Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.     

Identification of <Emphasis Type="Italic">CpTI</Emphasis> gene integration for 2-year-old transgenic poplars at DNA level

The putative transgenic hybrid triploid poplars [(P. tomentosa × P. bolleana) × P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors’ previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar.     

Introduction of mitochondrial DNA from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> into <Emphasis Type="Italic">Pleurotus pulmonarius</Emphasis> by interspecific protoplast fusion

Interspecific protoplast fusion between two monokaryotic strains (a methionine auxotrophic and chloramphenicol-resistant Pleurotus ostreatus strain and a wild-type strain of Pleurotus pulmonarius) was carried out to introduce mitochondrial DNA (mtDNA) from P. ostreatus into P. pulmonarius. Because mycelial colonies regenerated on minimum medium containing chloramphenicol only after the treatment of protoplast electrofusion, the regenerants were regarded as protoplast fusants. The fusants isolated from regenerated colonies were uninucleate, and their resistance of chloramphenicol seemed to be a stable trait. The fusants mated with P. pulmonarius but not with P. ostreatus, and showed almost identical random amplified polymorphic DNA (RAPD) patterns to that of the parental P. pulmonarius monokaryon. The sizes of mitochondrial genomes were estimated to be 81.5 kb for P. ostreatus monokaryon, 54.9 kb for P. pulmonarius monokaryon, and 84.5 or 86.0 kb for the fusants. BamHI and EcoRI digest patterns of the fusant mtDNAs were almost the same as the parental P. ostreatus monokaryon. From the above results, the fusants seemed to carry nuclei derived from the monokaryon of P. pulmonarius and mtDNA including the chloramphenicol-resistance gene from the P. ostreatus monokaryon, suggesting that the P. ostreatus mtDNA had been introduced into P. pulmonarius cells by interspecific protoplast fusion. Contribution No. 383 of the Tottori Mycological Institute     

Cloning and analysis of telomere-associated sequences of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Total genomic DNA was extracted from leaves of Ginkgo biloba L. by the method of hot CTAB. After determining quantification of DNA sample by microclorimetric spectrophotography, Arabidopsis-type telomere primer and Sau3A I cassette primer were used to isolate telomere-associated sequences from G. biloba L. by the method of cassette-ligation-mediated polymerase chain reaction (PCR). Using this method, two telomere-associated sequences, TAS1 and TAS2, were isolated. The authors preformed Southern hybridization ofEcoR I-treated G. biloba genomic DNA with each clone. The hybridization pattern showed that the clones obtained were derived from G. biloba genomic DNA. There are the Arabidopsis-type TTTAGG tandem repeats in telomeres of G.biloba.     

45S rDNA在7种竹子植物染色体上的定位

编码18-5.8-25S核糖体RNA的45S rDNA基因,是1个简单的多基因家族基因,一般以串联方式相连,对应于核仁组织区(NOR).首次利用荧光原位杂交技术(FISH),研究45S rDNA在散生竹类的毛竹和斑竹,混生竹类的茶秆竹、日本矮竹、菲白竹和铺地竹,以及丛生竹类的白绿竹染色体上的分布.结果表明:本研究所涉及的散生竹和混生竹的几个竹种,只观察到1对随体染色体的次缢痕区域有45S rDNA位点,而丛生竹类的白绿竹除随体染色体具有45S rDNA位点外,某些非随体染色体上也有不同拷贝数的45S rDNA位点存在.     

Analysis of genetic diversity for wild and captive green peafowl populations by random amplified polymorphic DNA technique

The genetic diversity of the populations for 14 wild green peafowls (Pavo muticus) and 18 captive green peafowls was investigated by using the technology of random amplified polymorphic DNA (RAPD). Totally 161 and 166 amplified bands were obtained by using 23 arbitrary primers to amplify the genomic DNA of wild and captive green peafowls respectively. The results showed that the average relative genetic distance of the wild and captive green peafowls populations was 0.0555 and 0.1355, respectively, and difference of the average relative genetic distances between the two populations was 0.1635. The Shannon diversity index for the wild and captive green peafowl populations was 0.4348 and 1.0163, respectively, which means that there exists significant difference in genetic diversity between the two populations, and the genetic diversity of wild green peafowl was low. The two populations originated from two different families according to analysis by the UPGMA method. This research can provide the theoretical basis for supervising genealogies management of peafowl populations.     

Root recovery of five tropical tree and shrub species by sieves of different mesh sizes

Accurate quantitative assessment of roots is key to understanding the belowground plant productivity as well as providing an insight of the plant-soil interactions. In this study, root recoveries by sieves of different mesh sizes (2.0, 1.0, 0.5 and 0.25 mm) were measured for five tropical tree and shrub species grown in monoculture stands: crotalaria (Crotalaria grahamiana Wight and Arn.), pigeonpea [Cajanus cajan (L.) Millsp.], sesbania [Sesbania sesban (L.) Merr.], tephrosia (Tephrosia vogelii Hook F.), siratro [Macroptilium atropurpureum (DC.) Urb.] and tithonia [Tithonia diversifolia (Hemsl.) Gray]. Root samples were take from 0-15 cm soil depth. Recovery of coarser roots (>1.0 mm) ranged from 70 to 93% and 90 to 98% of the cumulative root length and biomass respectively. The proportion of root length of the finer roots (<1.0 mm) was greater for pigeonpea (30%), tithonia (22%) and siratro (18%) compared with other species, but contributed negligibly to the cumulative total root biomass for all species. The use of 0.5 mm sieve improved the recovery of root length for most species but had little effect on root biomass. The 0.25 mm sieve was most effective in capturing finer roots (<0.5 mm) of pigeonpea which represented 16% of cumulative root length and 4% of root biomass recorded for this species. Recovery of roots of different diameter classes depended on species, suggesting that for an improved estimation of root parameters especially when sieves of large mesh sizes (>0.25 mm) are used, a correction factor could be useful for root length measurements but not root biomass measurements for a particular species in each site and for a specific study. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening method for wood extractives: direct cellulose thin-layer chromatography plate

A method for screening wood extractives was developed using cellulose thin-layer chromatography plate (Cell-TLC) separation and was directly applied to bioassays. Wood meal (60 mesh) from nangka (Artocarpus heterophyllus Lamk) heartwood was extracted with hot methanol. The crude extract was separated using a Cell-TLC plate (50 × 50mm). Two broad bands with R_f values of 0.46 and 0.96 were found, and the bands showed completely different effects against the pest termite Coptotermes formosanus Shiraki and the decay fungus Fomitopsis palustris. The band with R_f 0.46 was preferentially consumed by workers of C. formosanus, and it did not show any growth inhibition against F. palustris when the Cell-TLC plate was directly exposed to the organism. In contrast, the band with R_f 0.96 appeared to repel strongly the feeding by C. formosanus and inhibited the growth of F. palustris. It was concluded that the Cell-TLC system was applicable for screening wood extracts consisting of many compounds.     

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.     

Molecular evidence for natural interspecific hybridization in Prosopis

Interspecific hybridization can occur under natural conditions among species of the Prosopis genus, potentially giving rise to hybrid individuals with increased adaptation values. A study was conducted to assess the occurrence of interspecific hybridization among Prosopis in the Argentinian Chaco region and the species involved. The survey included seven hybrid individuals and their potential parents. Principal component analysis (PCA) was carried out based on 16 morphological characters. The molecular marker study was carried out by the comparison of RAPD patterns of amplification using six primers. A total of 91 RAPD fragments were analysed, yielding 33 species-specific markers, 24 bands that were common to two or three parental lines, and 22 bands of unknown origin. The dendrograms arising from Nei’s genetic distances derived from the molecular data showed a clustering pattern in agreement with the grouping obtained by the PCA of the morphological traits. The results demonstrate the occurrence of interspecific hybridization involving Prosopis alba, P. nigra and P. hassleri in the Argentinian Chaco.     

Aboveground biomass of three conifers in the Qianyanzhou plantation, Jiangxi Province, China

Regressive models of the aboveground biomass for three conifers in subtropical China—slash pine (Pinus elliottii), Masson pine (P. massoniana) and Chinese fir (Cunninghamia lanceolata)—were established. Regression analysis of leaf biomass and total biomass of each branch against branch diameter (d), branch length (L), d ³ and d ² L was conducted with functions of linear, power and exponent. A power law equation with a single parameter (d) was proved to be better than the rest for Masson pine and Chinese fir, and a linear equation with parameter (d ³) is better for slash pine. The canopy biomass was derived by adopting the regression equations to all branches of each individual tree. These kinds of equations were also used to fit the relationship between total tree biomass, branch biomass, foliage biomass and tree diameter at breast height (D), tree height (H), D ³ and D ² H, respectively. D ² H was found to be the best parameter for estimating total biomass. However, for foliage biomass and branch biomass, both parameters and equation forms showed some differences among species. Correlations were highly significant (P<0.001) for foliage biomass, branch biomass and total biomass, among which the equation of the total biomass was the highest. With these equations, the aboveground biomass of Masson pine forest, slash pine forest and Chinese fir forest were estimated, in addition to the allocation of aboveground biomass. The above-ground biomass of Masson pine forest, slash pine forest and Chinese fir forest was 83.6, 72.1 and 59 t/hm² respectively, and the stem biomass was more than the foliage biomass and the branch biomass. The underground biomass of these three forests which estimated with others’ research were 10.44, 9.42 and 11.48 t/hm², and the amount of carbon-fixed were 47.94, 45.14 and 37.52 t/hm², respectively. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(8): 1382–1388 [译自: 应用生态学报]     

湿地松样本量大小对性状遗传力估算的影响

[目的]在保证遗传力估算精确度较高的前提下,探讨对已有湿地松测定群体遗传力评估最经济有效的调查取样数量(样本量),为遗传选择提供最佳的遗传参数和选择策略。[方法]以61个湿地松22年生半同胞家系的胸径、树高等生长性状和木材基本密度、弹性模量等材性性状测定值为试验数据,利用ASReml-R软件混合线性模型的限制性极大似然估计法(REML)估算各性状在不同参试样本量下的遗传力及其标准误。通过比较分析在不同样本量下各性状遗传力及其标准误估算值的收敛性,讨论样本量对性状遗传力估算的影响,进而确定各性状遗传力评估所需的最少样本量。[结果]对于61个湿地松自由授粉家系测定林,当测定的家系数少于39个或者随机测量的单株数小于600株时,估算的遗传力极不稳定,标准误偏大,随着样本容量或家系容量的增加其精度与准确性逐渐增加;遗传力较低的性状其遗传力估计所需样本量普遍大于遗传力较高的性状。[结论]对本研究的测定群体而言,要获得精确度较高的遗传力估算值,所需测定的湿地松家系数应该大于39个或者随机测量的单株数大于600株;由于材性性状遗传力相对较高,其需要测定的样本量可相对少一些。本文为大型用材树种遗传力估算提供了一个具有参考价值的实例研究,结果及相应的研究方法对于类似遗传测定群体遗传参数估算具有参考意义。     

Gene flow pattern and mating system in a small population of Quercus semiserrata Roxb. (Fagaceae)

Pollen flow from external sources is important for the conservation of tree species in fragmented forests or small populations, because it can be sufficient to prevent differentiation among them, and appears to be able to prevent the loss of their genetic diversity through genetic drift. In this study, we examined the genetic heterogeneity of pollen pools accepted by each Quercus semiserrata seed parent at the Khun Wang Royal Agriculture Research Center, Thailand, both within and among two mast fruiting years (2005 and 2007), using paternity analysis and analysis of molecular variance (AMOVA). The mating systems of the trees were also examined using the multilocus mating system model (MLTR), after determining the genotypes at eight microsatellite loci of 26 seed-trees and 435 seeds from 8 seed-trees in the 2 mast fruiting years. The average distance of effective pollen flow within the plot was estimated to be 52.4 m, and 95% of effective pollen was dispersed within 200 m, indicating that effective pollen flow is highly localized and that most effective pollen is contributed by near-neighbor trees. The proportion of effective pollen that immigrated from external sources was estimated to be 26.2%. The AMOVA analysis based on the pollen haplotypes showed that the pollen pools, both total and for each reproductive year, significantly genetically differed among the seed parents. Using a mixed mating model, the estimate of biparental inbreeding for the total population (t_m − t_s) was 0.013, indicating that a low proportion of mating occurred among close relatives. The effective number of pollen donors (N_ep) was estimated to be 9.987 using the TwoGener model, or 10.989 using the mixed mating model. The effective number of pollen donors of seeds was higher in the mast fruiting year 2005 than in the other examined year, 2007. Consequently the allelic richness and genetic diversity of seeds produced in 2005 were higher than those produced in 2007. Overall, the results show that high outcrossing rates, high levels of gene flow from other populations and heterogeneity in the pollen received by an individual may enhance the ability of populations to maintain effective population sizes. Therefore, these processes may be sufficient to prevent loss of genetic diversity through genetic drift of Q. semiserrata at this study site.     

Investigation of the phenotypic and genetic properties of Brenneria nigrifluens strains,the causal agent of walnut bark canker in Kurdistan province,Iran

Phenotypic and genetic properties of Brenneria nigrifluens, the causal agent of walnut bark canker disease were studied using a combination of key biochemical tests, analysis of whole‐cell protein extracts and genetic fingerprints based on DNA primers corresponding to conserved motifs in bacterial repetitive (repetitive extragenic palindromic, enterobacterial repetitive intergenic consensus and BOX) elements and to the 16s rRNA gene. Phenotypic properties indicated that 11 strains showed a high degree of similarity and <5% of isolates showed different properties such as utilization of dulcitol and inositol. A majority of isolates produced rep‐PCR profiles similar (>80%) to a B. nigrifluens reference strain. This study showed that the majority of strains had 0–3 plasmids with estimated sizes ranging 9–32 Kb. Strains were clustered into two and three groups by whole‐cell protein analysis and rep‐PCR, respectively.     

Pronounced fluctuations of spruce bark beetle (Scolytinae: Ips typographus) populations do not invoke genetic differentiation

The dynamics of a recent outbreak of the spruce bark beetle (Ips typographus) in Switzerland was ruled by a devastating winter storm in 1999 and the drought and heat of the summer 2003. Starting from a similar level of population sizes, estimated as the rate of infested growing stock, beetle populations increased differently in magnitude and time among different regions in Switzerland. Accordingly, we expected local or regional genetic differentiation as a result of such repeated population expansion/breakdown dynamics. We analyzed 5 nuclear microsatellites of spruce bark beetles sampled from pheromone traps at 30 locations distributed over Switzerland. Our genetic results did not indicate any sign of population differentiation, structure, isolation by distance, or recent bottlenecks. This complete lack of genetic structure suggests that spruce bark beetles are highly mobile, precluding the formation of a spatial structure at neutral molecular markers. Thus, this molecular–genetic approach does not allow us to discriminate among regional gene pools and to identify the origin of expanding beetle populations.     

Inheritance and cross-resistance of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> insecticidal crystal protein Cry1Ac resistance in cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Hubner population from Tamil Nadu,India

Cotton bollworm, Helicoverpa armigera Hubner selected for five generations with Bacillus thuringiensis insecticidal crystal protein Cry1Ac in the laboratory developed 12.98-fold resistance. Resistance and susceptible populations were mass crossed to study the dominance of resistance. The Cry1Ac—selected (BCR) population showed 5.8-fold resistance to Cry1Aa and 5.04-fold resistance to Cry1Ab. The degree of dominance (D) was 0.34 and 0.40 for the R × S and S × R hybrids, respectively, which indicates incomplete recessive character of Cry1Ac resistance in the population. The estimated realized heritability (h ²) and response quotient (Q) of resistance for Cry1Ac were 0.52 and 0.15, respectively. This indicated the lower phenotypic variation in the selected population. The resistance risk assessment based on h ² indicated that the resistance would increased tenfold after <9 generations for Cry1Ac in the resistant population. The results show the ability of H. armigera to develop resistance against Cry1Ac and cross-resistance to Cry1Aa and Cry1Ab.     

Monitoring genetic stability inQuercus serrata Thunb. somatic embryogenesis using RAPD markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.     

Green manures for maize—bean systems in eastern Uganda: Agronomic performance and farmers' perceptions

Researchers worked with farmers in eastern Uganda to develop alternatives for soil management using crotalaria (Crotalaria ochroleuca), mucuna (Mucuna pruriens var. utilis), lablab (Dolichos lablab), and canavalia (Canavalia ensiformis) as green manures in short-term fallows. The participatory research was part of a community-based approach for systems improvement. Grain yields of maize (Zea mays) and bean (Phaseolus vulgaris) following one season of crotalaria fallow were 41% and 43%, respectively, more than following a two-season weedy fallow. Grain yields of maize following a one-season fallow with mucuna and lablab were 60% and 50% higher, respectively, as compared with maize following maize. Maize and bean yield were more, although effects were small, during the second and third subsequent seasons, indicating probable residual effects of the green manures. Mucuna and lablab were successfully produced by intersowing into maize at three weeks after sowing maize, although the yields of the associated maize crop were reduced by 24% to 28%. Farmers estimated the labor requirements for mucuna and lablab to be less than for crotalaria. Farmers independently experimented on how these species can be integrated into banana (Musa spp.), coffee (Coffea robusta), sweet potato (Ipomoea batatas), and cassava (Manihot esculenta) production systems. Farmers reported that the beneficial effects of the green manures included higher food-crop yields; weed suppression; improved soil fertility, soil moisture, and soil tilth; and erosion control. Mucuna and lablab were preferred because of reduced labor requirements and increased net benefits compared with continuous cropping. Farmer participation in the green manure research resulted in efficient generation and adaptation of green manure technology now being promoted in eastern and central Uganda.This revised version was published online in November 2005 with corrections to the Cover Date.