Genetic characterization of the endangered Kiso horse using 31 microsatellite DNAs

In order to contribute to conservation of the endangered Kiso horse, we clarified their genetic information using 31 microsatellite DNAs, and genotyped 125 horses, 83% of the existing breed. First, we clarified the current status of the horses. The horses were confirmed to have experienced rapid loss of population causing a bottleneck, and their effective population size was much smaller than their census size. Moreover, the number of alleles (6.3), observed heterozygosity (0.674), and expected heterozygosity (0.662) were in the same range as other endangered horses all over the world. Therefore, although their inbreeding level was not so severe (F(is): -0.017), the Kiso horse is surely one of the endangered. Second, we obtained genetic information of individuals. This information allowed us to understand the genetic distance of individuals, and might help in development of a reproductive strategy concerning the genetic distance between the mating pairs. Moreover, there appeared to be 4 subpopulations of Kiso horse, and this result was in good agreement with their historical background. Third, we confirmed that the parentage test for identification using the 31 microsatellite DNAs was highly reliable (probability of exclusion: 0.999999993). This identification increases the reliability of stud certification, and is also helpful for effective management. Understanding the genetic diversity within the population and the relationships among individuals is important to ensuring effective management for maintenance of genetic variation, and this study may help in conservation of the endangered Kiso horse.     

Inoculum effect of arbuscular mycorrhizal fungi on soybeans grown in long-term bare-fallowed field with low phosphate availability

ABSTRACT

Inoculation with arbuscular mycorrhizal fungi (AMF) can increase the growth of host plants, especially under condition of low phosphate (P) availability. Although this effect is shown relatively easily in simplified systems such as pot experiments, it is often hard to show in the field because of complicating factors such as competition with indigenous AMF. We conducted an AMF inoculation experiment with three Japanese soybean cultivars (Enrei, Misuzudaizu, and Akishirome) in an allophanic (Umbric Silandic) Andosol field under the long-term selective application of major nutrients (NPK and -P) and bare fallow. In the inoculation plots, introduced AMF were well colonized in soybean roots at flowering stage. In the -P plots, inoculation tended to increase the shoot dry weight of all the three soybean cultivars; this effect remained until harvest. Although a significant difference is not recognized, there was a tendency of residual effect on Enrei in the following year. In the NPK plots, inoculation did not significantly increase the shoot dry weight. We thought that in the -P plots, the long-term selective application of N and K and the long-term maintenance of bare fallow created the soil conditions of low P availability and poor native AMF. Thus, introduced AMF can benefit soybean growth and yield in the soil with low competitor AMF density and low phosphate availability.     

Possible roles and functions of LPL1 gene encoding lysophospholipase during early infection by Magnaporthe grisea

The rice blast fungus Magnaporthe grisea differentiates appressoria, which are required to attack its rice plant host. Clone A26, tentatively named LPL1, was previously found to be homologous to the known lysophospholipase genes from our subtractive cDNA library. The LPL1 protein had a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence, and the protein expressed in Escherichia coli had lysophospholipase activity. To clarify the functions and possible roles of LPL1, the gene was disrupted by targeted gene replacement. The ΔLPL1 mutants formed fewer appressoria on the hydrophobic surface of GelBond film, and the appressoria had reduced turgor pressure and penetration into cells of the leaf sheath. The ΔLPL1 mutants and wild-type differentiated normal appressoria on other artificial substrata such as polycarbonate plate and on rice leaf sheath. Cytological analysis of the appressoria indicated that ΔLPL1 mutants had a delay in the disappearance of lipid droplets. These findings imply that LPL1, phospholipid metabolism, or both are involved in glycerol biosynthesis and accumulation to generate turgor pressure in the appressorium. LPL1 was, however, dispensable for full pathogenicity, suggesting that other complementary pathways or similar genes related to phospholipid metabolism probably function in M. grisea.     

Inverse correlation between mRNA levels in GFP-silenced transgenic Nicotiana benthamiana and resistance to Potato virus X engineered to contain GFP sequence

Nicotiana benthamiana was transformed with a green fluorescent protein (GFP) gene driven by cauliflower mosaic virus 35S promoter. A GFP-silenced line and a nonsilenced line were selected after ultraviolet irradiation. GFP short-interfering RNAs (siRNAs) were detected in the silenced line but not in the nonsilenced line. T₁ progeny of the silenced line varied in GFP suppression patterns and were grouped into three types (I, II, III) based on the GFP suppression pattern. With Northern blot analysis, different levels of GFP mRNA accumulated, from a very low level in type I and II to an intermediate level in type III, in contrast to a much higher level in the nonsilenced line. Plants were also inoculated with Potato virus X engineered to contain the GFP sequence to evaluate the levels of virus resistance. None to a few GFP spots were observed on inoculated leaves in types I and II, whereas numerous spots and systemic infection appeared in type III. These results showed that virus resistance was inversely correlated with the levels of mRNA, suggesting that the strength of RNA silencing determines the extent of virus resistance.     

Impact of bark stripping by sika deer, Cervus nippon, on subalpine coniferous forests in central Japan

We investigated the effect of bark stripping by sika deer, Cervus nippon, on forest regeneration in subalpine coniferous forests on Mt. Ohdaigahara and in the Ohmine Mountains of central Japan. Bark stripping by sika deer occurred in five major tree species: Abies homolepis; Abies Veitchii; Tsuga diversifolia; Picea jezoensis var. hondoensis; and Chamaecyparis obtusa. The percentage of damaged trees on Mt. Ohdaigahara was higher than in the Ohmine Mountains, probably because of the higher deer density. On Mt. Ohdaigahara, the DBH distributions of stems for P. jezoensis var. hondoensis, A. homolepis, T. diversifolia andC. obtusa were bell-shaped with fewer smaller and larger trees. On the other hand, in the Ohmine Mountains the distributions for P. jezoensis var. hondoensis and A. Veitchii showed a reverse-J shaped with more smaller trees. Larger overstory conifers on Mt. Ohdaigahara were killed by bark stripping when 100% barked, although in the Ohmine Mountains ca. 50% of the trees survived even when 100% barked. After the disappearance of the overstory conifers on Mt. Ohdaigahara, the dwarf bamboo, Sasa nipponica, expanded into the forest floor due to changes in light reaching the forest floor. Since S. nipponica is the main forage of deer in this area, this increase caused a corresponding increase in the deer population, which in turn, could cause a further decline in the coniferous forests.     

Genetic diversity ofKandelia candel andBruguiera gymnorrhiza in the Southwest Islands, Japan

The genetic diversity of the mangrove tree speciesKandelia candel andBruguiera gymnorrhiza in the Southwest Islands of Japan was investigated. These islands are located at the periphery of the distribution area of the two species, and their populations are relatively small. Allozyme analyses of 17 loci inK. candel and 13 loci inB. gynmorrhiza revealed very little genetic variation in both species. At the species level, the proportion of polymorphic loci was 4.2%, the average effective number of alleles per locus was 1.02, and the average expected heterozygosity was 0.012 inK. candel. The corresponding figures forB. gymnorrhiza were 9.0%, 1.06 and 0.035, respectively. However, the coefficients of genetic differentiation among the populations were high (G _ST=0.165 forK. candel and 0.253 forB. gymnorrhiza). The genetic variation and habitat area ofK. candel on Amami Island is greater than on the islands Okinawa and Iriomote. The genetic variation and habitat area ofB. gymnorrhiza is greater on Iriomote Island than on the islands Okinawa and Amami. The level of genetic variation in both of the species might be related to their population sizes.     

Outdoor exposure tests of structural laminated veneer lumber (II): evaluation of the strength properties after nine years

To investigate the durability of structural laminated veneer lumber (LVL), outdoor exposure tests have been conducted since 1990 at a field-testing site at the Forestry and Forest Products Research Institute. This article is the second interim report on the results after 9 years of exposure. Seven kinds of structural LVL with no preservative treatment were subjected to the tests. Almost all the exposed specimens were decayed by a kind of brown rot fungi (Pseudomerulius aureus (Fr.) Julich). The degree of decay varied with wood species; grand fir and western hemlock LVL in particular showed weak resistance against the decay. All the specimens were stored for more than 1 year in a testing room conditioned at 20°C and 65% relative humidity. We then measured the ultrasonic velocity of the specimens by the Pundit method, penetration depth by the Pilodyn method, and bending strength by a conventional bending test. Correlation between nondestructive measurement factors and the density was strong even on LVL with many adhesive layers. The nondestructive testing method was found to be applicable to LVL as well as solid lumber. After the nondestructive measurements, each LVL was cut into three types of specimen (top: T, middle: M, and bottom: B) for the bending tests. The bending strength varied with the type of specimens. Correlation between modulus of elasticity and modulus of rupture was strong even in the decayed specimens.     

Hydraulic conductivity, photosynthesis and leaf water balance in six evergreen woody species from fall to winter

To confirm that freeze-thaw embolism is a primary stress for evergreen woody species in winter, hydraulic conductivity, photosynthesis and leaf water potential were measured during fall and winter in trees growing in a cool temperate zone (Nikko) and in a warm temperate zone (Tokyo). We examined two evergreen conifers that naturally occur in the cool temperate zone (Abies firma Siebold & Zucc. and Abies homolepis Siebold & Zucc.), and four evergreen broad-leaved woody species that are restricted to the warm temperate zone (Camellia japonica L., Cinnamomum camphora (L.) J. Presl, Ilex crenata Thunb. and Quercus myrsinaefolia Blume). In Tokyo, where no freeze-thaw cycles of xylem sap occurred, hydraulic conductivity, photosynthesis and water balance remained constant during the experimental period. In Nikko, where there were 38 daily freeze-thaw cycles by February, neither of the tracheid-bearing evergreen conifers showed xylem embolism or leaf water deficits. Similarly, the broad-leaved evergreen trees with small-diameter vessels did not exhibit severe embolism or water deficits and maintained CO(2) assimilation even in January. In contrast, the two broad-leaved evergreen trees with large-diameter vessels showed significantly reduced hydraulic conductivity and shoot die-back in winter. We conclude that freeze-thaw embolism restricts evergreen woody species with large-diameter vessels to the warm temperate zone, whereas other stresses limit the distribution of broad-leaved trees, that have small-diameter vessels, but which are restricted to the warm temperate zone.