Evaluation of genetic diversity of bread wheat landraces from Pakistan by AFLP and implications for a future collection strategy

We used amplified-fragment-length polymorphism (AFLP) markers to evaluate genetic variation in a set of bread wheat (Triticum aestivum L.) landraces and improved materials. Landraces collected from different geographic and agro-ecological zones in Pakistan in 1987, 1989 and 1991 were separated into two groups based on their geographic origins: northern (Himalaya) and south-western (Balochistan) Pakistan. Six AFLP primer combinations detected 453 AFLP markers in the 43 landrace accessions and four high-yield varieties (HYVs). Of these, 225 (49.67%) were rare (shared with < 5% of all accessions). Among these rare alleles, 23 (10.22%) were common in the Himalaya (shared with > 10% of accessions collected there) but were not found in Balochistan. We conclude that there is a higher probability of collecting rare alleles at overall, but which are in contrast locally common ones in the Himalayan region. Gene diversity was 0.17 in the Himalayan group and 0.15 in the Balochistan group. Considerable genetic variability was found in both groups. Accessions from different agro-ecological zones were indistinguishable by cluster analysis, indicating intensive seed trading within the country. Cluster analysis indicated that the landraces and the HYVs are genetically distinct; suggesting that genetic erosion of wheat landraces has been unlikely taken in place. This study provides an example of how analysis of existing materials and data, can serve as a basis for future collection planning and conservation policies.     

Basic characterization of avian β‐defensin genes in the Japanese quail,Coturnix japonica

In this study, we identified a cluster of 14 avian β‐defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and ‐13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1‐2) motif considered essential for the β‐defensin family. Each CjAvBDs also formed a sub‐group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long‐arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, ‐4, ‐5, ‐9, and ‐10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and ‐7 loci, whereas the CjAvBD101α, ‐101β, and ‐101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix ‐ Gallus.     

Callose in root apices of European chestnut seedlings: a physiological indicator of aluminum stress

We determined if callose formation in 1-cm-long root apices of European chestnut (Castanea sativa Mill.) seedlings is affected by naturally occurring high concentrations of aluminum (Al) under laboratory conditions and by low base saturation (BS) of soils under forest field conditions. Under controlled conditions in the laboratory, seedlings were treated with simulated soil solutions in the presence or absence (control) of Al. One day after exposing seedlings to a simulated soil solution containing 168 microM Al, callose formation in the root apices had increased significantly. After 7 days, callose concentration in root apices was significantly correlated with the concentration of free Al3+ ions recovered in the simulated soil solution and with the concentration of Al in fine roots. At four field sites in southern Switzerland, seedlings were grown for five months in either A-horizon soil with a BS above 17% or in a B-horizon soil with a BS below 5%. Callose concentrations were significantly higher in root apices in the B horizon than in the A horizon. Callose concentrations in root apices were negatively correlated with Ca/Al molar ratio in fine roots. We conclude that callose in the root apices of European chestnut could be a useful physiological parameter for assessing Al toxicity under both laboratory and field conditions.     

Increase in plasma total antioxidant capacity of grazing Japanese Black heifers and cows in forestland in Japan

Blood total antioxidant capacity (TAC) has become a key bio‐marker for animal health. Forest‐grazing cattle are known to forage various native plants that have high TAC. This study evaluated differences of plasma TAC between forest‐grazing (FG) and pasture‐grazing cattle (PG). Experiment 1 monitored the plasma TAC levels of 32 Japanese Black cattle. The level in PG did not change throughout the grazing period. However, that in FG, which increased from summer, was significantly higher than that in PG through fall (P < 0.05). In experiment 2, we used nine Japanese Black heifers and investigated their blood antioxidant parameters and the TAC in plants that the cattle consumed in late June and September. The plasma TAC levels in FG were significantly higher than those in PG in both periods (P < 0.05). Plasma levels of lipid peroxidation in FG tended to be lower than that in PG (P = 0.098). Furthermore, the TAC levels in various species of shrubs and trees consumed by FG were higher than those in pasture grasses. Results of this study show that plasma TAC of grazing Japanese Black cattle in forestland increase from summer through fall.     

Relationship between pulmonary arterial pressure and pulmonary thromboembolism associated with dead worms in canine heartworm disease.

To examine effects of thromboemboli due to dead worms on pulmonary arterial pressure (PAP), 20 to 50 dead heartworms were inserted into the pulmonary arteries of 4 heartworm uninfected dogs (uninfected group) and 11 dogs infected with heartworms (infected group). In the uninfected group, the mean PAP rose 1 week after worm insertion (10.9 to 166. mmHg), but it recovered by the 4th week. Clinical signs, hemodynamics and blood gas findings also deteriorated at the 1st week, but recovered at the 4th week. Angiographic and pathological findings indicated that blood flow recovered through the spaces between thromboemboli and vessel walls at the 4th week. The infected dogs were divided into three groups. In the infected-I group (5 dogs), the intimal lesions of the pulmonary arteries were slight, and clinical and laboratory findings showed changes similar to those of the uninfected group. In the infected-II group (4 dogs), the pulmonary arterial lesions were severe and the mean PAP was higher (25.7 mmHg) than in the uninfected group before worm insertion. An increase in PAP (34.1 mmHg) and worsening of clinical and laboratory findings were noticed till the 4th week. Thromboemboli adhered extensively to the vessel walls. Two dogs in the infected-III group died of severe dyspnea on the 9th and 10th day, and the mean PAP rose remarkably at the 1st week (from 19.4 to 28.2 mmHg). Severe pulmonary parenchymal lesions with edema or perforation were observed. From the above results, it was clarified that effects of dead worms on PAP and clinical signs depended on the severity of pulmonary arterial lesions before worm insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

Relationship among ovarian follicular status,developmental competence of oocytes,and anti‐Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti‐Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross‐sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.