Transmission of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Electrophoretic analysis of the transmission pattern of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of Lentinula edodes indicates that three of the four plasmids carried in parental monokaryons are effectively transferred and replicated in the protoplast fusants. The two monokaryons, 1158a and 1569a, carried different plasmids that could be distinguished by a single restriction digest. Electrophoresis of intact plasmids and restriction analyses indicate that all but one of the fusants carry three of the four possible plasmids, indicating that transmission of plasmids in protoplast fusions is principally biparental in L. edodes. Thus, heterocytoplasmic cells of L. edodes can be effectively constructed by protoplast cell fusion. In addition, plasmids of the same homology group cannot coexist in the heteroplasmic cells after protoplast cell fusion. Contribution No. 382 of the Tottori Mycological Institute     

Variation of fruiting body productivity in protoplast fusants between compatible monokaryons ofLentinula edodes

Mycelial protoplasts of two compatible monokaryons ofLentinula edodes were prepared, and electro-fusion was undertaken. Sixteen dikaryons isolated after the electrofusion were tested for their fruiting abilities in comparison to the two dikaryons obtained by reciprocal mycelial mating. There were significant differences in fruiting body yield among the fusants, and some of them produced more fruiting bodies than did normally mated dikaryons. Although these dikaryons were divided into five genetically different groups by isozyme analyses and a cluster analysis, we could not find clear differences among the groups in terms of fruiting body yield, mycelial growth rate, or degree of wood decay. It appeared that fruiting body yield correlated positively with the mycelial growth rate but negatively with the degree of wood decay.     

DNA fingerprinting of Populus trichocarpa clones using RAPD markers

Nine trees from a single, natural population of black cottonwood (Populus trichocarpa Torr. et Gray) in Alaska were screened for randomly amplified polymorphic DNA (RAPD) markers with ten different 10-base random oligonucleotide primers in order to evaluate the use of RAPD analysis for distinguishing black cottonwood clones. Nine primers amplified the genomic DNA targets; two primers were able to differentiate all clones. Eight clones were distinguished among the nine tree samples assayed. Two trees showed identical banding patterns with all primers used; therefore it is suggested that these trees are from the same clone. The RAPD fingerprinting method is simple and powerful-one primer can distinguish different clones, while the use of multiple primers reduces fingerprint similarity and resolves discrepancies.     

Study of sugi (Cryptomeria japonica D. Don) with male-sterility gene using CAPS markers

The frequency of open-pollinated male-sterile seedlings from a male-sterile mother tree in Cryptomeria japonica D. Don was determined in 765 open-pollinated seedlings. Pollen was not formed by 29 out of the 765 seedlings. To find pollen donor candidates for 29 male-sterile seedlings, a male-sterile tree was crossed with five individuals in the neighborhood of the mother tree. Analysis of microspores in male flowers of F₁ seedlings revealed that one of the five individuals had a heterozygous male-sterility gene. Paternity tests were conducted on the 29 male-sterile seedlings using eight kinds of CAPS markers in C. japonica. The pollen parents of 29 male-sterile seedlings were not only a tree in the neighborhood of the mother tree, but also other trees. Since it was found that the pollen parents of four of the male-sterile seedlings were not in the study plot, it was inferred that C. japonica trees that possess the male-sterility gene grow in other places and may be widely distributed.     

Methodological comparison of DNA extraction from Holcocerrus hippophaecolus （Lepidoptera： Cossidae） for AFLP analysis

Amplified fragment length polymorphism （AFLP） is a powerful DNA fingerprinting technique for studying genetic relationships and genetic diversity in insects. However, the crucial prerequisite for AFLP analysis is to extract DNA of high quality. In this study, we evaluate four different protocols （SDS method, improved SDS method, CTAB method and a complex method with SDS and CTAB） for isolating DNA from the seabuckthorn carpenter moth （Holcocerrus hippophaecolus （Lepidoptera： Cossidae））. The results indicate that the CTAB method does not produce DNA suitable for AFLP analysis. The SDS method and the complex method with SDS and CTAB are comparatively time-consuming and resulted in low yields of DNA and were therefore not used for AFLP assay. The improved SDS method is recommended for preparing DNA templates from H. hippophaecolus for AFLP analysis.     

Pollen dispersal in a hinoki (Chamaecyparis obtusa) seed orchard detected using a chloroplast DNA marker

Pollen dispersal was estimated in two test plots in a hinoki (Chamaecyparis obtusa) seed orchard using a chloroplast DNA marker, the spacer region between thetrnD andtrnY genes, and SSCP (single strand conformation polymorphism). In Plot 1, 2,020 seeds from 40 trees within 30 m of the marker tree were analyzed using the PCR-SSCP method. In Plot 2, 1,850 seeds from 37 trees were analyzed in the same manner. The results revealed that the maximum pollen dispersal distance in the two plots exceeded 25 m. Pollen dispersal appeared to be inversely proportional to the distance from the marker tree. The effective pollen dispersal was suggested to be less than about 20 m in a mature hinoki seed orchard. Adjacent trees had an excessive influence when the pollen density was increased by artificial flower stimulation. Therefore, it was suggested that seed production better resembles ideal random mating when carried out as naturally as possible. In conclusion, the SSCP chloroplast DNA marker was a useful tool for amassing basic information on pollen management in seed orchards of coniferous species.     

Possibility of grouping of Cyclobalanopsis species (Fagaceae) grown in Japan based on an analysis of several regions of chloroplast DNA

Four regions of chloroplast DNA were sequenced as a prospective genetic marker to identify Japanese representatives of Cyclobalanopsis: Quercus acuta, Q. sessilifolia, Q. salicina, Q. myrsinaefolia, Q. glauca, and Q. gilva. We found that Q. gilva was distinguished from other species based on both the trnL-trnF and trnT-trnL intergenic spacers. The evidence shows good coincidence with the fact that Q. gilva has several peculiar morphological features distinguishable from those of other species. There was no difference in trnL intron and matK. Both trnT-trnL and trnL-trnF intergenic spacers are capable of being used as genetic markers to identify Q. gilva among Cyclobalanopsis species.Part of this paper was presented at the 48th annual meeting of the Japan Wood Research Society, Shizuoka, April 3–5, 1998     

Diversity and genetic structure of teak (<Emphasis Type="Italic">Tectona grandis</Emphasis> L.f) in its natural range using DNA microsatellite markers

Teak (Tectona grandis L.f.) is considered to be an extraordinarily durable building timber with a worldwide reputation. Its widespread use has entailed the over-exploitation of natural forests and a large reduction in natural diversity. Fifteen microsatellite markers were used to study the genetic variability and structure of 166 teak trees distributed over the whole natural area of teak. Analysis showed that in the teak natural area there were four main centers of genetic variability. Two clusters were in India and could be considered as main centers of genetic diversity in teak. The third cluster mainly consisting of populations in Thailand and Laos was genetically very distinct from the Indian populations but presented only half as much allelic variability. A fourth cluster from Central Laos showed even less genetic variability. The use of SSR markers for conservation of teak forest diversity is discussed.     

Genetic diversity of sea buckthorn (Hippophae rhamnoides) populations in northeastern and northwestern China as revealed by ISSR markers

As a N₂-fixing tree species, sea buckthorn (Hippophae rhamnoides) is well adapted to arid regions and is utilized for multiple purposes in China. Current knowledge of genetic variability of H. rhamnoides is limited in terms of rangewide distributions. Eleven natural populations of sea buckthorn in northeastern and northwestern China were analyzed to detect genetic variation among and within populations, by use of ISSR (inter-simple sequence repeats) markers. Using eight primers, 207 polymorphic loci were observed, ranging in size from 250 to 2500 bp. The coefficient of gene differentiation (Gst = 0.0679) showed that the total molecular variance of 11 populations was mainly existed within populations. The genetic variation within and among the 11 populations was 93.21 and 6.79%, respectively. No significant correlation between genetic and geographic distances of the populations was found using ISSR markers. Our study provides a population-level genetic profile for further investigation and conservation of genetic diversity of sea buckthorn.     

Efficiency of extracting microarthropods from the canopy litter in a Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don) plantation: a comparison between the washing and Tullgren methods

We compared the efficiency of washing versus the Tullgren method for extracting microarthropods from dead leaves and branches in the canopy of Cryptomeria japonica trees. Oribatida and Collembola were consistently the numerically dominant taxa of microarthropods, but the relative abundance differed between the two extraction methods. Oribatida accounted for more than 70% of all the microarthropods collected by the washing method but less than 30% by the Tullgren method. Collembola accounted for less than 10% collected by the washing method but about 60% by the Tullgren method. The density of Oribatida was higher when collected by the washing method than by the Tullgren method, and vice versa for the density of Collembola. Our results suggest that the washing method is appropriate for collecting Oribatida and other microarthropods, whereas the Tullgren method is better for collecting Collembola from the canopy litter of C. japonica trees.     

Seedling growth and establishment in natural stands of yellow-cedar (Chamaecyparis nootkatensis) seedlings derived from the use of modified seed dormancy-breaking treatments

Following dispersal from the parent tree, seeds of yellow-cedar (Chamaecyparis nootkatensis[D. Don] Spach) exhibit low germination, primarily as a result of coat-imposed dormancy. Dormancy of the mature (intact) seed is effectively terminated by traditional warm/cold treatments. A chemical treatment using the anaesthetic 1-propanol combined with a three day warm water soak (30 °C), a two day GA₃ treatment and 60 d of moist chilling not only promotes high germinability of yellow-cedar seeds, but also elicits vigorous post-germinative growth following seedling emergence under nursery greenhouse conditions. Here we compare the effectiveness of the more traditional warm/cold treatments with the chemical treatment in terms of their capacity to elicit vigorous growth and establishment in natural stands following transplant of seedlings from a nursery greenhouse environment. Two seed lots (42313 and 43697) and open-pollinated seed from parent trees 13-6 and 19-8 showed equivalent seedling growth in natural stands following the chemical treatment and two traditional warm/cold treatments typically used for dormancy breakage by the forest industry and by the Ministry of Forests in British Columbia. The chemical protocol offers the advantage of reducing the time required to break seed dormancy. We have now demonstrated that it yields seedlings that exhibit vigorous growth and are capable of withstanding the vagaries of the environment.     

The penetration rate of laser pulses transmitted from a small-footprint airborne LiDAR: a case study in closed canopy, middle-aged pure sugi (Cryptomeria japonica D. Don) and hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stands in Japan

Penetration rates of laser pulses transmitted from a small-footprint airborne light detection and ranging (LiDAR) system in closed-canopy, middle-aged (40–50 years old) hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) and sugi (Cryptomeria japonica D. Don) plantations that had similar levels of canopy openness were studied. The number of transmitted pulses was 107 427 points/ha for the hinoki cypress stand and 122 883 points/ha for the sugi stand. The penetration rates of the first (P_f) and second pulses (P_s) that reached the ground were 0.1% and 1.0% in the hinoki cypress stand, and 2.6% and 5.5% in the sugi stand, respectively. Penetration rates for the total number of pulses (P_f+s) that reached the ground within each stand were 1.1% and 8.1%, respectively, and values for P_f+s, P_f, and P_s for sugi and hinoki cypress stands were significantly different (P < 0.001). The results of this study indicated that the generation of accurate digital terrain models in dense hinoki cypress stands with complex topographies using only the data from laser pulses with poor laser penetration rates is likely to be difficult.     

Characterization of lignin-carbohydrate linkages in the residual lignins isolated from chemical pulps of spruce (<Emphasis Type="Italic">Picea abies</Emphasis>) and beech wood (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>)

To investigate the linkage types between carbohydrates and lignin, residual lignins were isolated from three different unbleached pulps [kraft, alkaline sulfite anthraquinone methanol (ASAM), and soda with anthraquinone (AQ) and methanol] of spruce and beech wood and then characterized by oxidation with 2,3-Dichloro-5,6-dicyanobenzoquinone and followed by Prehm’s methylation. In residual lignins, sugar moieties were bound to lignins via benzyl ether bonds. In particular, galactose and mannose are predominantly linked to lignin fragments in residual lignins of spruce wood, while xylose and galactose are favored in the formation of LC bonds in the residual lignins of beech wood. In the case of hexoses, primary hydroxyl groups (C6 position) preferentially take part in benzyl ether linkages. Hydroxyl groups in the C2 and C3 positions of xylose participate in LC bonds and a small portion of arabinose was notably connected to lignin via the C5 position. Approximately seven or eight sugars were connected in soda/AQ/methanol residual lignin per 100 C9 lignin units, while the frequencies of LC bonds in kraft and ASAM residual lignins were distinctively less at one to three sugars per 100 C9 lignin units. The publication of this article was made possible by an Emachu Research Fund. The authors are grateful for the fund.     

Gas chromatographic and mass spectrometric (GC-MS) analysis of lignin-derived products from <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> treated in supercritical water

Sugi (Cryptomeria japonica D. Don) wood was treated with supercritical water (374°C, 22.1MPa), and fractionated into the water-soluble portion, the methanol-soluble portion, and the methanol-insoluble residue. The methanol-soluble portion mainly consisted of the lignin-derived products. To characterize the compounds in the methanol-soluble portion, gel permeation chromatographic (GPC) and gas chromatographic-mass spectrometric (GC-MS) analyses were performed. The GPC analysis indicated that the methanol-soluble portion contained lignin-derived monomeric and dimeric products. GC-MS analysis detected 31 products which were expected to be monomeric compounds, and 18 of these were identified to be guaiacol, methylguaiacol, ethylguaiacol, vinylguaiacol, eugenol, propylguaiacol, vanillin, cis-isoeugenol, homovanillin, trans-isoeugenol, acetoguaiacone, propioguaiacone, guaiacylacetone, 2-methoxy-4-(1-hydroxypropyl)phenol, homovanillic acid, 2-methoxy-4-(prop-1-en-3-one)phenol, coniferyl aldehyde, and ferulic acid. In addition, 22 dimeric products were detected, and 4 of these were believed to be compounds with biphenyl type (5-5), diphenylethane type (-1), stilbene type (-1), and phenylcoumaran type (-5) structures. These results clearly indicated that the methanol-soluble portion included various monomeric and dimeric compounds produced as a result of the cleavage of ether linkages and propyl chains of lignin.     

Predicting individual breeding values and making forward selections from open-pollinated progeny test trials for seed orchard establishment of interior Lodgepole Pine (<Emphasis Type="Italic">Pinus contorta</Emphasis> ssp. <Emphasis Type="Italic">latifolia</Emphasis>) in British Columbia

Ten-year height data from three open-pollinated lodgepole pine progeny test trials were analysed in order to select individuals for seed orchard establishment in the low and high elevation seed-planning units (TOLO and TOHI) of the new Thompson Okanagan seed planning-zone in interior British Columbia, Canada. The total test population had about 35,000 individuals from 362 families of 9 provenances and was divided into two portions, each serving as the base population for its appropriate seed-planning unit. For each population, various genetic parameters were estimated and individual breeding values were predicted. Combined-site estimates of individual, family, and within-family heritability ranged from 0.23 to 0.38, 0.77 to 0.84, and 0.17 to 0.29, respectively. Estimated coefficients of type-b genetic correlation were greater than 0.81, indicating little G × E interaction in this region. Provenance effects were large. Larch Hills and Inonoaklin River were the two superior provenances with an expected gain of 13% and 10% in stem volume at rotation age of 60 years in TOLO. Individual breeding values was not uniformly distributed among provenances and the top 100 individuals originated from 20 families in TOLO and 32 families in TOHI. Co-ancestry among these individuals should not be a serious concern in seed orchard establishment as the predicted genetic loss due to excessive inbreeding is only 1.06% in TOLO and 0.6% in TOHI. The status numbers of the two prospective seed orchards are 32 and 44, respectively, and both are well above the required minimum of 10 in British Columbia. Using unrelated clones for seed orchard establishment may result in reduced optimum genetic gain and thus is not a favourable option.