Genetic variation of Fraxinus angustifolia natural populations in Greece based on nuclear and chloroplast microsatellite markers

Assessment of genetic variation within and among populations is an essential parameter for the effective conservation of forest genetic resources. In this work, the genetic diversity within and among natural populations of the forest tree species Fraxinus angustifolia Vahl in Greece was studied using selected nuclear and chloroplast microsatellite DNA loci. Eight natural populations of F. angustifolia were identified in different locations of the mainland of Greece, and a total of 230 individuals were studied. High polymorphism was observed within populations, while the genetic differentiation among populations was moderate. Intra-population diversity was correlated with geographical coordinates, but no isolation by distance was observed. Of the three haplotypes identified, only one was dominant. Putative ancestral haplotypes were found at small spatial scales suggesting that population expansions could have originated in the region. This study located in sympatry haplotypes that in other parts of Europe are in allopatry, reinforcing the notion of population expansions from the south of Balkans including Greece. Suggestions for conservation and management of F. angustifolia are also reported.     

Use of DNA markers in forest tree improvement research

DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating genetic diversity in breeding populations, germplasm identification, verifying controlled crosses, and estimating seed orchard efficiencies. Because the number of DNA markers is potentially limitless, it should be possible to map individual quantitative trait loci (QTL) by linkage analysis with high-density maps. Finally, if such associations can be found, it may also be possible to design marker-assisted breeding strategies for forest trees.     

Genetic structure of the green oak leaf roller (Tortrix viridana L.) and one of its hosts, Quercus robur L.

An herbivorous insect, the green oak leaf roller (Tortrix viridana) and one of its host species, pedunculate oak (Quercus robur), were investigated for their genetic variation within and among populations on the basis of mitochondrial, chloroplast, and anonymous markers. Oaks and green oak leaf rollers from 10 oak stands in North Rhine-Westphalia (North-Western Germany) were examined. The genetic variation of pedunculate oaks is much lower within than among populations when assessed using chloroplast gene markers and higher among populations when assessed using anonymous markers. When using mitochondrial and AFLP markers, the genetic variation of the green oak leaf roller populations was higher within than among the populations, which suggests a high gene flow between the populations. Mantel tests on the gene diversities of oaks and the green oak leaf roller yielded a significant negative correlation for both marker types. To sum up, the reasons for the differences in the spatial patterns of the genetic variation of the host and herbivorous insect may be found in their different generation times, mechanisms and capacities for dispersal.The results for the green oak leaf roller revealed a higher migration rate than assumed before which has consequences for the prediction of the dynamics of future outbreak events.     

Indigenous genetic lineages of Fagus crenata found in the Izu Peninsula suggest that there was one of refugia for the species during the last glacial maximum

Regions believed to be refugia are known to have lineages indigenous to the regions. The Izu Peninsula, where genus Fagus existed during the last glacial period, was focused on to survey chloroplast DNA of Japanese beech (Fagus crenata Blume) that grew there. DNA sequence analysis of chloroplast genome regions of about 14 kb revealed two new haplotypes, O and E-1, in F. crenata populations in the Izu Peninsula and adjacent regions. Both haplotypes were conceivably derived from haplotype E. The result of the genetic analysis in this study in combination with the result of pollen analyses performed in the past clearly showed that refugia for F. crenata existed in the Izu Peninsula, and that F. crenata now in growth are lineages indigenous to the region.     

Genetic succession and spatial genetic structure in a natural old growth Cryptomeria japonica forest revealed by nuclear and chloroplast microsatellite markers

Spatial genetic structure and diversity of Cryptomeria japonica trees in old growth forest but selectively logged in approximately 300 years ago were analyzed using seven nuclear microsatellite markers and one chloroplast microsatellite marker. The individuals were sampled from a 4-ha natural forest plot (200 m × 200 m), which are consisted of three size classes including newly regenerated individuals such as regenerated individuals (REG class; stem diameter at breast height (DBH), 5–110 cm), remaining individuals (REM class; DBH ≥ 110 cm), and old stumps. Genetic diversity was similar between the three groups. These results suggest that genes from the old growth forest may have been passed on to the next generation (i.e., the current forest). DNA extraction and analysis from old stumps was partly successful and we could compare the genetic diversity between old stumps and living trees using four microsatellite loci. Patterns of spatial genetic structure detected by analyses of the nuclear and chloroplast markers differed substantially; all nuclear markers showed significant positive autocorrelation (P < 0.05) over short distances, but the chloroplast marker showed no significant autocorrelation over any distance. We concluded that Moran's I spatial autocorrelation pattern for the chloroplast microsatellite may have been influenced by the extensive pollen flow characteristic of this species, because chloroplast DNA of C. japonica is paternally inherited through pollen and is a wind-pollinated species that produces abundant pollen every few years. In contrast, Moran's I spatial autocorrelation pattern for nuclear microsatellites may reflect limited seed dispersal in gaps where the forest has regenerated and low levels of seed shadow overlap due to past logging. The average seed dispersal distance was 86.0 m at the study site and about 34.0% of the seeds were from the outside of the site, which revealed by parentage analysis. The individuals remaining after logging, and the extensive pollen flow from outside our study plot, may play important roles in maintaining the forests on this island. Gaps created by the selective logging activities appear to have been important for the regeneration of this species in the past but in the present time, natural disturbances such as typhoon and landslide are also important for gap creation.     

广东韶关马尾松种子园遗传多样性分析

从210个随机引物中筛选出的16个引物,共检测到118条带,其中多态性带95个,总多态位点比率为80.37%,充分说明了马尾松无性系间遗传背景的复杂性和多样性。采用Nei指数计算的建园无性系马尾松有效等位基因数平均值为1.3447,总基因多样性为0.2445,遗传多样度(h)平均值为0.2445,Shannon′s信息指数(I)平均值为0.3558,与一些松柏纲植物的RAPD数据资料相比,马尾松的遗传多样性处于较高水平,显示出该无性系马尾松种子园内各马尾松的遗传基础较宽,具备营建出遗传品质好、遗传多样性高、稳定性好的马尾松人工造林的能力,能满足林木育种的需要。     

Japanese beech (Fagus crenata) plantations established from seedlings of non-native genetic lineages

In order to examine whether seedlings of non-native genetic lineages had been planted, we compared chloroplast DNA haplotypes in plantations and neighboring natural populations of Fagus crenata in Nagano Prefecture, Japan. This region was chosen for study because there are abundant natural F. crenata populations with different haplotypes, and because the species is also frequently used for afforestation in the area. We sampled 159 trees from 30 populations of the species across most of its natural range in the region, and 136 trees from 20 plantations of the species, ranging in age from 3 to 17 years. Six single nucleotide polymorphisms and four insertions/deletions in two chloroplast DNA regions (trnL–trnF and trnK) were analyzed in each of the sampled individuals in order to determine their haplotypes. Four haplotypes (B, D, E and F) were detected in the natural populations, and these exhibited a clear geographical structure; in contrast, only two haplotypes (A and B) were found in the plantations. We found that 15 out of the 20 plantations—located in central and southern areas on the Pacific side of Honshu, where the natural populations contain haplotypes D, E, and F, and where the climate is characterized by dry, cold winters—had been established using seedlings with haplotypes A and B derived from the Sea of Japan side of the Japanese Islands, where it snows heavily. The risks associated with planting seedlings of inappropriate lineages are discussed.     

Analysis of mitochondrial DNA of an endangered beech species,Fagus hayatae Palibin ex Hayata

Mitochondrial DNA restriction fragment length polymorphisms were used to examine cytoplasmic diversity within a relic-like population of Fagus hayatae, located in northern Taiwan. Fifteen trees were surveyed for three restriction endonucleases (BamHI, EcoRI and HindIII) and five mitochondrial gene probes (atpA, atp6, atp9, coxI and coxII). The analysis failed to reveal any polymorphisms, an observation that suggests cytoplasmic uniformity in the F. hayatae population examined. It is also interesting to note that the chondriome type of our F. hayatae samples is very close to that characteristic of F. crenata populations in the southernmost area of Japan.     

Population management: potential impacts of advances in genomics

Even with advanced gene technologies continued population improvement remains a key foundation for future genetic gain in forest trees. Currently, this is served by maintaining genetic diversity while capturing genetic improvement, often through structuring populations into a genetic hierarchy, setting population sizes, and managing pedigrees and inbreeding. In the future, information from genomic technologies will complement classical approaches, such as common-garden field experiments, for characterizing the genetic base and measuring and monitoring genetic diversity. This will entail directly measuring DNA sequence diversity of both selectively neutral markers and functional genes. Calibrating neutral marker diversity against functional diversity will become easier with functional genomics. For maintaining pedigree and managing inbreeding, genomic information can be used to relax some traditional tenets of population management. With future knowledge of functional polymorphisms, the better understanding of the nature and origins of genetic variation should enhance management of populations to both conserve genetic diversity and exploit it by more efficient selection. Where fungal diseases threaten biotic crises, very large populations may be needed, the requisite sizes often being very uncertain. Gene discovery holds enormous promise, but depends heavily on comparative genomics, capitalizing on genomic information from Arabidopsis, Populus and Eucalyptus, and the increasing numbers of accessible ESTs. Much greater insights into non-codon mutational processes and rates should also guide population management. A key challenge, however, will be to utilize information and apply tools cost-effectively. Also, very detailed genomic information, exemplified by the poplar-genome sequencing, may allow earlier adaptation of technology and development of new information in angiosperms than in gymnosperms.     

Origins and diversity of the Portuguese Landrace of Eucalyptus globulus

The Portuguese Landrace of Eucalyptus globulus is of unknown origin, with the earliest plantings of this tree species dating back to the early 19th century. In Portugal it is currently a major seed source for plantations and is also used in breeding programs. Eucalyptus globulus is native to south-eastern Australia. The substantial genetic differentiation of chloroplast and nuclear DNA markers between different native geographic races of this species allowed us to uncover the Australian origins of the Portuguese Landrace and to study its genetic diversity. To achieve this, we sequenced a highly polymorphic region of chloroplast DNA from 47 Portuguese Landrace individuals, and genotyped 34 of these using seven nuclear microsatellites. We compared these individuals to those in a database comprising chloroplast DNA sequence profiles from 292 native trees and seven nuclear microsatellites from 372 native trees. The majority of the Portuguese Landrace samples had closest affinities, in both marker systems, to native trees from south-eastern Tasmania, but some had affinities to trees from south-eastern Victoria. The discrepancies in the affinities indicated by chloroplast versus nuclear DNA markers could be explained by inter-race hybridisation after introduction. The genetic diversity in the Portuguese Landrace was less than that found in native E. globulus at the species level, but was similar to the average diversity found in native races of the species. This study demonstrates the power of using independent marker systems to identify the origins and diversity of domesticated populations, by comparison with variation in native stands.     

Genetic evidence for paternal inheritance of the chloroplast in four Australian Callitris species (Cupressaceae)

The mode of chloroplast inheritance was investigated in the four Australian conifer species; Callitris columellaris, C. intratropica, C. gracilis and C. verrucosa (Callitroideae, Cupressaceae), as a basis for future use of chloroplast genetic markers for the genus. Open-pollinated seeds and the foliage tissue of the mother trees were collected from one population per species, in which two distinct chloroplast haplotypes at the trnL (UAA) 5′ exon–trnF (GAA) region were co-distributed. In total, the DNA sequences of the chloroplast fragments in 255 samples were determined, and the haplotypes of the germinated seedlings and the mother trees were compared. Among the 16 mother trees examined across all species, 12 trees were shown to have produced at least one offspring with a different haplotype from the mother, indicating the occurrence of paternal inheritance of chloroplast in the fertilization processes. In binomial GLMM analyses, the haplotype of mother trees in C. columellaris, C. gracilis, and C. verrucosa did not significantly affect those of their offspring, and the predicted frequency of seedlings’ haplotypes was compatible with the observed frequency in adult canopy trees. These findings provide a genetic support that paternal inheritance of chloroplast DNA via pollen dispersal would be dominant in the Callitris species.     

Genetic diversity and population structure of Raffaelea quercus‐mongolicae,a fungus associated with oak mortality in South Korea

Raffaelea quercus‐mongolicae is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak (Quercus mongolica) in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of R. quercus‐mongolicae. Restriction‐site‐associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of R. quercus‐mongolicae in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus‐mongolicae isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle‐vectored fungus support the hypothesis that this fungus was introduced to South Korea.     

Interfertile oaks in an island environment: I. High nuclear genetic differentiation and high degree of chloroplast DNA sharing between <Emphasis Type="Italic">Q. alnifolia</Emphasis> and <Emphasis Type="Italic">Q. coccifera</Emphasis> in Cyprus. A multipopulation study

The evergreen Quercus alnifolia and Q. coccifera form the only interfertile pair of oak species growing in Cyprus. Hybridization between the two species has already been observed and studied morphologically. However, little evidence exists about the extent of genetic introgression. In the present study, we aimed to study the effects of introgressive hybridization mutually on both chloroplast and nuclear genomes. We sampled both pure and mixed populations of Q. alnifolia and Q. coccifera from several locations across their distribution area in Cyprus. We analyzed the genetic variation within and between species by conducting analysis of molecular variance (AMOVA) based on nuclear microsatellites. Population genetic structure and levels of admixture were studied by means of a Bayesian analysis (STRUCTURE simulation analysis). Chloroplast DNA microsatellites were used for a spatial analysis of genetic barriers. The main part of the nuclear genetic variation was explained by partition into species groups. High interspecific differentiation and low admixture of nuclear genomes, both in pure and mixed populations, support limited genetic introgression between Q. alnifolia and Q. coccifera in Cyprus. On the contrary, chloroplast DNA haplotypes were shared between the species and were locally structured suggesting cytoplasmic introgression. Occasional hybridization events followed by backcrossings with both parental species might lead to this pattern of genetic differentiation.     

Phylogenetic relationship and genetic differentiation of Populus caspica and Populus alba using cpDNA and ITS noncoding sequences

Populus caspica Bornm.(section Leuce and subsection Albida), one of the most endangered endemic tree species in the Hyrcanian Forest in Iran, has numerous morphological characteristics that are closely similar to Populus alba; to clarify their taxonomic relatedness and genetic differentiation and thus inform conservation strategies, we used the noncoding regions of chloroplast DNA(cpDNA; trnL-F and trnH-psbA) and the internal transcribed spacer(ITS). Leaf samples were collected from six populations across northern Iran. cpDNA and ITS fragments were amplified by universal primers using the PCR technique and directed sequencing. The results showed that P. caspica is genetically differentiated from P.alba, and two ITS variants were detected within some P.caspica individuals. Conflicts between topologies from ITS and plastid genomes were observed. High differentiation of P. caspica from the other Populus species shown in this study confirmed the diverging taxonomic status of this endangered species. We recommend in situ conservation measures(e.g., protected areas) for at least several populations of this species, especially in the plain regions of the Hyrcanian forest.     

Population genetic structure and ecological niche modelling of the leafhopper Hishimonus phycitis

Witches’ broom disease of lime, caused by ‘Candidatus Phytoplasma aurantifolia’, is responsible for major losses of Mexican lime trees in Southern Iran, Oman and the United Arab Emirates. The causative phytoplasma is transmitted by the leafhopper, Hishimonus phycitis. We combined ecological niche modelling with environmental and genetic data for six populations of H. phycitis from Iran and one from Oman. The mitochondrial cytochrome c oxidase I (COI) gene and nine microsatellite DNA markers were used for the genetic analyses. Although the Oman population had specific haplotypes, the COI sequences were highly conserved among all populations studied. In contrast, the microsatellite data divided the populations from Iran and Oman into two separate clades. An analysis of molecular variance indicated a high level of variation within populations. The Mantel test showed no correlation between genetic and geographical distances. Gene flow values were small between the populations from Iran and north of Oman but significantly higher among the Iranian populations supporting the differentiation between Iran and Oman. In addition, we found that patterns of genetic divergence within Iranian populations were associated strongly with divergence in terms of their ecological niches. Data on six climatic variables, including elevation, were used to create ecological niche models. Our results suggest that the genetic differentiation of H. phycitis may be attributable to climatic conditions and/or geographical barriers.     

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]     

Maternally and paternally inherited molecular markers elucidate population patterns and inferred dispersal processes on a small scale within a subalpine stand of Norway spruce (Picea abies [L.] Karst.)

The within-population spatial structure of genetic diversity is shaped by demographic processes, including historical accidents such as forest perturbations. Information drawn from the analysis of the spatial distribution of genetic diversity is therefore inherently linked to demographic-historical processes that ultimately determine the fate of populations.All adult trees and saplings in a 1.4-ha plot within a mixed Norway spruce (Picea abies [L.] Karst) stand were characterised by means of chloroplast (paternally inherited) markers, and a large sub-sample of these were genotyped at mitochondrial (maternally inherited) molecular markers. These data were used to analyse the spatial distribution of genetic variation and to compare the patterns corresponding to the two marker types. The plot presented non-homogeneous local stem density in the younger cohorts, and the indirect effect of this source of variation on the spatial genetic structure was investigated. Results suggest that (i) spatially limited seed dispersal induced patchiness in genotype distribution, while pollen flow had a homogenising effect; (ii) deviations from random spatial structure were stronger in the demographically most stable portions of the stand, while they were weaker where sudden bursts of regeneration occurred; (iii) spatially overlapping adult and sapling cohorts displayed the same spatial genetic structure (stronger on stable areas, weaker in portions of the stand undergoing events of intense regeneration), which was substantiated by the influence of local demographic processes. Regeneration dynamics as modulated by demography thus influences the distribution of genetic diversity within the stand both in the younger life stages and in the adult population.     

Polymorphism and heredity of cpDNA and mtDNA in the Section Leuce of <Emphasis Type="Italic">Populus</Emphasis>

The chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of 16 Populus species (Section Leuce) and their F1 generation were detected using PCR-RFLP technique. The results show that cpDNA in the F1 generation of 22 hybrid combinations was inherited maternally, which supported the conclusions of the study of plasmid cytology. The mtDNA fragments amplified by PCR were consistent with the restriction maps in all hybrid combinations and no polymorphism was detected, indicating that the Section Leuce is highly conserved in mitochondrial gene sequences. These results provided direct evidence of maternal chloroplast inheritance in Populus tomentosa, P. bolleana, P. davidiana, P. adenopoda, P. tomentosa × P. bolleana, P. alba × P. glandulosa and P. alba × P. tomentosa.     

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.     

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]