Populationsgenetische Fragmentanalyse von europäischen Heterobasidion annosum s. s.-Stämmen

Die fünf Arten des Heterobasidion annosum s. l.-Komplexes sind in Nordamerika und Eurasien dafür bekannt große Schäden in Nadelforsten zu verursachen. In Deutschland sind mit H. annsosum s. s., H. parviporum und H. abietinum alle drei eurasischen Weißfäuleerreger sympatrisch vertreten. Besonders in der Norddeutschen Tiefebene fallen H. annsoums s. s.-Stämme durch ansteigende Befallsherde und hohe Pathogenität auf. Seit Jahren untersucht die Nordwestdeutsche forstliche Versuchsanstalt (NW-FVA) Göttingen die Zusammenhänge der sogenannte „Ackersterbe“ in einzelnen Regionen. In diesem Zusammenhang sollte die vorliegende Arbeit den molekular-genetischen Aspekt behandeln und die inner- und zwischenartlichen Strukturen sowie das Beziehungsgefüge von H. annosum s. s. und H. parviporum-Individuen aus 18 europäischen Ländern darstellen. Für die molekularen Untersuchungen wurde die AFLP-Analyse (amplified fragment length polymorphism) ausgewählt. Die Vorteile der AFLP liegen in der großen Anzahl polymorpher und selektionsneutraler Marker, der guten Reproduzierbarkeit und der damit verbundenen Robustheit dieser Methode. Basierend auf acht AFLP-Primerpaarkombinationen mit 209 Individuen wurde eine binäre Matrix erstellt. Diese bildete die Grundlage für die molekulargenetischen Analysen und folgende Fragestellungen: Können die Heterobasidion-Individuen in Netzwerk- und Dendrogramm-Analysen den herkunftsspezifischen Populationen zugeordnet werden und lässt sich ein geografisches Muster erkennen? Wie groß ist das Ausmaß der genetischen Diversität innerhalb und zwischen den untersuchten europäischen Heterobasidion annosum s. l.-Populationen? Ist das gesteigerte Schadbild in der norddeutschen Tiefebene auf eine mögliche invasive Heterobasidion-Art zurückzuführen? Wie sieht die Populationsstruktur von Heterobasidion annosum s. l. in Europa bzw. Deutschland aus und gibt es Hinweise auf Subpopulationen oder Hybridisierungsereignisse? Die von der AFLP-Analyse erzeugten 888 informativen Marker wurden in Neighbor-Joining- und UPGMA-Phänogrammen, Median-joining Netzwerk und einem Principal coordinates analysis (PCoA)-Koordinatensystem dargestellt. Übereinstimmend zeigten sich die Arten H. annosum und H. parviporum eindeutig distinkt. Die Boostrapunterstützung der meisten H. annosum s. s.-Individuen erwies sich jedoch innerartlich als überwiegend schwach. Häufig kam es zu Gruppierungen der Individuen gemäß ihrer regionalen Herkunft. Seltener bildeten die Populationen gemeinsame Cluster entsprechend ihrer Länderzugehörigkeit. Eine zonale Gruppierungsstruktur der Individuen nach Süd-, Mittel-, und Nordeuropa konnte in keiner Analyse beobachtet werden. Mit der Analysis of Molecular Variance (AMOVA) wurde mit der AFLP-Methode eine genetische Varianz zwischen den beiden untersuchten Arten von 72 % nachgewiesen. Intraspezifisch ließ H. annosum s. s eine sehr hohe genetische Varianz innerhalb der Populationen (73%) und eine geringe Differenzierung zwischen diesen erkennen. Lediglich die deutschen Populationen zeigten eine gewisse Abgrenzung zueinander (33%). Als Ursache für die hohe individuelle genetische Vielfalt wird zum Einen das dichte Vorkommen der H. annosum s. s.- Wirte in Europa angesehen. Darüber hinaus sind gebietsfremde Sporeneinträge durch überregionale Forstarbeiten, globale Holzimporte und die für H. annosum s. s. nachgewiesenen weiten Sporenflüge zu erklären. Das Populationsannahmemodell von STRUCTURE generierte für den Datensatz beider Arten eine optimale Populationsanzahl von ΔK=4. Drei der Subpopulationen wurden H. annosum s. s. zugeordnet. Eine Korrelation dieser genetischen Cluster mit ihrem geografischen Ursprung oder der Wirtsbaumart war nicht feststellbar. Ein möglicher Zusammenhang der Subpopulationen mit weiteren ökologischen Parametern wie z. B. Substratgrundlage, Pathogenität, Sporulationsverhalten und sich änderte klimatische Umweltbedingungen konnten in dieser Studie nicht untersucht werden. Des Weiteren zeigte die STRUCTURE-Analyse, dass einige H. parviorum-Individuen Anteile eines H. annosum s. s.-Clusters führten. Um zu klären, ob es sich hierbei um einen erebten und konservierten Polymorphisums, oder um Introgression handelt, wären weiterführende Analysen notwendig.

Friends or relatives? Phylogenetics and species delimitation in the controversial European orchid Genus ophrys

Background and Aims Highly variable, yet possibly convergent, morphology and lack of sequence variation have severely hindered production of a robust phylogenetic framework for the genus Ophrys. The aim of this study is to produce this framework as a basis for more rigorous species delimitation and conservation recommendations. Methods Nuclear and plastid DNA sequencing and amplified fragment length polymorphism (AFLP) were performed on 85 accessions of Ophrys, spanning the full range of species aggregates currently recognized. Data were analysed using a combination of parsimony and Bayesian tree-building techniques and by principal coordinates analysis. Key Results Complementary phylogenetic analyses and ordinations using nuclear, plastid and AFLP datasets identify ten genetically distinct groups (six robust) within the genus that may in turn be grouped into three sections (treated as subgenera by some authors). Additionally, genetic evidence is provided for a close relationship between the O. tenthredinifera, O. bombyliflora and O. speculum groups. The combination of these analytical techniques provides new insights into Ophrys systematics, notably recognition of the novel O. umbilicata group. Conclusions Heterogeneous copies of the nuclear ITS region show that some putative Ophrys species arose through hybridization rather than divergent speciation. The supposedly highly specific pseudocopulatory pollination syndrome of Ophrys is demonstrably 'leaky', suggesting that the genus has been substantially over-divided at the species level.

Intercontinental dispersal prior to human translocation revealed in a cryptogenic invasive tree

In this study, complementary species-level and intraspecific phylogenies were used to better circumscribe the original native range and history of translocation of the invasive tree Parkinsonia aculeata. Species-level phylogenies were reconstructed using three chloroplast gene regions, and amplified fragment length polymorphism (AFLP) markers were used to reconstruct the intraspecific phylogeny. Together, these phylogenies revealed the timescale of transcontinental lineage divergence and the likely source of recent introductions of the invasive. The sequence data showed that divergence between North American and Argentinean P. aculeata occurred at least 5.7 million years ago, refuting previous hypotheses of recent dispersal between North and South America. AFLP phylogenies revealed the most likely sources of naturalized populations. The AFLP data also identified putatively introgressed plants, underlining the importance of wide sampling of AFLPs and of comparison with uniparentally inherited marker data when investigating hybridizing groups. Although P. aculeata has generally been considered North American, these data show that the original native range of P. aculeata included South America; recent introductions to Africa and Australia are most likely to have occurred from South American populations.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn(-)) for utilization of methyl phosphonate (MePn) and that Phn(+) variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn(+) variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn(+) revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn(+) and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

Mapping quantitative trait loci for flag leave senescence as a yield determinant in winter wheat under optimal and drought stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

Adaptive divergence and speciation among sexual and pseudoviviparous populations of Festuca

Pseudovivipary is an environmentally induced flowering abnormality in which vegetative shoots replace seminiferous (sexual) inflorescences. Pseudovivipary is usually retained in transplantation experiments, indicating that the trait is not solely induced by the growing environment. Pseudovivipary is the defining characteristic of Festuca vivipara, and arguably the only feature separating this species from its closest seminiferous relative, Festuca ovina. We performed phylogenetic and population genetic analysis on sympatric F. ovina and F. vivipara samples to establish whether pseudovivipary is an adaptive trait that accurately defines the separation of genetically distinct Festuca species. Chloroplast and nuclear marker-based analyses revealed that variation at a geographical level can exceed that between F. vivipara and F. ovina. We deduced that F. vivipara is a recent species that frequently arises independently within F. ovina populations and has not accumulated significant genetic differentiation from its progenitor. We inferred local gene flow between the species. We identified one amplified fragment length polymorphism marker that may be linked to a pseudovivipary-related region of the genome, and several other markers provide evidence of regional local adaptation in Festuca populations. We conclude that F. vivipara can only be appropriately recognized as a morphologically and ecologically distinct species; it lacks genetic differentiation from its relatives. This is the first report of a ‘failure in normal flowering development’ that repeatedly appears to be adaptive, such that the trait responsible for species recognition constantly reappears on a local basis.

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stressresistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

Genetic diversity and specialisation of Eudarluca caricis on some graminaceous Puccinia species

Eudarluca caricis is a common hyperparasite of rusts. A total of 100 cultures were isolated from six Puccinia species or forms growing on 10 species of British grasses at two sites approximately 3 km apart. 82 isolates collected in 2005 were partially sequenced at the ITS locus, and amplified fragment length polymorphism profiles generated for 86 isolates from 2005 and 12 from 2007. Partial ITS sequences of most isolates grouped closely, in a clade with previously reported graminaceous Puccinia isolates and a number of Melampsora isolates. A second clade was very distinct and contained mostly isolates from P. poarum on Poa trivialis. All isolates had distinct AFLP haplotypes. The P. poarum isolates were very distinct from isolates collected from other rusts at the same site. Isolates from P. brachypodii f. sp. arrehenatheri growing on Arrhenatherum elatius in 2005 and 2007 at the same location were distinct (P < 0.001). Isolates from each rust or grass in one year and site were more similar than expected from overall variation between isolates (P<0.001). Isolates from P. coronata on different grasses clustered together (with isolates from P. brachypodii f. sp. poae-nemoralis), suggesting partial host rust specialisation in E. caricis.

Genética e mapeamento molecular da resistência parcial à ferrugem da folha da aveia (Avena sativa L.)

A ferrugem da folha é a moléstia de maior importância econômica para a cultura da aveia e a resistência qualitativa, geralmente utilizada para o seu controle, apresenta pouca durabilidade. A utilização de resistência parcial, caracterizada pelo progresso lento da moléstia, tem sido reconhecida como alternativa para obtenção de genótipos com resistência mais durável. Os objetivos deste trabalho foram determinar o progresso da ferrugem, o controle genético da resistência, e identificar marcadores moleculares associados a essa resistência, em várias gerações e anos. Populações F2, F3, F4, F5 e F6 do cruzamento UFRGS7/UFRGS910906 (sucetível/parcialmente resistente) (1998, 1999 e 2000) e F2 do cruzamento UFRGS7/UFRGS922003 (1998), foram avaliadas a campo quanto à porcentagem de área foliar infectada, para determinar a área sob a curva do progresso da doença (ASCPD). Mapeamento molecular, com marcadores AFLP (amplified fragment length polymorphism), foi realizado em F2 e F6, do primeiro cruzamento, identificando marcadores associados à resistência quantitativa (“quantitative resistance loci” ou QRLs). Resultados de três anos evidenciaram alta influência do ambiente na expressão da resistência, apresentando, entretanto, variabilidade genética para resistência parcial nas populações segregantes. A distribuição de freqüências do caráter ASCPD nas linhas recombinantes F5 e F6 foi contínua, indicando a presença de vários genes de pequeno efeito em seu controle. Estimativas de herdabilidade variaram de moderada a alta. O mapa molecular F2 foi construído com 250 marcadores, em 37 grupos de ligação, e o mapa F6 com 86 marcadores em 17 grupos de ligação. Cinco QRLs foram identificados na F2 e três na F6. O QRL identificado na F6, pelo marcador PaaMtt340 apresentou consistência em dois ambientes.

Caracterização fenotípica e genotípica de isolados de Ornithobacterium rhinotracheale do Brasil.

Ornithobacterium rhinotracheale é uma bactéria associada com doença respiratória, decréscimo no crescimento, condenação de carcaças e mortalidade em galinhas e perus. Esta bactéria tem sido isolada em vários países e, recentemente, foi isolada no Brasil pelo nosso grupo que também estabeleceu um protocolo de reação em cadeia da polimerase (PCR) para a sua detecção e identificação e determinou a prevalência de anticorpos contra esta bactéria em plantéis comerciais de frangos e de matrizes da Região Sul do Brasil. O presente trabalho teve o objetivo de caracterizar isolados de O. rhinotracheale através de sorotipificação, resistência a antimicrobianos e single-enzyme amplified fragment length polymorphism (SAFLP). Vinte e sete isolados foram compatíveis com esta espécie através de isolamento em ágar sangue com gentamicina, coloração de Gram, teste de aglutinação em lâmina e reação em cadeia da polimerase. Dezenove isolados foram classificados como sorotipo A, seis não puderam ser sorotipificados com o painel de soros existentes e dois pertenceram ao sorotipo C. Vinte e cinco isolados foram sensíveis à norfloxacina, amoxicilina, doxiciclina, lincomicina e cefalotina, dois isolados foram resistentes à neomicina e 18 foram resistentes à sulfametoxazol/trimetoprima. Na análise de SAFLP, 22 isolados apresentaram padrão idêntico e os cinco isolados restantes foram classificados em cinco padrões distintos. Os resultados da sorotipificação indicaram que o sorotipo A de O. rhinotracheale é predominante em criações comerciais no Brasil. Os isolados brasileiros foram sensíveis à maioria dos antimicrobianos testados. O poder discriminatório do teste de suscetibilidade a antimicrobianos foi maior do que a sorotipificação e SAFLP, porém o método de SAFLP gerou um maior número de padrões, sugerindo que possa ser utilizado como ferramenta em estudos epidemiológicos.

Pathogenicity, colony morphology and diversity of isolates of Guignardia citricarpa and G-mangiferae isolated from Citrus spp.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização molecular através da técnica fAFLP de isolados de Diaporthe citri

A citricultura é um mercado em expansão, principalmente no Estado de São Paulo, cuja importância na balança comercial já é reconhecida. Como em qualquer espécie cultivada, o crescimento das áreas de cultivo favorecem também o crescimento de problemas fitossanitários. Desta forma, as espécies de citros são afetadas por diversas doenças destacando-se entre elas a melanose, causada por Diaporthe citri (Wolf.), à qual a grande maioria das variedades comerciais são suscetíveis. O conhecimento da diversidade intra-específica é de grande importância, já que esta poderá auxiliar na seleção de variedades com resistência. O objetivo deste trabalho foi avaliar a variabilidade genética em isolados de Diaporthe citri, originários de diferentes locais, variedades e partes da planta, utilizando marcadores moleculares. Marcadores do tipo AFLP (Amplified Fragment Length Polymorphism) foram utilizados para caracterização de dez isolados do patógeno. Os DNAs genômicos extraídos da massa micelial foram utilizados nas reações de amplificação. A técnica fluorescent AFLP permitiu a distinção dos isolados estudados, tendo sido classificados em quatro grupos distintos. Contudo, estes grupos não foram formados em razão da região geográfica, parte da planta ou variedade.

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis, Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.

Genetic relationships among Arachis species based on AFLP

Amplified Fragment Length Polymorphism (AFLP) was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6% denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI - ACC / Mse I - CTG, while the lowest was 108, with Eco RI - ACT / Mse I - CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species.