Ecological and genetic aspects of grape phylloxera Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) performance on rootstock hosts

Performance and genetic variability of clonal lineages derived from one Californian and one German population of grape phylloxera, Daktulosphaira vitifoliae Fitch were studied on their natal grape rootstock host and on three novel hosts over four generations in an aseptic dual culture system. The ability of D. vitifoliae to adapt to new hosts was measured by changes in fitness (rm) over four generations. The performance of a given clonal lineage changed over successive generations, depending upon the host plant and the phylloxera group. Analysis of amplified fragment length polymorphism-polymerase chain reaction (AFLP-PCR) banding patterns from 40 individual parthenogenetic D. vitifoliae revealed equal levels of genetic variation both among the four clonal lineages analysed and within the different generations of one lineage. Analysis of molecular variance (AMOVA) showed no significant differences between the D. vitifoliae lineages reared on different host plants, nor was a correlation between host performance and genotype found.     

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insect's biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.     

An in vitro assessment of phylloxera (Daktulosphaira vitifoliae Fitch) (Hom., Phylloxeridae) life cycle

Phylloxera single founder lineages were maintained in an aseptic, dual-culture system to investigate the phylloxera life cycle, focusing on holocycle induction. Holocyclic morphs were developed on the grape rootstock 41B and they produced vital sexuales. The morphological development of sexual stages was studied and an assessment of genetic variation within a single founder lineage was performed. Twenty individual phylloxera of differing stages (apterous parthenogenetic, pre-sexual nymphs, sexuparae and sexuals) were genetically fingerprinted using amplified fragment length polymorphism-polymerase chain reaction technology. Four individuals displayed differing banding patterns, exhibiting three different independent genotypes. No influence of the morphal stage on these patterns could be detected. This study did not achieve phylloxera matings. However, the system could serve as a tool for future studies, performing controlled and systematic crosses and back-crosses to examine Mendelian genetics.     

Fine-scale genetic structure of grape phylloxera from the roots and leaves of Vitis

Patterns of variation at microsatellite loci suggest that root populations of the pest grape phylloxera (Daktulosphaira vitifoliae) are largely parthenogenetic in Australian vineyards. To investigate reproduction in leaf galling phylloxera and the association between these individuals and phylloxera on roots, we examined in detail genetic variation in phylloxera from a vineyard block. Some genotypes found on leaf galls within this block were not present on roots, whereas others spanned both zones. There was no evidence that genotypes on roots were the product of sexual reproduction in leaf galls. mtDNA variation was not associated with the location of the phylloxera clones. The spatial distribution of genotypes within a root population was further investigated by intensively sampling phylloxera from another vineyard block. Join-count spatial autocorrelation statistics were used to explore fine-scale spatial structure. Clones were nonrandomly distributed within the block and there was evidence that the distribution of clones followed rows. These findings suggest firstly that there is limited dispersal of root and leaf feeding phylloxera, and secondly that factors, other than vine host, are likely to be important and contribute to clonal structure within populations.     

Dynamic grapevine clones��an AFLP-marker study of the Vitis vinifera cultivar Riesling comprising 86 clones

Grapevine cultivars are planted in worldwide viticulture and are asexually propagated. Horticultural clones are asexually derived from a single individual, and clonal variation can only occur through mutations. Molecular markers are an important tool for the differentiation and identification of clones and mutations. For breeding purposes and clonal selection, knowledge upon the variability of a given clone is essential. The aim of this study was to assess amplified fragment length polymorphism (AFLP) markers for classifying mutations in 86 Riesling clones of Vitis vinifera and to enhance our understanding on the dynamic of grapevine clones analysed by AFLP fingerprints. AFLP markers detected 135 polymorphic bands of a total amount of 305 bands. AFLP markers detected two different types of mutations: single-event mutations, only detected once in one clone, displaying the variation of the grape genome and specific loci mutations where the mutation could be found frequently in the set of clones and therefore stand for the stability of grapevine genome. A general grouping of clones according to age, sub-clonal lineage or origin could not be determined by the set of AFLP markers employed.     

Genetic diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age

Perennial grasses constitute a major group of species showing a dramatic decline of biodiversity in successional plant communities. Using AFLP markers, we examined 12 populations of the expansive grass Brachypodium pinnatum differing in habitat age (30–50, ca. 100 and >300 years old) in order to determine whether clonal diversity of populations, genetic variation, and the relative importance of clonal propagation versus sexual reproduction change with grassland age. Five AFLP primer combinations gave a total of 517 bands, 79% of which were polymorphic. 314 different multilocus lineages were distinguished among the 453 samples analyzed. The number of genotypes (G) and clonal richness (R) decreased with habitat age, while the distribution of the frequency of genets changed from many clones of similar size to dominance by one or a few large clones. We consider these results to give evidence of significant role of sexual reproduction in the early phases of colonization and prevalence of clonal growth and competitive exclusion of less adapted genotypes in the later ones. However, habitat age had only marginal effect on genetic diversity, as percentage of polymorphic loci (PPL) within all the populations analyzed was similar, viz. 38.6–43.5%.     

Impact of multispores in vitro subcultivation of Glomus sp. MUCL 43194 (DAOM 197198) on vegetative compatibility and genetic diversity detected by AFLP

Vegetative compatibility and amplified fragment length polymorphism (AFLP) genotyping of in vitro multispores clonal lineages, issued from the same ancestor culture of the arbuscular mycorrhizal fungal strain MUCL 43194 and subcultured several generations in different locations, was assessed. Vegetative compatibility was studied by confronting the germ tubes of two spores from the same or different clonal lineages and stained with nitrotetrazolium blue–Trypan blue and diamidinophenylindole to detect hyphal fusions and nuclei, respectively. Further AFLP analysis of single spores was performed to assess the genetic profile and Dice similarity between clonal lineages. Germ tubes of spores distant by as many as 69 generations were capable of fusing. The anastomosis frequencies averaged 69% between spores from the same clonal lineage, 57% between spores from different clonal lineages, and 0% between spores belonging to different strains. The AFLP patterns showed similarities averaging 92% within clonal lineages and 86% between clonal lineages. Each spore presented unique genotype and some of them shared more markers with spores from different lineages than within the same lineage. We showed that MUCL 43194 maintained self-recognition for long periods of subcultures in vitro and that spores involved in compatibility tests had different genotypes. Our findings suggest that MUCL 43194 maintains genetic diversity by means of anastomoses.     

Identification of new polymorphic microsatellite markers in the NA1 and NA2 lineages of Phytophthora ramorum

Phytophthora ramorum is a recently introduced pathogen in Europe and North America consisting of three clonal lineages. Due to the limited intralineage genetic variation, only a few polymorphic markers are available for use in studies involving the epidemiology and evolution of P. ramorum. A total of 159 primer pairs for candidate polymorphic SSR loci were tested with universal labeling. Four polymorphic microsatellite loci were identified within the NA1 lineage and one within the NA2 lineage, demonstrating the power and flexibility of the screening technique. The markers may significantly increase the number of genotypes that can be identified and as such can help better characterize the North American lineages of P. ramorum.     

Variation in performance on two grape cultivars within and among populations of grape Phylloxera from wild and cultivated habitats

When an indigenous insect becomes a pest, comparisons of performance of pest and non-pest populations on crop plants and of genetic variation in that performance may provide insight into the evolution of pest populations. To measure such genetic variation, 8–15 clones of the grape phylloxera (Daktulosphaira vitifoliae Fitch) were collected from wild grapevines in each of 3 geographically isolated sites (populations) and from commercial vineyards in northern California. A complete life table was made for clonal replicates from populations collected from wild grapevines on each of two commercial grape cultivars, the susceptibleVitis vinifera (L.) cultivar Cabernet Sauvignon, and the phylloxera-resistant rootstock ‘AxR # 1’. Variation in mean performance on these two hosts was partitioned among clones within collection sites and among sites. Performance measures included an individual analog to the intrinsic rate of increase (r), age at first oviposition, fecundity in the first ten days of reproduction, total fecundity, and longevity. The overall performance of phylloxera from the wild grapevines on the resistant cultivar AxR # 1 was greater than or equal to that on the susceptible cultivar Cabernet Sauvignon. There was significant variation among clones within populations from wild grapes in the rate of increase on ‘AxR # 1’ and marginally significant clonal variation in some of the component paramters. There was no significant variation among clones within populations on ‘Cabernet Sauvignon’ and no significant differences between populations on either crop in any trait. In a second experiment we compared the relative performance of 15–17 clones from wild grapevines and from commercial vineyards when reared on ‘Cabernet Sauvignon’ and ‘AxR # 1’. Phylloxera from commercial vineyards had much higher overall performance on ‘Cabernet Sauvignon’ than did phylloxera from the wild grapevines. Phylloxera from the commercial vineyard also had higher performance on ‘Cabernet Sauvignon’ than on ‘AxR′ 1’ but the performance of the phylloxera from wild and commercial grapes did not differ on ‘AxR # 1’. Our results show that there is genetic variation in traits related to performance on a resistant rootstock within these indigenous non-pest populations of phylloxera, but not among them. The pattern of performance of pest and non-pest populations on two commercial cultivars suggests that current levels of phylloxera performance on crop cultivars are the result of adaptation to those cultivars which has occurred while phylloxera has been associated with viticulture. Implications of these results for understanding the recent adaptation of phylloxera to ‘AxR # 1’ in California are also discussed.     

Genetic diversity of Chinese alligator (Alligator sinensis) revealed by AFLP analysis: an implication on the management of captive conservation

Chinese alligator (Alligator sinensis) is one of the most critically endangered species among 23 extant crocodiles in the world. To prevent the extinction of the species, a captive propagation started at early 1980s, and the total number of alligator was brought up to 10 thousands from dozens of founder in 2000. But several genetic investigations showed those alligators were under an extremely low genetic diversity status with few detectible polymorphic loci. To get more insight into its genetic diversity for the management of captive Chinese alligator, AFLP was adopted to characterize variations in the population. Total of 347 bands were generated from 47 individuals using 3 primer combinations, of which 203 (58.50%) were polymorphic, and 35 AFLP phenotypes were revealed from those individuals. Comparing the results between RAPD and AFLP analysis on almost same sample set clearly indicated that AFLP is more efficient in revealing polymorphic loci, especially in those populations with extremely low genetic diversity. In present three assays, electrophoresis profile also displayed 3 individuals possessing very highly polymorphic AFLP phenotypes that were never been found by RAPD and mtDNA D-loop sequencing, implicating that we should offer these individuals more breeding opportunities to maintain the genetic diversity in the population and restrict those carrying few polymorphic loci from reproduction.     

Genetic variation of Avicennia marina (Forsk.) Vierh. (Avicenniaceae) in Vietnam revealed by microsatellite and AFLP markers

Genetic variation of Avicennia marina in the costal area of Vietnam was examined using microsatellite and AFLP markers. By using five microsatellite loci a total of 21 alleles were detected. The average number of alleles per locus per population ranged from 1.667 to 3.000. The observed heterozygosity varied from 0.180 to 0.263, with an average of 0.210 indicating relatively low level of genetic variation comparing to the previous studies on A. marina in the worldwide range. The expected heterozygosity was larger than the observed heterozygosity leading to positive inbreeding coefficients in all the six populations. Highly significant departures from Hardy-Weinberg Equilibrium were detected in four populations. AFLP analysis revealed a total of 386 loci, of which 232 (60.1%) were polymorphic. In congruent with microsatellite markers relatively low levels of genetic variation were detected at both gene and nucleotide levels (H = 0.086; pi = 0.0054). Reduced level of genetic variation was found in the central population, and in the southern populations. Both microsatellite and AFLP markers revealed large genetic differentiation (F(ST) = 0.262 and 0.338, respectively) indicating strong genetic structure among regional populations. Pairwise genetic distance by AFLP showed two populations in the north and the other two in the south are closely related each other.     

吡虫啉对褐飞虱DNA甲基化多态性的影响

为了探知基因组甲基化是否参与了昆虫抗药性, 本研究在室内对褐飞虱 Nilaparvata lugens连续9个世代的3龄若虫施用吡虫啉, 用AFLP检测褐飞虱抗性产生过程中DNA甲基化多态性的变化。利用25对AFLP引物共获得120个位点, 其中15个位点呈现甲基化多态性, 共获得78条多态性条带。根据多态性条带在不同世代样本中出现的多少计算多态性条带比例, 其中最高比例出现在G5代（10.26%）, 最低比例出现在G6代（1.28%）。多态性条带在不同世代间比例的变化趋势表明, 褐飞虱对吡虫啉的筛选产生快速应答。在筛选早期（G1, G2和G3）世代间DNA甲基化多态性比例差异相对较小, 变化范围在3.85%～6.41%之间; 在筛选中期（G4, G5和G6）世代间比例差异较大, 变化范围在1.28%～10.26%之间; 在筛选后期（G7, G8和G9）世代间比例差异相对较小, 变化范围在5.13%～7.69%之间。结果说明, 吡虫啉的连续施用能够诱导褐飞虱基因组产生甲基化变异, 初步揭示甲基化在褐飞虱抗药性产生过程中参与了基因组的表达调控。     

Molecular evidence for a founder effect in invasive house finch (Carpodacus mexicanus) populations experiencing an emergent disease epidemic

The impact of founder events on levels of genetic variation in natural populations remains a topic of significant interest. Well-documented introductions provide a valuable opportunity to examine how founder events influence genetic diversity in invasive species. House finches (Carpodacus mexicanus) are passerine birds native to western North America, with the large eastern North American population derived from a small number of captive individuals released in the 1940s. Previous comparisons using amplified fragment length polymorphism (AFLP) markers found equivalent levels of diversity in eastern and western populations, suggesting that any genetic effects of the founder event were ameliorated by the rapid growth of the newly established population. We used an alternative marker system, 10 highly polymorphic microsatellites, to compare levels of genetic diversity between four native and five introduced house finch populations. In contrast to the AFLP comparisons, we found significantly lower allelic richness and heterozygosity in introduced populations across all loci. Three out of five introduced populations showed significant reductions in the ratio of the number of alleles to the allele size range, a within-population characteristic of recent bottlenecks. Finally, native and introduced populations showed significant pairwise differences in allele frequencies in every case, with stronger isolation by distance within the introduced than native range. Overall, our results provide compelling molecular evidence for a founder effect during the introduction of eastern house finches that reduced diversity levels at polymorphic microsatellite loci and may have contributed to the emergence of the Mycoplasma epidemic which recently swept the eastern range of this species.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Population genetic structure of an aquatic herb Batrachium bungei (Ranuculaceae) in the Hengduan Mountains of China

Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic variation of 14 populations of Batrachium bungei (Ranunculaceae) from the Hengduan Mountains of China, which is a global biodiversity region that is poorly studied. A total of 75 bands with 52 polymorphic loci were generated from the ten primers used in this study. A total of 155 different genotypes or clones were identified among the 442 samples. A low level of genetic diversity within population and high genetic differentiation among populations were revealed in this study. Factors, such as inbreeding, clonal growth, bottlenecks, population isolation and founder events during postglacial recolonizations, might have played important roles in shaping the population structure of B. bungei.     

Characterization of a genetic resource collection for Miscanthus (Saccharinae,Andropogoneae, Poaceae) using AFLP and ISSR PCR

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat markers were employed to characterize a genetic resource collection of Miscanthus, a grass under trial in Europe as a biomass crop. The 26 polymorphic markers produced by two ISSR fingerprinting primers were able to discriminate taxa and identify putative clones. AFLP fingerprints were fully reproducible and produced a larger number of markers for the three primer pairs tested, of which 998 were polymorphic (representing 79.3% of all bands). AFLP markers distinguished species, infra-specific taxa (varieties and cultivars) and putatively clonal material. They were also used to assess the inter-relationships of the taxa, to investigate the origin of important hybrid plants and to estimate the overall level of genetic variation in the collection. They were useful for assessing the species status of certain taxa such as M. transmorrisonensis, an endemic from Taiwan that was clearly distinct from M. sinensis; whereas other taxa of disputed species status, such as M. condensatus and M. yakushimanum were not genetically distinct from M. sinensis. The AFLP markers detected a high degree of infra-specific variation and allowed subdivisions of the genetic resource collection to be made, particularly within M. sinensis.     

Grape phylloxera (Daktulosphaira vitifoliae) – a review of potential detection and alternative management options

The management options for grape phylloxera, Daktulosphaira vitifoliae, a monophagous insect pest of Vitis species are reviewed. Although in a worldwide context, grape phylloxera is managed predominantly by the use of resistant rootstocks developed through conventional breeding of hybrid crosses of American Vitis species, this management aspect is largely excluded from the review so that emerging technologies in the field of detection, quarantine and alternative management are discussed. In some viticulture regions of the world, where grape phylloxera's geographic distribution is limited (e.g. Australia), the pest is managed through a combination of surveillance, detection and quarantine. Although some alternative management options for grape phylloxera exist they have received relatively limited research attention because of the relative success of resistant rootstocks. The resilience of resistant rootstocks as the primary management option could also be challenged in the future by host‐plant interactions with diverse grape phylloxera clonal lineages and by potential impacts of climate change on both grapevine and grape phylloxera distribution. A range of control options exist which could be integrated into an improved management system for grape phylloxera. Priority areas for future evaluation and further development include early detection techniques, investigation into the use of biological control agents and development of an integrated approach to grapevine phylloxera management.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Development of microsatellite primers of the largest seagrass, Enhalus acoroides (Hydrocharitaceae)

? Premise of the study: Microsatellite primers were developed for the seagrass Enhalus acoroides to investigate genetic variation and identify clonal structure. ? Methods and Results: Four polymorphic loci and 32 monomorphic loci were developed in E. acoroides. Two to four alleles per locus were observed at the polymorphic loci across 60 individuals of two E. acoroides populations. The observed and expected heterozygosities within populations ranged from 0.100 to 0.5667 and from 0.0977 to 0.5079, respectively. ? Conclusions: Our study revealed very low polymorphism in E. acoroides, even at the polymorphic loci. Nevertheless, these primers are a useful tool to study genetic variation, clonal structure, and mating system.     

Genome wide AFLP markers support cryptic species in Coniophora (Boletales)

Numerous fungal morphospecies include cryptic species that routinely are detected by sequencing a few unlinked DNA loci. However, whether the patterns observed by multi-locus sequencing are compatible with genome wide data, such as amplified fragment length polymorphisms (AFLPs), is not well known for fungi. In this study we compared the ability of three DNA loci and AFLP data to discern between cryptic fungal lineages in the three morphospecies Coniophora olivacea, Coniophora arida, and Coniophora puteana. The sequences and the AFLP data were highly congruent in delimiting the morphotaxa into multiple cryptic species. However, while the DNA sequences indicated introgression or hybridization between some of the cryptic lineages the AFLP data did not. We conclude that as few as three polymorphic DNA loci was sufficient to recognize cryptic lineages within the studied Coniophora taxa. However, based on analyses of a few (three) sequenced loci the hybridization could not easily be distinguished from incomplete lineage sorting. Hence, great caution should be taken when concluding about hybridization based on data from just a few loci.