Application of random amplified polymorphic DNA (RAPD) to detect genetic variation in Norway spruce

The RAPD assay is a screening method for genetic variation; in Norway spruce biology research, it may find many applications. We have investigated into its suitability for Norway spruce genetics by optimizing the protocol, testing for stability in clones, especially tissue culture clones, assessing the variation in a seed sample, and checking for correct Mendelian segregation in haploid megagametophytes. The RAPD assay produced numerous genetic markers quickly. The data obtained gave insight into the genetic make-up of clones and seed.

---

Zusammenfassung Die RAPD Methode, ein Schnelltest für die genetische Variation, wurde für die europäische Fichte getestet, wo viele Aspekte der Biologie der Baumart damit untersucht werden könnten. Das Protokoll wurde optimiert, und genetische Stabilität in Klonen, besonders aus Gewebekultur, die genetische Variation in einer Samenprobe und die Mendel-konforme Aufspaltung in haploiden Megagametophyten wurde untersucht. Zahlreiche genetische Marker konnten gefunden werden, wodurch ein Einblick in die genetische Zusammensetzung des untersuchten Materials gegeben war.

     

Characterization of Cryphonectria parasitica strains by random amplified polymorphic DNA (RAPD) technique and conventional methods

In recent years damage to Austrian Castanea sativa populations caused by Cryphonectria parasitica has increased. A total of 34 isolates out of 13 observation plots of this phytopathogen in the south-east of Austria were examined for laccase activity, virulence, number of vegetative compatibility groups and for the existence of hypovirulence associated dsRNA. Furthermore, the applicability of the random amplified polymorphic DNA (RAPD) technique for investigating population structure was tested. Small dsRNA fragments were found in two Austrian strains. The same two strains exhibited reduced virulence. Seven vegetative compatibility groups were found in the areas examined. RAPD analysis proved to be an efficient method for distinguishing between different C. parasitica genotypes.     

用随机扩增多态DNA (RAPD)技术分析野生与笼养绿孔雀种群的遗传多样性

利用随机扩增多态DNA(RAPD)技术对野生14个和笼养18个绿孔雀(Pavomuticus)个体进行了种群遗传多样性分析。用23个随机引物对野生与笼养绿孔雀基因组DNA扩增,分别获得161和166个扩增片段。计算发现野生与笼养绿孔雀的种群内平均相对遗传距离分别是0.0555和0.1355,两种群间的平均相对遗传距离为0.1635;野生绿孔雀和笼养绿孔雀种群的Shannon多样性指数平均分别是0.4348和1.0163,表明有显著性差异。以上分析都显示野生绿孔雀的遗传多样性很低。利用UPGMA法聚类显示两个种群都是分别来源于两个家系,可据此进行绿孔雀的繁育管理。图1表3参5。     

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.     

Use of random amplified polymorphic DNA (RAPD) for detection of genetic variation and proof of the heterothallic mating system in Hypoxylon mediterraneum

The efficiency of RAPD analysis in the evaluation of genetic variability in Hypoxylon mediterraneum has been tested on 49 monoascospore isolates of the fungus from different localities. A large genetic variability was detected within single stromata and among monoascospore isolates in a restricted area. The large variability in restricted populations can be explained by the heterothallic mating system and the life cycle of the fungus and its ecological adaptations. RAPD markers can discriminate monoascospore isolates both in the single stromata and among stromata. The application of RAPD markers for epidemiological studies is discussed.     

Genetic variation of the resin top fungus in Finland as determined by random amplified microsatellites (RAMS)

The analysis of random amplified microsatellite (RAMS) markers among aecia of the causal agent of the resin top disease (Peridermium pini) on Scots pine (Pinus sylvestris) suggested that the genetic variation between the populations of this rust fungus is low in Finland. The method used allowed the identification of heterozygotic aecia in two loci, where the degree of heterozygosity was, however, low. The RAMS patterns of the Finnish aeciospores, other aeciospores from Thetford, UK and Cronartium flaccidum from Italy were highly similar suggesting that the autoecious and heteroecious forms of these rust fungi would be genetically closely related.     

Novel in silico EST-SSR markers and bioinformatic approaches to detect genetic variation among peach(Prunus persica L.)germplasm

Because there are thousands of peach cultivars,cultivar classification is a critical step before starting a breeding project.Various molecular markers such as simple sequence repeats(SSRs) can be used.In this study,67 polymorphic primers produced 302 bands.Higher values for SI index(1.903) suggested higher genetic variability in the genotype under investigation.Mean values for observed alleles(Na),expected heterozygosity(He),effective alleles(Ne),Nei's information index(h),and polymorphic information content(PIC) were 4.5,0.83,5.45,0.83,and 0.81,respectively.The dendrogram constructed based on Jaccard's similarity coefficients outlined four distinct clusters in the entire germplasm.In addition,an analysis of molecular variance(AMOVA) showed that70.68% of the total variation was due to within-population variation,while 29.32% was due to variation among populations.According to this research,all primers were successfully used for the peach accessions.The EST-SSR markers should be useful in peach breeding programs and other research.     

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.     

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.     

Multiallelic and multilocus simple sequence repeats(SSRs) to assess the genetic diversity of a Salix spp. germplasm collection

Salix L.(willow) is the largest genus of the family Salicaceae and plays an important role in riparian habitats,wetlands and in shrub tundra.Due to the different implications for the species belonging to this family,it is fundamental to identify molecular tools characterizing relevant clones.A set of six multilocus and multiallelic simple sequence repeat(SSRs) markers are presented,leading to390 polymorphic fragments considered as single dominant markers and able to discriminate successfully 92 S.alba L.from 24 Salix spp.The polymorphic fragments have been used to perform genetic diversity studies,and to investigate population structures and cluster analysis in a germplasm collection.The results highlight the capability of the six SSRs to be powerful genetic resources in applied forestry research,both to distinguish S.alba clones from Salix spp.and to perform genetic population studies for breeding programs.     

A population genetic study in a scattered forest species, wild service tree [Sorbus torminalis (L.) Crantz], using RAPD markers

Wild service tree [Sorbus torminalis (L.) Crantz] is a forest tree widespread in Europe, characterized by a scattered distribution. Its hermaphroditic flowers are pollinated by insects, and outcrossing is the prominent mating system, also due to the presence of gametophytic self-incompatibility. Genetic diversity and differentiation of 22 populations from northwestern Italy were investigated by means of variation scored at 53 polymorphic RAPD (Random Amplified Polymorphic DNA) loci. Populations differed in genetic variation, with Shannon diversity index ranging from 0.166 to 0.469. According to Analysis of Molecular Variance (AMOVA), most of the genetic variation was found within populations (61.78%) with a significant proportion of variance attributable to genetic differences between regions (23.60%) and between populations (14.62%). Evidence for isolation by distance was found in the set of populations sampled. The effect of habitat fragmentation on genetic variation was also evaluated. The efficacy of RAPD markers in analysing genetic variation, and the contribution of the results in terms of the preservation of biodiversity are discussed.     

The infestation of Vicia faba L. (Fabaceae) by Aphis fabae (Scop.) (Homoptera: Aphididae) under the influence of Lamiaceae (Ocimum basilicum L. and Satureja hortensis L.)

Abtract The system Vicia faba–Aphis fabae fabae was studied under the influence of Ocimum basilicum (basil) and Satureja hortensis, in a wind tunnel, in the greenhouse and in field experiments. In the wind tunnel at 20°C both Lamiaceae were deterrent for A. fabae, and S. hortensis proved to be more deterrent than O. basilicum. In experiments in the greenhouse at low temperatures (average minimum 14.6°C, average maximum 24.1°C), A. fabae colonised first and significantly more intense Vicia fabae (field beans) not surrounded by O. basilicum or S. hortensis. At high temperatures (average minimum 18.0°C, average maximum 38.5°C) this relation was inverted: Vicia faba surrounded by the two Lamiaceae were preferred for colonisation (Ocimum basilicum significantly). It showed that pots with Lamiaceae were no obstacle for the aphids to reach Vicia faba. In strip cropping in the field, the repellent effect of Ocimum basilicum proved to be stronger than of Satureja hortensis. In 2002 there was observed only a tendency of lower aphid attack of field beans intercropped with Lamiaceae, while in 2004 and 2005 the infestation of Vicia faba by Aphis fabae was significantly lower in plots intercropped with basil. In plots with Satureja hortensis as intercrop, Vicia faba were significantly lower infested, after 3 weeks. The differences between the results of the wind tunnel/greenhouse at low temperatures, and the field experiments concerning the deterrence by Satureja hortensis cannot be explained. But basing on our results with Ocimum basilicum and those published by other authors, it is recommended to follow up intercropping, after a sincere analysis in every case, in favour of agronomists.     

Performance and genetic diversity of 23 provenances of northern red oak( Quercus rubra L.) after 25 years of growth in South Korea

Growth traits and genetic diversity of 23 provenances of Quercus rubra introduced from North America were analyzed in a provenance trial established with a randomized block design in Hwaseong,Gyeonggi,South Korea in 1993.Growth variables and survival at age 25 were compared with results from early stages.Height,DBH,volume and stem straightness of Q.rubra was better than those of the domestic oak(Quercus accutissima).Growth of the Dunham Island provenance from New York was the best among the 23 provenances that of the Eagle River provenance from Wisconsin was poorest.Survival rate at age25 was on average 52%.The longitude of seed origin and growth of provenance were consistently significantly negatively correlated at all ages.Growth of coastal provenances was superior to that of the inland provenances,which were separated by the Appalachian Mountains.Genetic diversity and genetic distance among the provenances were evaluated using micro satellite markers.The allelic frequencies showed high polymorphism in 10 microsatellite loci,and 292 alleles were found.Of 10 loci,two were commonly found in the 23 provenances.The mean allelic diversity and heterozygosity observed among the provenances were similar to those from the native populations of Q.rubra in North America.Nei's genetic distance among the 23 was estimated and showed that a clear trend between geographic and genetic distances,indicating that some provenances have high genetic diversity with superior growth performance.     

Parasitoids of Saperda populnea (L.) (Coleoptera: Cerambycidae) on aspen (Populus tremula L.) in Bulgaria

The parasitoids of Saperda populnea (L.) (Col.: Cerambycidae) were studied in Bulgaria during the period 1997?–?2001. Galls with pest larvae were collected from young aspen trees at eight locations (Sofia, Kokaliane, Plana, Churek, Gorni Lom, Gintzi, Dolno Kamartzi and Klisura) and examined under laboratory conditions. Four species were reared from 1118 galls containing overwintering pest larvae: Iphiaulax impostor (Scop.) (Hym.: Braconidae), Dolichomitus populneus (Ratz.), Schreineria populnea (Gir.) (Hym.: Ichneumonidae) and Billaea irrorata (Meig.) (Dipt.: Tachinidae). All of the parasitoids were solitary. Schreineria populnea was found as a new parasitoid of the host in Bulgaria. B. irrorata emerged mainly from late-stage S. populnea larvae; the remaining species from both early- and mid-stage larvae. In the parasitoid complex of S. populnea, the most numerous were B. irrorata (59.8?%) and D. populneus (29.4?%). The percentage of host larval parasitism varied from 2.4 to 33.3, with an average of 9.7?%. B. irrorata was the most important in reducing the pest number. The average mortality of S. populnea caused by the tachinid was 5.8?%, and the highest mortality observed in individual study was 20.2?%.     

Morphological parameters and genetic diversity of progenies from seed production areas and unimproved stands of teak (Tectona grandis L.f.) in India

We compared morphological and genetic attributes of prog-eny obtained from three seed production areas (SPAs) and three corre-sponding unimproved stands (UIS) of teak (Tectona grandis L.f) at three bro...     

The use of the white poplar(Populus alba L.)biomass as fuel

We determined the calorific value of white poplar(Populus alba L.) woody biomass to use it as firewood. The value of 19.133 MJ kg-1obtained experimentally shows that the white poplar can be quite successfully used as firewood. Being of a lower quality in comparison with usual beech firewood, the white poplar has similar calorific value. The white poplar has a calorific density of 30.7 % lower than that of current firewood. That is why the price of this firewood from white poplar is lower accordingly. Also, the prognosis of calorific value on the basis of the main chemical elements, being very close to the experimental value(+2.6 %), indicates an appropriate value can be achieved to be used for investigation with the chemical element analysis.     

Chloroplast DNA variation of northern red oak (Quercus rubra L.) in Indiana

Chloroplast DNA (cpDNA) variation was examined in 48 northern red oaks at 14 sites representing contrasting glacial histories and age structures within the state of Indiana in the United States. PCR-RFLP of three intergenic regions revealed five haplotypes. Haplotype I was common to seven sites and was the most frequent (17 trees). Haplotype II was common to five sites and was nearly as frequent as haplotype I (16 trees). Haplotypes III, IV and V were equally infrequent and did not occur together. Genetic diversity resided among rather than within populations (G_ST = 0.73 ± 0.14). This preliminary survey shows that cpDNA diversity will be a useful tool for the investigation of ancient seed dispersal patterns in northern red oak.     

DNA sequence variation and development of SNP markers in beech (Fagus sylvatica L.)

European beech (Fagus sylvatica L.) is one of the most important deciduous tree species in Central Europe. The potential of beech to adapt to climate change, higher temperatures, and less precipitation in the summer months is still unknown. Most studies in beech used microsatellite, AFLP (amplified fragment length polymorphism), or isozyme markers, which have only a restricted potential to analyze adaptation. Only few studies investigated genes probably involved in the adaptation to drought stress and bud phenology in beech. In this study, SNP (single nucleotide polymorphisms) markers were developed in order to analyze adaptation and their technical advantages compared to microsatellites and AFLPs were discussed. Partial sequences of ten candidate genes probably involved in drought stress and/or bud phenology were identified at the genomic level, and SNPs and indels (insertions/deletions) in coding and non-coding regions were analyzed. Plant material was sampled along a precipitation gradient in Germany. In total, 8,145?bp were sequenced and analyzed, 4,038?bp were located in exon and 4,107?bp in intron regions. 63 SNPs and 11 indels were detected, which are differently distributed over the studied gene regions. The nucleotide diversity ranged from 0 to 6.62 (π?×?10^?3) and is comparable to other tree species, whereas the mean nucleotide diversity (2.64) for F. sylvatica is comparatively low. These results will help to investigate the genetic basis of drought stress and bud burst and to conduct association mapping in natural populations. Furthermore, the detected SNPs can also be used for population genetic studies.     

The molecular identification and genetic diversity of economically important wireworm species (Coleoptera: Elateridae) in Canada

Click beetle larvae (wireworms) are significant crop pests in Europe and North America. In Canada, there are ~30 economically important species which are morphologically difficult to identify, but for which sequence data are lacking. Accurate knowledge of damage-causing species and the population genetics and phylogeography of elaterids will provide insight into their sustainable management. Here, we use interspecific variation in mitochondrial 16S rRNA as a robust method of identification, consider the intra- and interspecific genetic variation of some important Canadian wireworm pests and assess the genetic structure and isolation by distance for a re-emerging major pest species, Hypnoidus bicolor Eschscholtz. Wireworms were sampled from Canada and the USA, identified as morphospecies, and sequenced at the 16S rRNA region (294–442 bp). Within some species unusually high intraspecific genetic distances between samples suggested the possibility of cryptic wireworm species or misidentifications, though this was <1 % for most species. Phylogenetic analyses gave some indication of the likely identity of these ambiguous samples. There was a significant correlation between genetic and geographic distance and significant genetic structuring within and between H. bicolor populations, which appeared to be composed of two species comprising several haplotypes. These data provide a starting point for determining the distribution of damage-causing species throughout Canada. The inclusion of data from other nuclear and mitochondrial loci, and use of sequence data from known adult samples, would further aid identification and relationships of wireworm species.