DNA marker-assisted evaluation of potato genotypes for potential resistance to potato cyst nematode pathotypes not yet invading into Japan

One of major objectives of crop breeding is conferring resistance to diseases and pests. However, large-scale phenotypic evaluation for many diseases and pests is difficult because strict controls are required to prevent their spread. Detection of disease resistance genes by using DNA markers may be an alternative approach to select potentially resistant accessions. Potato (Solanum tuberosum L.) breeders in Japan extensively use resistance gene H1, which confers nearly absolute resistance to potato cyst nematode (Globodera rostochiensis) pathotype Ro1, the only pathotype found in Japan. However, considering the possibility of accidental introduction of the other pathotypes, breeding of resistant varieties is an important strategy to prevent infestation by non-invading pathotypes in Japan. In this study, to evaluate the prevalence of resistance genes in Japanese genetic resources, we developed a multiplex PCR method that simultaneously detects 3 resistance genes, H1, Gpa2 and Gro1-4. We revealed that many Japanese varieties possess not only H1 but Gpa2, which are potentially resistant to other pathotypes of potato cyst nematode. On the other hand, no genotype was found to have the Gro1-4, indicating importance of introduction of varieties having Gro1-4. Our results demonstrate the applicability of DNA-marker assisted evaluation of resistant potato genotypes without phenotypic evaluation.     

Incorporation of fallow weed increases phosphorus availability in a farmer’s organic rice fields on allophanic Andosol in eastern Japan

ABSTRACT

We investigated the amount of soil phosphorus (P) in a farmer’s paddy fields under organic farming (OF) for various periods from 0 to 22 years as well as other farmers’ fields under conventional farming. All the fields are located in allophanic Andosol with long history of P fertilizer application, and some of them have been converted to OF across years. After conversion to OF, P was supplied only with winter fallow weeds mainly Foxtail (Alopecurus aequalis), rice residues (rice bran and straw), and guano. We determined total-P (Tot-P) and plant-available P (Av-P), which consists of Truog-P (Tru-P) and Bray-2-P under reducing condition with ascorbic acid (Asc-P), in soils of each field. For both Av-Ps, the ratio to Tot-P increased across years under OF following quadratic functions with both linear and quadratic terms being statistically significant. The ratios showed little changes for the initial 15 (Tru-P) or 10 (Asc-P) years and increased rapidly thereafter. These temporal changes in Av-P were consistent with the rapid increase of the amount of P accumulated in the winter fallow weeds and incorporated in the fields after beginning of OF. These results led us to the hypothesis that the incorporation of winter weeds has contributed to the increase of Av-P in the organic fields across years. We tested this hypothesis by investigating temporal changes of Av-P after suspending the weed incorporation for 2 consecutive years in plots of the organic fields. Both Av-Ps were significantly greater in plots with continued weed incorporation (CWI) than those in plots with its suspension. We further found that the increase of Asc-P in plots with CWI was 4.9-fold the input of total P in the incorporated weeds. This suggests that the incorporation of winter fallow weeds enhanced soil-P availability beyond the supply of P accumulated in the weeds.     

Effects of the presence of grazing‐experienced heifers on the development of foraging behavior at the feeding station scale for first‐grazing season calves

In this study, effects of grazing‐experienced heifer presence on foraging behavior development at the feeding station (FS) scale for first‐grazing season calves were determined. A group of four calves grazing alone (C), and another comprising four calves (Wc) and three grazing‐experienced heifers (Wh), were alternately stocked every day for 2 h on the same pasture for 26 days. The foraging time budget for Wc was significantly longer than that for C (P < 0.05) on day 7, and was similar to that for Wh on all days. Bite rate per FS was significantly higher for Wc (15.5 bite/min) than for C (13.2 bite/min) from day 1 to 14 (33.4 vs. 29.0 bite/min, respectively; P < 0.01) and significantly lower than that for Wh on all days. The number of steps taken between adjacent FSs by Wc (4.7) was significantly lower than that for C (7.2) on days 1 and 14 (2.1 vs. 2.9 steps, respectively; P < 0.01) and was similar to Wh on all days. This suggests that grazing‐experienced heifers have positive effects on the foraging behavior development of new‐season grazing calves through 14 days after the start of stocking.     

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein‐4 increases oocyte diameter,but impairs subsequent developmental competence

Bone morphogenetic protein‐4 (BMP‐4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo‐grown oocytes. We herein investigated whether an extended IVG culture with BMP‐4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte‐granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP‐4 (10 ng/mL), while a 12 day culture with BMP‐4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP‐4 (83.2%), but was significantly lower without BMP‐4 (58.9%) than the control (83.0%). Prolong‐cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP‐4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP‐4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP‐4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

Effect of increased concentrate allotment before evening grazing on herbage intake,nitrogen utilization and rumen fermentation in dairy cows grazed on perennial ryegrass pasture

Two experiments were conducted to elucidate the effect of increased concentrate allotment before evening grazing on herbage intake, nitrogen utilization and rumen fermentation in dairy cows. In experiment 1, nine lactating cows were grazed in the morning and evening sessions (2.5 h each). The cows were allocated to treatments of three concentrate allotment levels before the evening grazing session by altering proportions to daily total offered; 25%, 50% and 75%. Daily herbage dry matter intake quadratically decreased with increasing the concentrate allotment levels (P < 0.05). Nitrogen utilization was similar among treatments. To investigate diurnal changes in rumen fermentation, a second experiment was conducted where six ruminally cannulated non‐lactating dairy cows grazed in the morning and evening sessions (3 h each) were subjected to the same treatments as experiment 1. Total volatile fatty acid concentration in the rumen linearly increased with increasing the concentrate allotment levels throughout the pre‐evening grazing session to the post‐morning grazing session (P < 0.01). The results indicate that dairy cows reduce daily herbage intake but do not alter nitrogen utilization with increasing concentrate allotment before evening grazing. © 2016 Japanese Society of Animal Science     

Seasonal foraging patterns of forest‐grazing Japanese Black heifers with increased plasma total antioxidant capacity

Forest‐grazing enables the intake of high total antioxidant capacity (TAC) plants that might be beneficial for the TAC status of cattle. This study evaluated the relation between the seasonal foraging patterns of forest‐grazing Japanese Black (JB) heifers or the TAC levels in shrubs and trees and the changes of plasma TAC. We examined 12 JB heifers, four each of which were allocated to forest‐grazing (F), pasture‐grazing, and pen‐housed groups. The plasma TAC level in F heifers on July 26, August 13, 30 and September 17 were significantly higher than those on April 27 and June 4 (P < 0.05). In F group, the mean rates of foraging frequency (FF) of shrubs and trees during July 5–8 and September 13–16 were much higher than that during May 31–June 3 (P < 0.05). The rate of FF of grass significantly decreased later in the season (P < 0.05). The mean TAC levels in these shrubs and trees were higher than those in grasses, concentrates, and timothy hay. Results suggest that an important factor in the increase of plasma TAC in forest‐grazing cattle might be the increased foraging of TAC‐rich shrubs and trees during summer–fall.