Ribotyping of EntomopathogenicVerticittium lecanii in Japan

To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.     

Cloning and characterization of cDNA encoding a prohibitin-like protein from Theileria orientalis

A cDNA clone encoding a prohibitin-like protein (Toprh) was isolated from a piroplasm cDNA library of Theileria orientalis and its nucleotide sequence was determined. An open reading frame, encoding a polypeptide of 278 amino acid residues, was found in Toprh cDNA sequence. An intron of 89 bp was identified when this cDNA clone was compared with the Toprh gene in the genome of T. orientalis. The deduced amino acid sequence of Toprh shares 93.8, 93.1 and 69.1% identities with the prohibitins of T. parva (from chromosome 1), T. annulata (from chromosome 1), and Plasmodium falciparum, (from chromosome 10), respectively. By Western blot analysis, Toprh was found to be expressed in the piroplasm stage of the parasites.     

Genetic diagnosis of claudin-16 deficiency and sex determination in bovine preimplantation embryos

Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.     

Detection of novel gammaherpesviruses in wild animals of South Africa

Herpesviral DNA was detected in 24/261 DNA samples that were extracted from whole blood of 13 wild animal species in South Africa. Herpesviral DNA was shown in 22 impalas (Aepyceros melampus) and 2 springboks (Antidorcas marsupialis). All of DNA sequences detected in impalas were identical, whereas two DNA sequences detected in springboks were different each other. These three sequences showed 44 to 72% homology to the corresponding gene of the Gammaherpesvirinae, indicating that these three viruses should be unrecognized novel gammaherpesviruses. The putative novel herpesviruses were tentatively designated as Aepyceros melampus (impala) herpesvirus 1 (ImHV-1), Antidorcas marsupialis (springbok) herpesviruses 1 (SpHV-1) and 2 (SpHV-2). ImHV-1 seems to be a relatively independent virus. SpHV-1 belongs to a group of ruminant lymphotropic herpesviruses and SpHV-2 is closer to a malignant catarrhal fever virus group. Potential threat of these herpesviruses to domestic animals should be considered.     

Expression patterns of the implantation-associated genes in the uterus during the estrous cycle in mice

The mRNA expression patterns of EGF, HB-EGF, Amphiregulin, EGF receptor, IGF-1, CSF-1, IL-1 alpha, IL-1 beta, IL-1 receptor type 1, IL-1 receptor antagonist, LIF, COX-1, COX-2, Mucin-1, calcitonin, and rat USAG-1 mouse homologue, all of which are involved in the process of conceptus implantation to the endometrium, were examined during the estrous cycle by means of real-time quantitative PCR. COX-2, HB-EGF, LIF, Mucin-1, CSF-1, IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist were temporally regulated during the estrous cycle and highly expressed during the estrous stage. In the case of COX-1, EGF, IGF-1, and EGF receptor, the highest mRNA expression was during the diestrous stage. In contrast, the rat USAG-1 mouse homologue mRNA expression did not change during the estrous cycle. These results indicate that rat USAG-1 mouse homologue expression at implantation might be specifically regulated by embryonic factors rather than the maternal environment.     

Coumestrol decreases intestinal alkaline phosphatase activity in post-delivery mice but does not affect vitamin D receptor and calcium channels in post-delivery and neonatal mice

In this study, we investigated the effects of administration of coumestrol during pregnancy on calcium (Ca) metabolism in post-delivery maternal and neonatal mice. From 6.5 to 16.5 days post coitus (dpc), pregnant females were administered daily doses of coumestrol (200 microg/kg body weight/day). One day after parturition, blood samples and the kidneys, liver, jejunum and duodenum were obtained from each of maternal mouse, and blood samples and the kidneys and liver were obtained from neonatal mice. Coumestrol did not have any significant effect on the Ca and inorganic phosphorus concentrations in the sera of the maternal and neonatal mice. No notable effects of coumestrol were observed in relation to Vitamin D receptor expression in the maternal and neonatal mice by immunohistochemical analysis. Coumestrol did not affect the Vitamin D receptor and epithelial calcium channel and 2 mRNA levels in any of the organs investigated. Enzyme histochemical analysis showed that coumestrol decreased intestinal alkaline phosphatase activity in the maternal jejunum and duodenum. In the duodenum, coumestrol decreased expression of intestinal alkaline phosphatase, c-fos and vascular endothelial growth factor at the mRNA level. However, we did not observe any significant effects of coumestrol on the expression of these genes. In conclusion, coumestrol decreased intestinal alkaline phosphatase activity in the small intestines of maternal mice at the level used in the present study, and the mechanisms underlying this effect are different for the jejunum and duodenum.     

Estimation of variance and genomic prediction using genotypes imputed from low‐density marker subsets for carcass traits in Japanese black cattle

The influence of genotype imputation using low‐density single nucleotide polymorphism (SNP) marker subsets on the genomic relationship matrix (G matrix), genetic variance explained, and genomic prediction (GP) was investigated for carcass weight and marbling score in Japanese Black fattened steers, using genotype data of approximately 40,000 SNPs. Genotypes were imputed using equally spaced SNP subsets of different densities. Two different linear models were used. The first (model 1) incorporated one G matrix, while the second (model 2) used two different G matrices constructed using the selected and remaining SNPs. When using model 1, the estimated additive genetic variance was always larger when using all SNPs obtained via genotype imputation than when using only equally spaced SNP subsets. The correlations between the genomic estimated breeding values obtained using genotype imputation with at least 3,000 SNPs and those using all available SNPs without imputation were higher than 0.99 for both traits. While additive genetic variance was likely to be partitioned with model 2, it did not enhance the accuracy of GP compared with model 1. These results indicate that genotype imputation using an equally spaced low‐density panel of an appropriate size can be used to produce a cost‐effective, valid GP.     

Identification of quantitative trait loci affecting corpora lutea and number of teats in a Meishan x Duroc F2 resource population

Understanding of the genetic control of female reproductive performance in pigs would offer the opportunity to utilize natural variation and improve selective breeding programs through marker-assisted selection. The Chinese Meishan is one of the most prolific pig breeds known, farrowing 3 to 5 more viable piglets per litter than Western breeds. This difference in prolificacy is attributed to the Meishan's superior prenatal survival. Our study utilized a 3-generation resource population, in which the founder grandparental animals were purebred Meishan and Duroc pigs, in a genome scan for QTL. Grandparent, F1, and F2 animals were genotyped for 180 microsatellite markers. Reproductive traits, including number of corpora lutea (number of animals = 234), number of fetuses per animal (n = 226), number of teats (n = 801), and total number born (n = 288), were recorded for F2 females. Genome-wide significance level thresholds of 1, 5, and 10% were calculated using a permutation approach. We identified 9 QTL for 3 traits at a 10% genome-wise significance level. Parametric interval mapping analysis indicated evidence of a 1% genome-wise significant QTL for corpora lutea on SSC 3. Nonparametric interval mapping for number of teats found 4 significant QTL on chromosomes SSC3 (P < 0.01), SSC7 (P < 0.01), SSC8 (P < 0.01), and SSC12 (P < 0.05). Partial imprinting of a QTL affecting teat number (P < 0.10) was detected on SSC8. Using the likelihood-ratio test for a categorical trait, 2 QTL for pin nipples were detected on SSC2 and SSC16 (P < 0.01). Fine mapping of the QTL regions will be required for their application to introgression programs and gene cloning.