RAPD markers related to sex locus in Populus tomentosa

By using the methods of random amplified polymorphic DNA (RAPD) and bulked segregate analysis (BSA), we identified markers that are linked to the sex determination in the dioecious Populus tomentosa. Male and female bulks were created through rough mixing equal amounts of its five individual DNA. A total of 88 primers were screened. Twelve primers produced clear patterns with at least one band that appeared to be polymorphic between the two bulks. Subsequently, five male and female individuals were analyzed with those 12 primers, and only S60 (ACCCGGTCAC) could generate a common 1800 bp DNA fragment in all five male individuals and male pool but not in any female individuals. It can be concluded that the gender of P. tomentosa is most likely connected to the S60-1800 bp DNA fragment and RAPD markers. S60, therefore, can be used for selecting the gender of P. tomentosa. __________ Translated from Journal of Central South University of Forestry & Technology, 2008, 28(3): 80–83 [译自: 中南林业科技大学学报]     

Genetic diversity of Betula luminifera populations at different elevations in Wuyi Mountain and its association with ecological factors

The random amplified polymorphic DNA (RAPD) technique was used to evaluate the genetic diversity and population structure of 91 genets from four wild populations of Betula luminifera at different elevations in the National Nature Reserve of theWuyi Mountain, Fujian Province, China. Eighteen random primers (from 139 primers) produced a total of 199 scorable amplified fragments, of which 174 (87.44%) were polymorphic across all individuals. The genetic diversities of B. luminifera at the population level and species level were PPL = 60.05%, h = 0.2242, I = 0.3181 and PPL = 87.44%, h = 0.3442, I = 0.4899, respectively. The value of differentiation (G _st= 0.3486) and analysis of molecular variance (AMOVA) indicated that there was a relatively high genetic differentiation among populations, and about one-third of the genetic variation occurred among populations. Pearson correlation analysis further revealed that the genetic diversity within populations had significant or very significant correlation with the elevation, climatic factors (annual average temperature and annual precipitation) and soil nutrient factors (total nitrogen, C/N ratio and organic matter). Mantel tests show that there was a significant correlation between the genetic distances among populations and the distance of elevation, and the divergence of soil nutrient factors. The results of the present study suggested that the relatively high genetic differentiation among populations of B. luminifera at different elevations might be caused by ecological factors and gene flow. __________ Translated from Scientia Silvae Sinicae, 2008, 44(3): 50–55 [译自: 林业科学]     

Genetic diversity of Quercus glandulifera var. brevipetiolata populations in three forest communities with different succession stages

In order to understand the relationship between population succession and its genetic behavior, random amplified polymorphic DNA (RAPD) technique was used to analyze the genetic diversity of Quercu glandulifera var. brevipetiolata populations in three forest communities with different succession stages (coniferous forest, coniferous and broad-leaved mixed forest, evergreen broad-leaved forest). The results showed that 145 repetitive loci were produced in 60 individuals of Q. glandulifera using 11 primers, among which 120 loci were polymorphic, and the total percentage of polymorphic loci was 82.76% with an average of 64.14%. Estimated by the Shannon information index, the total genetic diversity of the three populations was 0.4747, with an average of 0.3642, while it was 0.3234, with an average of 0.2484, judged from the Nei index. Judged from percentage of polymorphic loci, Shannon inform at ion index and Nei index, the genetic diversity followed a decreasing order: coniferous forest > broad-leaved mixed forest > evergreen broad-leaved forest. Analysis of molecular variance (AMOVA) showed that 69.73% of the genetic variance existed within populations and 30.27% of the genetic variance existed among populations. The coefficient of gene differentiation (GST) was 0.2319 and the gene flow (N _m) was 1.6539. The mean of genetic identity among populations of Q. glandulifera was 0.8501 and the mean of genetic distance was 0.1626. The genetic identity between the Q. glandulifera population in the coniferous forest and that in the coniferous and broadleaved mixed forest was the highest. UPGMA cluster analysis based on Nei’s genetic distance showed that the population in the coniferous forest gathered with that in the coniferous and broad-leaved mixed forest firstly, then with that in the evergreen broad-leaved forest. The genetic structure of Q. glandulifera was not only characteristic of the biological characteristics of this species, but was also influenced by the microenvironment in different communities. __________ Translated from Journal of Northwest Forestry University, 2008, 23(1): 18–22 [译自: 西北林学院学报]     

Genetic diversity of Lithocarpus harlandii populations in three forest communities with different succession stages

By using random amplified polymorphic DNA (RAPD) technique, this paper studied the genetic diversity and genetic differentiation of Lithocarpus harlandii populations in three forest communities (coniferous forest, coniferous and broad-leaved mixed forest, and evergreen broad-leaved forest) with different succession stages in Tiantai Mountain in Zhejiang Province. The results showed that a total of 173 repetitive loci were produced in 60 individuals of L. harlandii by 12 random primers, among which, 152 loci were polymorphic, and the total percentage of polymorphic loci was 87.86%. The average percentage of polymorphic loci of the populations was 65.32%, and their total genetic diversity estimated by Shannon information index was 0.4529, with an average of 0.3458, while that judged from Nei’s index was 0.3004, with an average of 0.2320. The percentage of polymorphic loci, Shannon information index, and Nei’s index of the populations were in the sequence of coniferous forest community > coniferous and broad-leaved mixed forest community > evergreen broad-leaved forest community. Analysis of molecular variance (AMOVA) showed that 72.85% of genetic variance was found within the populations, and 27.15% of genetic variance resided among the populations. The coefficient of gene diferentiation was 0.2277, and the gene flow was 1.6949. The genetic structure of L. harlandii was influenced not only by the biological characteristics of this species, but also by the microenvironment of different communities. The mean of genetic identity among three populations of L. harlandii was 0.8662, and the mean of their genetic distance was 0.1442. The genetic similarity between coniferous and broad-leaved mixed forest community and evergreen broad-leaved forest community was the highest, while that between evergreen broad-leaved forest community and coniferous forest community was the lowest. The unweighted pair group method with arithmeticmean (UPGMA) cluster analysis based on Nei’s genetic distance showed that conierous and broad-leaved mixed forest community first gathered with evergreen broad-leaved forest community, and then with coniferous forest community. __________ Translated from Chinese Journal of Ecology, 2007, 26(4): 509–514 [译自: 生态学杂志]     

Genetic diversity of natural Hepatacodium miconioides populations in Zhejiang Province

Hepatacodium miconioides is the Class II protected plant species in China. This paper studies the genetic diversity and differentiation of its nine natural populations in Zhejiang Province by using random amplified polymorphic DNA (RAPD) technique. Twelve random primers were selected in the amplification, and 164 repetitive loci were produced. The percentage of polymorphic loci in each H. miconioides population ranged from 14.60% to 27.44%, with an average of 20.73%. Among the test populations, Kuochangshan had the highest percentage of polymorphic loci, Simingshan took the second place, and Guanyinping had the lowest percentage. As estimated by Shannon index, the genetic diversity within H. miconioides populations accounted for 27.28% of the total genetic diversity, while that among H. miconioides populations accounted for 72.72%. The genetic differentiation among H. miconioides populations as estimated by Nei index was 0.715,7. This figure was generally consistent with that estimated by Shannon index, i.e., the genetic differentiation among populations was relatively high, but that within populations was relatively low. The gene flow among H. miconioides populations was relatively low (0.198,7), and the genetic similarity ranged from 0.655,7 to 0.811,9, with an average of 0.730,6. The highest genetic distance among populations was 0.422,9, while the lowest was 0.208,3. All the results showed that there was a distinct genetic differentiation among H. miconioides populations. The genetic distance matrix of nine test populations was calculated using this method, and the clustering analysis was made using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis suggested that the nine populations of H. miconioides in Zhejiang Province could be divided into two groups, the eastern Zhejiang group and the western Zhejiang group. __________ Translated from Chinese Journal of Applied Ecology, 2005, 16(5): 795–800 [译自: 应用生态学报, 2005, 16(5): 795–800]     

Association of Sphaeropsis sapinea with insect‐damaged red pine shoots and cones

The association of the shoot blight and canker pathogen Sphaeropsis sapinea with red pine (Pinus resinosa) shoots and cones damaged by insects (especially Dioryctria sp.) was investigated. Samples from a single plantation approximately 35 years old, in Sauk Co., Wisconsin and also from three plantations, between approximately 40 and 50 years old, located in an area of pine shoot moth activity in the preceding year in Adams Co., Wisconsin were visually examined. Samples were arbitrarily collected from trees felled in the first plantation in May. Pycnidia of S. sapinea and insect damage were observed on 56 of 91 (62%) of closed cones and 17 of 165 (7%) of previous year's shoots. In the absence of insect damage, pycnidia of the pathogen were identified only on eight of 91 (9%) closed cones and never on previous year's shoots. In each of the other three plantations, 10 trees were located at intervals along transects in mid‐June; one branch from the lower half of the crown per tree was pruned off, and both current and previous year's shoots were examined. Insect damage and S. sapinea pycnidia were too rare on current year's shoots to draw any conclusions. Insect damage occurred on 20–40% of over 2000 previous year's shoots that were examined, but pycnidia of the pathogen were identified on only about 5%. Although infrequent, S. sapinea was identified in association with insect‐damaged previous year's shoots from these three plantations three times more frequently than those without insect damage. Random amplified polymorphic DNA (RAPD) markers from eight randomly selected isolates were consistent with the A group of S. sapinea, which can be aggressive on red pine. This ability to exploit insect‐damaged shoots may facilitate long‐term persistence of S. sapinea at low disease incidence and severity. The potential role of insect wounds as infection courts and insects as vectors of this important pathogen of pines deserves further study.     

Isozyme and RAPD polymorphisms in Heterobasidion annosum in Italy

Isozyme and random amplified polymorphic DNA (RAPD) polymorphisms were used to study variability in a group of 41 isolates from the Italian population of Heterobasidion annosum. The isolates belonged to the intersterility groups P and S, and particularly to the group that is most widely distributed in Italy, group F. Isozyme analysis was effective in identifying the three intersterility groups and revealed a high degree of genetic divergence within the P group isolates; the mannose phosphate isomerase (MPI-2) locus was diagnostic in the attribution of isolates to the more correlated F and S groups. RAPDs were detected following amplification by the polymerase chain reaction (PCR). 74 RAPD fragments, obtained through amplifications with eight primers, were scored. Isolates from the 3 intersterility groups were clearly divergent based on analysis of RAPD markers. However, a similarity index calculated for the isolates within the F population indicated a high uniformity of the isolates collected throughout the Italian peninsula.     

Population genetic structure of <Emphasis Type="Italic">Juniperus phoenicea</Emphasis> (Cupressaceae) in the western Mediterranean Basin: gradient of diversity on a broad geographical scale

• Introduction, Material and Methods  

The genetic structure and diversity of ten natural populations of Juniperus phoenicea L. from the western part of the species range have been studied using random amplified polymorphic DNA (RAPD) markers.     

An effective medium for isolating Sphaeropsis sapinea from asymptomatic pines

The pathogen Sphaeropsis sapinea can persist in stems and branches of asymptomatic pines and can later induce disease when triggered by host stress. Several experiments were conducted to test if:(i) medium amended with tannic acid (TA) can increase the frequency of cultural detection of this shoot blight and canker pathogen from asymptomatic red pine (Pinus resinosa) stems, and (ii) S. sapinea can persist in asymptomatic red pine in the field following artificial inoculation. TA (0.5% w/v) in 2% (w/v) water agar proved to be the best medium for isolation of S. sapinea among a larger number of tested media. The addition of TA had little or no effect on the growth of two group A and two group B isolates of S. sapinea. However, when TA was added, 11 other fast‐growing fungal isolates from stems/branches of red or jack pines (P. banksiana) were inhibited and grew more slowly (p < 0.05) than both S. sapinea groups. The TA‐amended medium improved cultural detection of S. sapinea from 2‐year‐old, asymptomatic red pine nursery seedlings compared with two other methods used for the cultural detection of S. sapinea (32%vs. 8.5% and 18% recovery; p < 0.001 and p = 0.031, respectively). A field test using the TA‐amended media established that S. sapinea can persist asymptomatically in red pine trees for at least 1 year. This medium significantly reduces the frequency of false‐negatives from asymptomatic field material.     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

Spatial distribution pattern and dynamics of the primary population in a natural Populus euphratica forest in Tarim Basin, Xinjiang, China

One 50 m × 50 m standard plot was sampled in a natural forest of Populus euphratica in Awati County, situated at the edge of the Tarim Basin, Xinjiang Uygur Autonomous Region, China. The field investigation was conducted with a contiguous grid quadrate method. By means of a test of variance/mean value ratio, aggregation intensity index and theoretical distribution models, the spatial distribution pattern and the dynamics of primary populations in P. euphratica forest were studied. The results showed that the spatial distribution pattern of two dominant arbor populations conformed to clumped distribution. The aggregation intensity of the P. euphratica population was higher than that of P. pruinosa population. The spatial distribution pattern of two companion plant populations in the shrub layer also conformed to clump type, though the aggregation intensity of Tamarix chinensis was higher. In the herb layer, the distribution patterns of Glycyrrhiza uralensis and Asparagus persicus conformed respectively to a clumped pattern and a random pattern. The results of a Taylor power method test and Iwao’s regression model also verified that both P. euphratica and P. pruinosa populations belong to a clumped pattern. Although the distribution pattern of P. pruinosa population at different development stages all belonged to a clumped distribution pattern, the aggregation intensity dropped gradually along with age development. The distribution patterns of the P. euphratica population at different development stages changed from random type to clumped type, and further to random type. The differences in spatial distribution patterns of different populations at different development stages were related not only to ecological and biological characteristics of each species in the communities in the light of competitive exclusion principle among the populations, but were also closely related to the habitats in which the species lived in. __________ Translated from Journal of Southwest Forestry College, 2007, 27(2): 1–5 [译自: 西南林学院学报]     

Diversity of soil microorganisms in natural Populus euphratica forests in Xinjiang, northwestern China

To better understand the distribution of soil microorganisms in Populus euphratica forests in Xinjiang, northwestern China, we studied and compared the populations and numbers of bacteria, fungi and actinomycetes in the soil at four different age stages of natural P. euphratica forests, i.e., juvenile forests, middle-aged forests, over-mature forests and degraded forests. Results showed that there were clear differences in the amount of microorganism biomass and composition rates across the four forest stages. Dominant and special microorganisms were present in each of the four different soil layers. The vertical distribution showed that the microorganism biomass decreased with increasing soil depth. The population of microorganisms was the lowest at 31–40 cm of soil depth. The microorganisms consisted of bacteria, actinomycetes, as well as fungi. Bacteria were the chief component of microorganisms and were widely distributed, but fungi were scarce in some soil layers. Aspergillus was the dominant genus among the 11 genera of fungi isolated from the soil in different age stages of P. euphratica forests. __________ Translated from Journal of Beijing Forestry University, 2007, 29(5): 127–131 [译自: 北京林业大学学报]     

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.     

Small-scale genetic diversity in oneseed hawthorn (<Emphasis Type="Italic">Crataegus monogyna</Emphasis> Jacq.)

We have examined the pattern of random amplified polymorphic DNA (RAPD) variation among six populations of Crataegus monogyna from northern Italy, extending over an area of about 20,000 km². The aim was to evaluate local differentiation in relation to geographical distance. Of the 73 loci analysed, 65 were polymorphic, and there were no bands at complete fixation at the population level. The mean genetic diversity was 0.291, with the values for individual populations ranging between 0.252 and 0.333. According to the analysis of molecular variance most of the genetic variation was found within populations (about 80%), with a significant proportion being attributable to genetic differences between populations. No evidence for isolation by distance was found in the set of populations sampled. The efficacy of RAPD markers in analysing genetic variation, and the contribution of the results in terms of the preservation of biodiversity and defining the most appropriate strategies for collecting forest reproductive material, are discussed.     

Genetic uniformity characterizes the invasive spread of Neofusicoccum parvum and Diplodia sapinea in the Western Balkans

In the past decade, trees and shrubs in the Western Balkans region have been damaged by canker and die‐back disease caused by Botryosphaeriaceae species. These pathogens include Neofusicoccum parvum and Diplodia sapinea. In this study, we determine genetic diversity and structure between populations of N. parvum and D. sapinea from Serbia and Montenegro (Western Balkans) using DNA sequence data of the internal transcribed spacer rDNA, translation elongation factor 1‐alpha, β‐tubulin‐2 and microsatellite markers. The relationship of both pathogens was compared for populations from the Continental (CR) and Mediterranean (MR) regions and for isolates of D. sapinea from Cedrus spp. and Pinus spp. Neofusicoccum parvum and D. sapinea were shown to have a low gene and genotypic diversity across the regions and hosts. All genotypes of D.  sapinea found on Pinus spp. were also present on Cedrus spp. The CR and MR populations of both species were found to be only slightly separated from one another by a geographical barrier. Low genetic diversity and dominance of N. parvum and D. sapinea on non‐native trees suggests that these species have most likely been introduced into Western Balkans, possibly through the movement of infected plants.     

韩国、美国和中国栗疫病菌的遗传变异分析

用RAPD方法,对来自韩国、美国和中国的26个栗疫病菌菌株进行了遗传变异分析.使用筛选的12个随机引物,共扩增了115个0.19～3.1 kb大小的扩增片段,其中多态性片段占61.7%.聚类分析结果,相似系数为0.92时,26个菌株分为两大组,一组由21个菌株组成,包括大部分韩国菌株和美国菌株;另一组包括部分韩国菌株和中国菌株.表明美国菌株和大部分韩国菌株的遗传相似性很高,部分韩国菌株有较大的变异,而中国菌株则表现出了遗传上的远缘关系.     

Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.