Analysis of Vietnamese rice germplasm provides an insight into Japonica rice differentiation

Genetic differentiation among landraces of rice in northern Vietnam that lie in the centre of genetic diversity of Asian cultivated rice was analysed using DNA markers, and morphological and physiological traits to obtain an insight into the genetic differentiation of Asian rice. Principle coordinate analysis, based on nuclear RFLP data, divided Vietnamese rice varieties into three groups, designated A, B and C. Chloroplast DNA variation discriminated group A, which corresponded to the Indica rice varieties, from both groups B and C, which corresponded to the Japonica rice varieties. Physiological characteristics for Indica‐Japonica and tropical‐temperate Japonica discrimination suggested groups A and B consisted of typical Indica and Japonica varieties, respectively, while the characteristics of group C were distinct from those of the other two groups. This suggests that the genetic differences between groups B and C represent a new aspect of genetic variation that has not been previously reported in Japonica rice. Groups C and B corresponded with upland and lowland rice, respectively. Eight marker loci were associated with upland‐lowland differentiation in Japonica rice. Highlighted chromosomal regions might have been selected during the history of cultivation under different cultural conditions, and thus contain genes related to adaptation.     

Polymorphism of two waxy proteins in the emmer group of tetraploid wheat, Triticum dicoccoides, T. dicoccum, and T. durum

Gel-electrophoretic analyses detected polymorphism of two waxy (Wx) proteins, Wx-Al and Wx-Bl, in 334 accessions of the emmer group of tetraploid wheat, Triticum dicoccoides, T. dicoccum and T. durum. The null allele for the Wx-Al protein (Wx-Alb) was found in one accession of T. dicoccoides and seven of T. dicoccum, but it was not present in those T. durum accessions analysed. The null allele for the Wx-Bl protein (Wx-Blb) did not occur in three emmer-wheat species. Wx-Al and Wx-Bl proteins showing alteration in mobility in SDS-PAGE gel or in isoelectric points were found in five accessions and considered to be the products of new alleles, Wx-Ald, Wx-Ale and/or Wx-Bld. Densitometric analysis of Wx protein bands revealed that Wx-Al was present in smaller amounts than Wx-B1 in almost all accessions.     

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.     

Effects of heat stress on production,somatic cell score and conception rate in Holsteins

We examined the effects of heat stress (HS) on production traits, somatic cell score (SCS) and conception rate at first insemination (CR) in Holsteins in Japan. We used a total of 228 242 records of milk, fat and protein yields, and SCS for the first three lactations, as well as of CR in heifers and in first‐ and second‐lactation cows that had calved for the first time between 2000 and 2012. Records from 47 prefectural weather stations throughout Japan were used to calculate the temperature–humidity index (THI); areas were categorized into three regional groups: no HS (THI < 72), mild HS (72 ≤ THI < 79), and moderate HS (THI ≥ 79). Trait records from the three HS‐region groups were treated as three different traits and trivariate animal models were used. The genetic correlations between milk yields from different HS groups were very high (0.91 to 0.99). Summer calving caused the greatest increase in SCS, and in the first and second lactations this increase became greater as THI increased. In cows, CR was affected by the interaction between HS group and insemination month: with summer and early autumn insemination, there was a reduction in CR, and it was much larger in the mild‐ and moderate‐HS groups than in the no‐HS group.     

Intestinal Absorption of Fucoidan Extracted from the Brown Seaweed,Cladosiphon okamuranus

The aim of this study was to examine the absorption of fucoidan through the intestinal tract. Fucoidan (0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL) was added to Transwell inserts containing Caco-2 cells. The transport of fucoidan across Caco-2 cells increased in a dose-dependent manner up to 1.0 mg/mL. It reached a maximum after 1 h and then rapidly decreased. In another experiment, rats were fed standard chow containing 2% fucoidan for one or two weeks. Immunohistochemical staining revealed that fucoidan accumulated in jejunal epithelial cells, mononuclear cells in the jejunal lamina propria and sinusoidal non-parenchymal cells in the liver. Since we previously speculated that nitrosamine may enhance the intestinal absorption of fucoidan, its absorption was estimated in rats administered N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water. Rats were fed 0.2% fucoidan chow (BBN + 0.2% fucoidan rats), 2% fucoidan chow (BBN + 2% fucoidan rats) and standard chow for eight weeks. The uptake of fucoidan through the intestinal tract seemed to be low, but was measurable by our ELISA method. Fucoidan-positive cells were abundant in the small intestinal mucosa of BBN + 2% fucoidan rats. Most fucoidan-positive cells also stained positive for ED1, suggesting that fucoidan was incorporated into intestinal macrophages. The uptake of fucoidan by Kupffer cells was observed in the livers of BBN + 2% fucoidan rats. In conclusion, the absorption of fucoidan through the small intestine was demonstrated both in vivo and in vitro.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

Genetic distance and classification of domestic animals using genetic markers

Genetic distance is used to classify and elucidate the evolutionary relationship between populations, such as species, which have been diverging for a long period. The properties of genetic distance measures in livestock classification have hitherto not been investigated. We used simulation studies to compare the accuracy of eight genetic distance measures to classify recently diverged populations. The effect of varied population sizes from generation to generation were also taken into account. Comparatively better results are obtained from Nei's standard distance with two alleles at each locus and from Nei's D_A distance with four alleles at each locus under various conditions. However, the difference in accuracy among distance measures was small and very high correlations were shown. The number of alleles at each locus had a greater effect on accuracy than the number of loci or type of distance measures. It indicates that highly polymorphic markers, such as DNA microsatellite, are powerful tool to improve the accuracy in classification of domestic animals. The six distance measures had a linear relationship with generation number at constant population size. None of the distance measures, however, showed a linear relationship when population size varied. The effect of fluctuating population size on the average distance was obvious over the first few generations but declined when the number of generations was increased.