RAPD analysis of genetic relationships among Sphaeropsis sapinea isolates

Genetic relationships were studied among 23 isolates of Sphaeropsis sapinea collected from China, the United States, England, South Africa and Chile by using a random amplification of a polymorphic DNA (RAPD) analytical method. One hundred and 35 DNA fragments were amplified with 12 random primers by a polymerase chain reaction PCR technique and 96.3% were polymorphic. The genetic dendrogram based on RAPD analysis showed that the S. sapinea isolates could be divided into three types. Isolate CWS41 from Chile was separated genetically as the first type that was different from other isolates and isolates F2 and J2 from China comprised the second group. The third RAPD group accommodated other isolates including the B morphotype isolate CWS43 from the United States. __________ Translated from Journal of Nanjing Forestry University, 2006, 30(1): 13–16 [译自: 南京林业大学学报]     

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 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 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]     

Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

A polymerase chain reaction-restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus. __________ Translated from Journal of Nanjing Forestry University, 2005, 30(4): 5–9 [译自: 南京林业大学学报]     

Genetic diversity of Populus euphratica populations in northwestern China determined by RAPD DNA analysis

Five Populus euphratica Oliver populations in northwestern China were analyzed using RAPD DNA markers to determine genetic diversity among and within populations. One hundred-and-five polymorphic bands were observed, ranging in size from 250 bp to 1700 bp, using 10 primers. Only one population on the north side of the Tianshern Range had a unique band common to all individuals that was not found in individuals from populations in the Tarim River valley. Intra-population genetic diversity was high in two populations along the Tarim River and low in the other three populations. There was no significant correlation between genetic distances and geographic distances. The result of correspondence analysis shows that the individuals from the three populations with low genetic diversity are isolated from each other. The result of cluster analysis based on genetic distance shows that the population in the Tianshern Range is genetically distant from the other populations. These results suggest that the Tianshern Range population was genetically isolated from the other populations.     

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.     

Improvement of wood properties by urea-formaldehyde resin and nano-SiO2

In order to improve wood properties of triploid clones of Populus tomentosa, urea-formaldehyde (UF) resin was compounded with nano-SiO₂, coupling agents and flame retardants in different ways to prepare five kinds of modifiers. The poplar wood samples were impregnated with the modifiers and heated to prepare UF-SiO₂-wood composites. The antiswelling efficiency, resistance of water absorption, oxygen index and hardness of the composites were measured. Results show that all of the modifiers reduced water absorption of poplar wood and enhanced flame resistance and hardness. Nano-SiO₂ showed a marked effect in improving the hardness of wood. In addition, all of the modifiers, except UF-C-SiO₂-polymer, improved the dimensional stability of poplar wood. The UF resin and nano-SiO₂ compound improved general properties of poplar wood. __________ Translated from Journal of Beijing Forestry University, 2006, 28(2): 123–128 [译自: 北京林业大学学报]     

基于cpDNA rps16序列分析兰考泡桐与白花泡桐和毛泡桐的遗传关系

【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。     

Effect of Regeneration Method on RAPD-Based Genetic Variation of Cyclobalanopsis glauca (Fagaceae)

Cyclobalanopsis glauca is a dominant species of evergreen broad-leaved forests in mainland China. This study compares the genetic variation of an artificially regenerated population with its donor population and two other wild populations, by using RAPD markers. A total of 74 clear, reproducible bands were scored for 12 RAPD primers; 72 were polymorphic (P = 97.3%). AMOVA revealed that most genetic variation was within populations and only 10.35% was among populations. Various measures indicated that there is no difference in genetic diversity between the planted and the original populations. Φ_ST between the planted offspring population and the donor population was larger than those between the planted and other two natural populations, indicating that artificial regeneration might lead to biased genetic composition, given that temporal differentiation is usually lower than spatial differentiation. This divergence may be due to unequal seed production among the maternal individuals and viability differences among seeds.     

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.     

An analysis of the pattern of genetic variation in Vitellaria paradoxa using RAPD markers

Vitellaria paradoxa C.F.Gaertn. is one of the most economically and socially important tree species in the Sudano-Sahelian region. Little is known of the pattern of variation within its natural range. Eight populations covering most of the natural range from Senegal to Uganda were sampled and leaves of 118 individual trees were collected. An analysis of molecular diversity was carried out using random amplified polymorphic DNA (RAPD) markers. Fifteen random primers generated 67 polymorphic and 15 monomorphic RAPD loci ranging from size 1670 bp to 280 bp. Shannon's diversity index varied from Central Africa/Ndele (0.374) to Uganda/Amoya (0.350) but the differences between populations were smaller than the population standard errors. Correspondence analysis of unrooted neighbour-joining trees suggested that genetic distances between populations were correlated with geographic distances. This trend was confirmed by a Mantel test giving a coefficient of correlation between genetic and geographic distances of R = 0.88 (P = 0.0001). Result of AMOVA (analyses of molecular variance) showed that 14.8% (P = 0.002) of the RAPD variation was distributed among populations. Nested analysis of variance indicated that variance between the western and eastern groups of population represented 8.7% (P = 0.001) of the total variation and the variation amongst populations within group was 9.5% (P = 0.001). Eighty two percent of the variation was explained by variation amongst individuals within populations. The origin of genetic structure and level of diversity may be explained by the glacial refugia, the biological traits of Vitellaria paradoxa and by the impact of semi-domestication. Based on these results, sampling options of the natural populations are suggested for in or ex situ conservation. For the development of Vitellaria paradoxa breeding population, the sampling should consist of many individual trees selected within a few populations to capture a large proportion of variation.This revised version was published online in November 2005 with corrections to the Cover Date.     

Genetic Diversity of RAPD Mark for Natural Davidia involucrata Populations

The genetic diversity and genetic variation within and among populations of five natural Davidia involucrata populations were studied from 13 primers based on random amplified polymorphic DNA (RAPD) analysis. The results show that natural D. involucrata population has a rich genetic diversity, and the differences among populations are significant. Twenty-six percent of genetic variation exists among D. involucrata populations, which is similar to that of the endangered tree species Liriodendron chinense and Cathaya argyrophylla in China, but different from more widely distributed tree species. The analysis of the impacts of sampling method on genetic diversity parameters shows that the number of sampled individuals has little effect on the effective number of alleles and genetic diversity, but has a marked effect on the genetic differentiation among populations and gene flows. This study divides the provenances of D. involucrata into two parts, namely, a southeast and a northwest provenance. Translated from Scientia Silvae Sinicae, 2004, 40(4) (in Chinese)     

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]     

Cloning and RNAi construction of a LEAFY homologous gene from Populus tomentosa and preliminary study in tobacco

PtLFY, a LEAFY (LFY) gene, was cloned from Populus tomentosa (LM50) by PCR. Sequencing analysis indicated that PtLFY was 2 629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa (LM50 and 5082). The pBI121-Ptalfy (reverse)-intron-Ptlfy-GUS-nos was constructed using RNA interference (RNAi) technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed. [Supported by the National Natural Science Foundation of China (Grant No. 30371175) and Postdoctoral Foundation of China (Grant No. 2002032041)]     

Genetic analysis and conservation of endangered medicinal tree species <Emphasis Type="Italic">Taxus wallichiana</Emphasis> in the Himalayan region

Taxus wallichiana is one of the most important medicinal tree species of the Himalayan region. Leaf and bark of the species yield an important drug called taxol, which is used for treatment of many types of cancer. There is a serious threat to the existence of the species due to over exploitation in its native habitat. Adequate information on the nature and the extent of genetic diversity in this important species is required for developing suitable strategy for its conservation. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to assess genetic variation in nine natural populations of T. wallichiana from western part of the Himalayan ranges. Both the markers revealed low genetic diversity in these populations. Average heterozygosity for AFLP and RAPD were 0.3715 and 0.3072, respectively. Φ_ST values derived from molecular variance were 0.0855 and 0.1005 for AFLP and RAPD, respectively, whereas the corresponding G_ST values were 0.1796 and 0.2140. Most part of the genetic variation was present within the populations. However, between population variation was low but statistically significant, which suggested that the sampled populations might not constitute a single panmictic population. Cluster analysis and Mantel’s correlation revealed that genetic differentiation broadly followed geographic distribution of the populations. T. wallichiana thus urgently needs to be conserved using both in situ and ex situ conservation approaches.     

Variation of leaf characteristics in Populus tomentosa Carr.

An investigation was conducted to determine the extent of variations among nine provenances of Populus tomentosa Carr. in terms of leaf characteristics. A total of 263 accessions were studied under field conditions in the National Gene Bank of P. tomentosa in 2003. All of the accessions were characterized by 17 indices from 1 to 2-dimension constructions. Variance analysis of all characteristics showed that there were significant differences among the nine provenances and among individuals within each provenance. This study reveals that the evaluated germplasm appears to have a wide genetic base and high potential for further genetic improvements and it also indicates that abundant gene resources of P. tomentosa have been collected and preserved in the National Gene Bank. [Supported by the “Tenth Five-year Plan” National Key Project in Science and Technology (Grant No. 2002BA515B0303) and the National “863” Project (Grant No. 2002AA241071)]     

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.