Detection of candidate polymorphisms around the QTL for fat area ratio to rib eye area on BTA7 using whole‐genome resequencing in Japanese Black cattle

In our previous study, we performed genome‐wide association study (GWAS) to identify the genomic region associated with Fat area ratio to rib eye area (FAR) and detected a candidate in BTA7 at 10–30 Mbp. The present study aims to comprehensively detect all polymorphisms in the candidate region using whole‐genome resequencing data. Based on whole‐genome resequencing of eight animals, we detected 127,090 polymorphisms within the region. Of these, 31,945 were located within the genes. We further narrowed the polymorphisms to 6,044 with more than five allele differences between the high and low FAR groups that were located within 179 genes. We subsequently investigated the functions of these genes and selected 170 polymorphisms in eight genes as possible candidate polymorphisms. We focused on SLC27A6 K81M as a putative candidate polymorphism. We genotyped the SNP in a Japanese Black population (n = 904) to investigate the effect on FAR. Analysis of variance revealed that SLC27A6 K81M had a lower p‐value (p = .0009) than the most significant SNP in GWAS (p = .0049). Although only SLC27A6 K81M was verified in the present study, subsequent verification of the remaining candidate genes and polymorphisms could lead to the identification of genes and polymorphisms responsible for FAR.     

Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

Ratio of rice reflectance for estimating leaf blast severity with a multispectral radiometer

 Rice reflectance was measured to determine the spectral regions most sensitive to leaf blast infection with a multispectral radiometer. As disease severity increased, reflectance also increased in the 400–500 nm (blue), 570–700 nm (red), and 900–2000 nm regions but decreased in the 500–570 nm and 700–900 nm regions. The increased reflectance in the blue and red regions may be attributed to decreased chlorophyll and carotenoid contents in response to the blast infection. The maximum and minimum reflectance differences occurred at 680 nm and 760 nm for the nondiseased and diseased rice, respectively. The spectral location of maximum sensitivity was 675 nm regardless of disease severity. Rice reflectance ratios were evaluated as indicators of leaf blast severity. Two ratios, R550/R675 (reflectance at 550 nm divided by reflectance at 675 nm), and R570/R675 quantified the significant disease severity. These wavelengths were selected based on the sensitivity minima and maxima. The ratios of nondiseased rice plants varied depending on growth stage. The variation in ratios must be considered when they are used to estimate leaf blast severity. Received: April 2, 2002 / Accepted: August 12, 2002     

Methanogenic archaeal communities developed in paddy fields in the Kojima Bay polder, estimated by denaturing gradient gel electrophoresis, real-time PCR and sequencing analyses

Methanogenic archaeal communities inhabiting the paddy field soils in the Kojima Bay polder were investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), real-time PCR and sequencing analyses. Soil samples of the plow and subsoil layers were collected in 2006 from four paddy fields that were reclaimed between 1692 and 1954. The DGGE band patterns of the targeted 16S rRNA genes amplified from the extracted DNA from the samples were different from the patterns from the paddy field soils in diluvial and alluvial areas. The numbers of targeted 16S rRNA genes, which were involved with methanogenic archaeal and other archaeal sequences, were approximately 10⁷–10⁸ and 10⁶ g⁻¹ dry soil in the plow and subsoil layers, respectively. Sequences of methanogenic archaeal 16S rRNA genes belonging to Methanocellales (Rice cluster I), Methanosarcinales and Methanobacteriales were obtained from the major DGGE bands. Whereas sequences in Methanomicrobiales, which were predominant methanogens in the diluvial and alluvial paddy fields, were not recovered. Known halophilic and methylotrophic methanogens, which are characteristic of saline and marine environments, were not detected. These results indicate that distinctive methanogenic archaeal communities have developed in the paddy field soils in the Kojima Bay polder.     

The g.841G>C SNP of FASN gene is associated with fatty acid composition in beef cattle

The objective of the current study is to evaluate the association between fatty acid composition and fatty acid synthase gene polymorphisms as responsible mutations. For this purpose, we selected seven previously reported single nucleotide polymorphisms (SNPs) in FASN gene, including one within promoter region (g.841G>C) and six non‐synonymous SNPs (g.8805C>T, g.13126C>T, g.15532A>C, g.16024A>G, g.16039C>T, g.17924A>G), and genotyped them in Japanese Black cattle. Genotyping results revealed that g.8805 C>T and g.17924 A>G were monomorphic loci. Genome‐wide association analysis including the other five SNPs revealed that only g.841G>C showed significant associations with the percentages of C14:0, C14:1, C16:1 and C18:1 at 5% genome‐wide significance level. In order to further evaluate the effect, we genotyped g.841G>C using additional three populations, including two Japanese Black populations and a Holstein cattle population. g.16024A>G was also genotyped and included in the analysis because it has been reported to be associated with fatty acid composition in Japanese Black cattle. In the result of analysis of variance, g.841G>C showed stronger effects on fatty acid percentage than those of g.16024A>G in all populations. These results suggested that g.841G>C would be a responsible mutation for fatty acid composition and contribute to production of high‐grade beef as a selection marker in beef cattle.     

Fine mapping the number of corpora lutea quantitative trait loci on SSC3: Analysis of the porcine follicle‐stimulating hormone receptor gene

In females, follicle‐stimulating hormone (FSH) targets a FSH receptor (FSHR) expressed only on granulose cells, inducing maturation of the ovarian follicles. We hypothesized that genetic variants in the FSHR gene influence litter size by affecting the number of corpora lutea. We fine‐mapped a region of Sus Scrofa chromosome 3 that contains quantitative trait loci for corpora lutea. Polymorphisms were detected in the exons and 5′ flanking region of the porcine FSHR gene, a positional candidate for the statistically most significant of the quantitative trait loci. Finally, 248 F₂ animals from a Duroc and Meishan cross were genotyped for three FSHR SNPs at positions 74, 532 and 1166, and these were correlated with the phenotypes of litter size and corpus luteum number. Three haplotypes were identified: M1 (G/G/C), M2 (C/A/T) and D (C/A/C). In the F₂ population, the M1 haplotype was associated with a greater number of corpora lutea (P < 0.01) and also seemed to be associated with increased litter size, although the association was not significant (P = 0.2571). Some polymorphisms resulting in amino acid substitutions in these genes were excluded from the polymorphisms possibly responsible for the number of corpora lutea.     

Age and growth of yellowstriped butterfish, <Emphasis Type="Italic">Labracoglossa argentiventris</Emphasis>, around Izu Oshima Island

ABSTRACT:   Age and growth of the yellowstriped butterfish, Labracoglossa argentiventris , around Izu Oshima Island were studied using a total of 1450 fish. Age was determined by counting the edge of the opaque zones as a ring mark on sectioned sagittal otoliths. Formation of the first ring was observed during spring or summer, corresponding to 1.5 years after hatching. Thereafter, one ring was formed each year in the same season as the previous year. The growth of the butterfish was rapid until 2 years of age. The maximum likelihood method was applied to the age and length data for estimating parameters in von Bertalanffy, Gompertz, and Logistic growth models. The selected model, based on the Akaike Information Criterion, was the von Bertalanffy growth model, which indicated differential asymptotic length and variance by sex.     

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.     

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.     

Quantitative trait loci for leg weakness traits in a Landrace purebred population

Leg weakness in pigs is a serious problem in the pig industry. We performed a whole genome quantitative trait locus (QTL) analysis to find QTLs affecting leg weakness traits in the Landrace population. Half-sib progeny ( n  = 522) with five sires were measured for leg weakness traits. Whole genome QTL mapping was performed using a half-sib regression-based method using 190 microsatellite markers. No experiment-wide significant QTLs affecting leg weakness traits were detected. However, at the 5% chromosome-wide level, QTLs affecting leg weakness traits were detected on chromosomes 1, 3, 10 and 11 with QTL effects ranging from 0.07 to 0.11 of the phenotypic variance. At the 1% chromosome-wide level, QTLs affecting rear feet score and total leg score were detected on chromosomes 2 and 3 with QTL effects of 0.11 and 0.13 of the phenotypic variance, respectively. On chromosome 3 and 10, some QTLs found in this study were located at nearby positions. The present study is one of the first reports of QTLs affecting fitness related traits such as leg weakness traits, that segregate within the Landrace population. The study also provides useful information for studying QTLs in purebred populations.