For surface culture acetification, the most consumed was proline, arginine being the main nitrogen source for submerged acetification systems. On the other hand, it was also observed that the nitrogen requirement of the bacteria is proportional to the time spent in the acetification process.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

Genomic breeding value estimation using genetic markers, inferred ancestral haplotypes, and the genomic relationship matrix

With the introduction of new single nucleotide polymorphism (SNP) chips of various densities, more and more genotype data sets will include animals genotyped for only a subset of the SNP. Imputation techniques based on unobserved ancestral haplotypes may be used to infer missing genotypes. These ancestral haplotypes may also be used in the genomic prediction model, instead of using the SNP. This may increase the reliability of predictions because the ancestral haplotype may capture more linkage disequilibrium with quantitative trait loci than SNP. The aim of this paper was to study whether using unobserved ancestral haplotypes in a genomic prediction model would provide more reliable genomic predictions than using SNP, and to determine how many loci in the genomic prediction model would be redundant. Genotypes of 8,960 bulls and cows for 39,557 SNP were analyzed with a hidden Markov model to associate each individual at each locus to 2 ancestral haplotypes. The number of ancestral haplotypes per locus was fixed at 10, 15, or 20. Subsequently, a validation study was performed in which the phenotypes of 3,251 progeny-tested bulls for 16 traits were used in a genomic prediction model to predict the estimated breeding values of at least 753 validation bulls. The squared correlation between genomic prediction and deregressed daughter performance estimated breeding value, when averaged across traits, was slightly higher when 15 or 20 ancestral haplotypes per locus were used in the prediction model instead of the SNP genotypes, whereas the prediction model using a genomic relationship matrix gave the lowest squared correlations. The number of redundant loci [i.e., loci that had less than 18 jumps (0.1%) from one ancestral haplotype to another ancestral haplotype at the next locus], was 18,793 (48%), which means that only 20,764 loci would need to be included in the genomic prediction model. This provides opportunities for greatly decreasing computer requirements of genomic evaluations with very large numbers of markers.     

Haplotypes responsible for early embryonic lethality detected in Nordic Holsteins

Widespread use of a limited number of elite sires in dairy cattle breeding increases the risk of some deleterious allelic variants spreading in the population. Genomic data are being used to detect relatively common (frequency >1%) haplotypes that never occur in the homozygous state in live animals. Such haplotypes likely include recessive lethal or semilethal alleles. The aim of this study was to detect such haplotypes in the Nordic Holstein population and to identify causal genetic factors underlying these haplotypes. Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA) genotypes for 26,312 Nordic Holstein animals were phased to construct haplotypes. Haplotypes that are common in the population but never observed as homozygous were identified. Two such haplotypes overlapped with previously identified recessive lethal mutations in Holsteins—namely, structural maintenance of chromosomes 2 (HH3) and brachyspina. In addition, we identified 9 novel putative recessive lethal-carrying haplotypes, with 26 to 36 homozygous individuals expected among the genotyped animals but only 0 to 3 homozygotes observed. For 2 out of 9 homozygous-deficient haplotypes, insemination records of at-risk mating (carrier bull with daughter of carrier sire) showed reduced insemination success compared with not-at-risk mating (noncarrier bull with daughter of noncarrier sire), supporting early embryonic mortality. To detect the causative variant underlying each homozygous-deficient haplotype, data from the 1000 Bull Genome Project were used. However, no variants or deletions identified in the chromosome regions covered by the haplotypes showed concordance with haplotype carrier status. The carrier status of detected haplotypes could be used to select bulls to reduce the frequency of the latent lethal mutations in the population. If desired, at-risk matings could be avoided.     

乳蛋白多态性和盐分布对牛乳凝乳性能的影响

研究不同基因型乳蛋白对牛乳凝乳特性的影响规律。采集1 071 头荷斯坦奶牛血样,分析κ-酪蛋白（κ-casein,κ-CN）和β-乳球蛋白（β-lactoglobulin,β-LG）的基因型,在明确基因型的基础上,采集样品开展牛乳凝乳能力评价。在初步筛选的基础上,选择凝乳性能好、凝乳性能差和不凝乳样品各至少30 份,重复3 次,开展凝乳流变学特性、蛋白多态性及矿物离子分布分析。通过动态流变仪、电感应耦合等离子体质谱仪、毛细管电泳、高效液相色谱技术分析不同凝乳等级牛乳的凝乳时间,胶体钙、镁、磷含量差异,不同基因型导致蛋白多态性及含量对牛乳凝乳能力的影响。结果显示,在所有奶牛组中,β-LG的AB基因型（占比48.48%）最常见,但AA型基因（30.97%）的原料乳凝乳效果较好;κ-CN的BB基因型（12.00%）凝乳效果较好,较AA、AB等其凝乳时间更短和凝胶强度更强。凝乳性能好的样品中CN含量及胶体钙含量较高,pH值较凝乳性能差和不凝乳样品低,凝乳时间与κ-CN含量呈反比,酪蛋白和乳清蛋白组成和基因频率的变化会影响牛乳凝乳性能的变化。     

Novel polymorphisms of the growth hormone gene and their effect on growth traits in Chinese goats

The polymorphisms of the growth hormone (GH) gene were analyzed in 686 individuals from four goat populations, Three haplotypes (A, B and C) and three observed genotypes (AA, AB and AC) were detected at the P2 locus, and three haplotypes (E, F and G) and three observed genotypes (EE, EF and EG) were also detected at the P4 locus. In addition, five single nucleotide polymorphisms (SNPs)—A112G, C142T (Gly > Ser), C214T (P2 locus), C266A (Pro > His) and C214T (P4 locus, Arg > Trp), were identified by GH gene sequencing and PCR-SSCP analysis. The SNPs loci were in Hardy–Weinberg disequilibrium in three goat populations (P < 0.05). Association of polymorphisms with growth traits was done in BG, F1 and F1 populations, which were shown to be associated with growth traits in three goat populations. The SNPs in the goat GH gene had significant effects on growth traits (P < 0.05). suggesting that the GH gene is a strong candidate gene that affects growth traits in goat.     

低温贮藏下生肉丸中6种氨基酸与N-亚硝胺形成的关系探讨

为初步确定氨基酸与N-亚硝胺之间的转化关系,本研究以精瘦肉为原料,在肉丸中分别添加1 mg/kg赖氨酸、精氨酸、丙氨酸、脯氨酸、酪氨酸、缬氨酸,低温(4 ℃)贮藏9 d,利用气相色谱-串联质谱联用(GC-MS/MS)方法定期测定肉丸中N-亚硝胺含量,探究氨基酸与N-亚硝胺间的转化关系,并分析五味子对N-亚硝胺的产生以及外源氨基酸与N-亚硝胺之间关系的影响。结果表明,肉丸中检出N-亚硝基二甲胺(NDMA)、N-亚硝基哌啶(NPIP)、N-亚硝基吡咯烷(NPYR)、N-亚硝基二正丁胺(NDBA)、N-亚硝基二苯胺(NDPHA)五种N-亚硝胺。精氨酸、脯氨酸可促进N-二甲基亚硝胺(NDMA)、N-亚硝基二正丁胺(NDBA)的生成,其中精氨酸的添加使NDMA含量最高增加了73.18%,脯氨酸的添加使NDBA含量最高增加了156.18%;同时发现五味子使添加精氨酸的NDMA含量最高降低了28.37%,使添加脯氨酸的NDBA含量最大降幅达到29.47%,NPIP含量最大降幅达到48.62%。综上,精氨酸可提高肉制品中NDMA的含量,脯氨酸可提高NDBA的含量;五味子的添加总体上降低了NPIP含量,但差异不显著;五味子对添加精氨酸的NDMA含量有降低作用,同时对添加脯氨酸的NDBA含量也有降低作用。     

Improvement of the expression level of β-glucosidase from Agrobacterium sp. in Escherichia coli by rare codon optimization

To achieve high level expression of β-glucosidase from Agrobacterium sp. (Abg) in an Escherichia coli expression system, 6 rare codons at +3, +11, +158, +308, +314, and +318 of abg gene were replaced with favored codons using site-directed mutagenesis. The rare codon replacements of +3 (CCC) and +11 (CCC) positions enhanced the expression level of Abg by 2- and 3.6-fold, respectively. The double mutant, Abg-DM, where both of +3 and +11 rare codon positions were modified showed a 5.2-fold higher expression level than the original abg gene. Circular dichroism (CD) spectra of the over-expressed Abg and original Abg enzymes were identical, indicating that Abg was properly folded during over-expression.     

Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, ?F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.     

Genetic variation in the kappa-casein gene (CSN3) of Chinese yak (Bos grunniens) and phylogenetic analysis of CSN3 sequences in the genus Bos

Variants of κ-casein (CSN3) have been extensively studied in cattle and 13 alleles have been identified at the protein and DNA levels to date. Evolution of some of these alleles and a possible common ancestor remain unclear. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of CSN3 exon IV in domesticated yak revealed a 2-allele polymorphism showing migration patterns different from known cattle variants. The PCR products of both yak CSN3 SSCP alleles were sequenced. All yak had nucleotide sequences coding for Thr in AA position 136 (identical to bovine CSN3*A) and Ala in position 148 (identical to bovine CSN3*B). The sequencing results were confirmed by PCR-RFLP analysis using HindIII and TaqI. A 12-bp insertion in the coding region, representing a repeated nucleotide and AA motif, was found in 1 yak allele. The duplication corresponds to the codons for AA 147 to 150 (Glu-Ala-Ser-Pro) or 148 to 151 (Ala-Ser-Pro-Glu), which are repeated identically. In 21 yak samples genotyped by PCR-SSCP analysis, frequencies for the insertion variant and the short variant were about 68 and 32%, respectively. The loss of the insertion may have led to the ancestral CSN3 allele from which all currently known variants of CSN3 in the genus Bos evolved. This is the first report of polymorphisms in the yak CSN3 gene and may be helpful for future studies on genetic variation within and between yak populations or on associated traits.     

The contents of the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-l-α,β-diaminopropionic acid), and other free and protein amino acids in the seeds of different genotypes of grass pea (Lathyrus sativus L.)

The free and protein amino acids of nine different genotypes of grass pea (Lathyrus sativus L.) seeds were analysed by HPLC with pre-column PITC (phenyl isothiocyanate) derivatisation. Among the free amino acids, homoarginine was quantitatively the most important (up to 0.8% seed weight) and stable while the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-l-α,β-diaminopropionic acid) showed highest variation (0.02–0.54%) in the nine genotypes examined. Among protein amino acids, glutamic acid was quantitatively most significant, followed by aspartic acid, arginine, leucine, lysine and proline. The sulphur amino acid, methionine, showed the lowest concentration in all the L. sativus genotypes, and also in lentil (Lens culinaris) and in soybean (Glycine max) seeds analysed at the same time.