Microsatellite DNA markers for Plasmopara viticola,the causal agent of downy mildew of grapes

Microsatellite loci were isolated from Plasmopara viticola (Oomycetes), the causal agent of downy mildew of grape, one of the most damaging fungal diseases of grapevine worldwide. Seven polymorphic loci were obtained from an enriched partial genomic library. A low genetic diversity was observed at all loci, with a mean observed allele number of 3.75 and an observed heterozygosity ranging from 0.074 to 0.547. Cross‐amplification tests on three closely related taxa indicated that two loci could be used in other Oomycetes species. These microsatellite loci were proved to be useful for population genetic analysis.     

Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.     

Glasshouse evaluation of fungicides for the control of sunflower downy mildew (Plasmopara halstedii)

A precise, reproducible and easy-to-handle glasshouse test is described for the evaluation of the systemic activity of chemicals for the control of Plasmopara halstedii, the downy mildew pathogen of sunflower. Four-day-old sunflower germlings were inoculated by immersing them in a zoosporangium suspension. Seedlings were then immersed in appropriate concentrations of the chemicals to be tested. Plants were grown in a glasshouse and assessed on three occasions to determine successively antisporulant, curative (systemic fungistatic), and eradicant effects. Sporulation in general was inhibited by lower concentrations than those required to exert an eradicant effect. There was a highly significant correlation between the ED₅₀ values for visually recognised disease symptoms (stunting, dampingsff and leaf chlorosis) and for both curative and eradicant effects. Among 13 compounds tested, metalaxyl, RE 26745, furalaxyl, LAB 149202F and cymoxanil showed sufficient eradicant activity, to justify field evaluation for eradication of seed infections.     

Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virions†

Eight pathotypes of Plasmopara halstedii were screened to investigate the occurrence of virions and the potential viral influence on the pathogenicity of the sunflower downy mildew pathogen. In 23 of 26 P. halstedii isolates derived from eight countries in Europe, North America and South America, virions were detected by transmission electron microscopy. By contrast, there were no ultrastructural indications of virus‐like particles in eight other related Oomycetes. The virions of representative P. halstedii isolates were morphologically and biochemically characterized and compared among each other. Regardless of their host's pathotypes, the geographical origin of the isolate and the sensitivity towards the fungicide metalaxyl, the viral characters obtained were uniform. The virions were isometric and measured approximately 37 nm in diameter. One polypeptide of c. 36 kDa and two segments of single‐stranded RNA (3.0 and 1.6 kb) were detected. Both viral RNA segments were detected by capillary electrophoresis in the three remaining P. halstedii isolates where virions were undetectable by transmission electron microscopy. Virus‐specific primers for the 1.6 kb‐segment were synthesized and used to determine and compare a partial sequence of the viral coat protein among virions of different P. halstedii pathotypes. In all tested isolates, fragments of 0.7 kb were amplified which were directly sequenced. Sequence variation was insignificant. As both less aggressive and more aggressive P. halstedii isolates contained virions, the presence or absence of virions could not explain the diverse aggressiveness of the downy mildew pathogen towards sunflower. Moreover, the results indicated that pathogenicity of P. halstedii was not related to variation in morphological or biochemical characters of the virions.     

Positional cloning of a candidate gene for resistance to the sunflower downy mildew, Plasmopara halstedii race 300

The resistance of sunflower to Plasmopara halstedii is conferred by major resistance genes denoted Pl. Previous genetic studies indicated that the majority of these genes are clustered on linkage groups 8 and 13. The Pl6 locus is one of the main clusters to have been identified, and confers resistance to several P. halstedii races. In this study, a map-based cloning strategy was implemented using a large segregating F2 population to establish a fine physical map of this cluster. A marker derived from a bacterial artificial chromosome (BAC) clone was found to be very tightly linked to the gene conferring resistance to race 300, and the corresponding BAC clone was sequenced and annotated. It contains several putative genes including three toll-interleukin receptor-nucleotide binding site-leucine rich repeats (TIR-NBS-LRR) genes. However, only one TIR-NBS-LRR appeared to be expressed, and thus constitutes a candidate gene for resistance to P. halstedii race 300.     

A QTL analysis of sunflower partial resistance to downy mildew ( Plasmopara halstedii) and black stem ( Phoma macdonaldii) by the use of recombinant inbred lines (RILs)

Partial resistance to downy mildew (Plasmopara halstedii) and to black stem (Phoma macdonaldii) in sunflower were investigated under natural field infection and a controlled growth chamber respectively. Genetic control for resistance to the diseases was determined in recombinant inbred lines (RILs) and their two parents, ’PAC-2’ and ’RHA-266.’ The experiments were undertaken in a randomized complete block design with two replications, in a field severely infected by downy mildew and in a controlled growth chamber with plants inoculated with an agressive French isolate of P. macdonaldii. Each replication consisted of three rows, 4.6-m long, giving 48 plants per RIL or parent in the field and 15 plants in the growth chamber. Genetic variability was observed among the RILs for resistance to both diseases. When 10% of the selected RILs were compared with the mean of the two parents genetic gain was significant for partial resistance to the diseases. Four putative QTLs for resistance to downy mildew on linkage groups 1, 9 and 17 were detected using composite interval mapping. The QTLs explained 54.9% of the total phenotypic variance. Major QTLs (dmr1–1 and dmr1–2) for resistance were found on linkage group 1 with up to 31% of the phenotypic variability explained by two peaks. QTL analysis of resistance to black stem showed seven QTLs on linkage groups 3, 6, 8, 9, 11, 15 and 17. The detected QTLs together explain 92% of the phenotypic variation of the trait. Crosses between RILs contrasted for their resistance to downy mildew and black stem, and exhibiting molecular polymorphism in detected QTLs, will be made in order to focus more-precisely on the genomic region of interest. Received: 28 February 2001 / Accepted: 14 June 2001     

A set of polymorphic EST‐derived markers for Picea species

A pooled DNA method was used to produce fully informative EST (expressed sequence tag)‐derived markers for the Picea genus. Nine markers were produced from 10 cDNA identified as candidates for cold tolerance or embryogenesis. Indels and SNPs (single nucleotide polymorphisms) were characterized from sequences obtained from pools of 10 individuals for each of the three species: Picea glauca (white spruce), Picea mariana (black spruce) and Picea abies (Norway spruce). Indels were present in 28% of the sequences and SNPs with a frequency greater than 10% were present on average in 1.2% of the positions.     

Polymorphic microsatellite markers for the Douglas‐fir pathogen Phaeocryptopus gaeumannii,causal agent of Swiss Needle Cast disease

Ten polymorphic microsatellite markers were isolated from Phaeocryptopus gaeumannii, causal agent of the Douglas‐fir foliage disease Swiss Needle Cast. The primer sets were tested on 60 isolates that had, with more conservative markers, previously segregated into three reproductively isolated lineages comprising nine genotypes. The microsatellite data yielded 46 multilocus genotypes that segregated into three groups identical to the three lineages previously recovered. Microsatellite genotypic diversity ranged from 0.85 to 0.99 within lineages and was lowest where both pathogen and host are exotic.     

Characterization of expressed sequence tag‐derived microsatellites from the kuruma prawn,Marsupenaeus japonicus

The kuruma prawn, Marsupenaeus japonicus, is a commercially important benthic marine crustacean in East Asia. Understanding the species’ population structures will be very important for its proper stock assessment and management strategy. Herein, 13 polymorphic microsatellite loci originating from expressed sequence tag libraries of M. japonicus were isolated and characterized. Number of alleles per locus ranged from two to 15, observed and expected heterozygosities ranged from 0.250 to 0.906 and from 0.310 to 0.932, respectively. The adequate level of variability within the population renders these microsatellite loci ideal markers for population genetic analysis of M. japonicus.     

Molecular markers for systematic identification and population genetics of the invasive Ponto‐Caspian freshwater gammarid Dikerogammarus villosus (Crustacea,Amphipoda)

The Ponto‐Caspian amphipod, Dikerogammarus villosus, is an invasive species of many European rivers. First, we show that size difference of nrDNA ITS1 allows discriminating D. villosus from Dikerogammarus bispinosus, a closely related but morphologically hardly distinguishable species. Second, we present two types of polymorphic markers for D. villosus, three microsatellites and two single nucleotide polymorphisms (SNPs) of mtDNA COI gene, which were scored by polymerase chain reaction‐single strand conformational polymorphism (PCR‐SSCP). These markers will be very useful in studying population genetics of D. villosus.     

Isolation of polymorphic tetranucleotide microsatellite markers for the silky short‐tailed bat Carollia brevicauda

We isolated 10 polymorphic microsatellite markers for the silky short‐tailed bat, Carollia brevicauda, from genomic libraries enriched for (AAGG)_n repetitive elements. The number of alleles ranged from six to 25 per locus with the observed heterozygosity ranging from 0.29 to 0.95. These markers will be useful for analysis of questions concerning population genetic structure and models of speciation. Results of cross‐species amplification in Carollia castanea and Carollia perspicillata are also reported.     

Identification of non-TIR-NBS-LRR markers linked to the <Emphasis Type="Italic">Pl5</Emphasis>/<Emphasis Type="Italic">Pl8</Emphasis> locus for resistance to downy mildew in sunflower

The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.     

Isolation of polymorphic tetranucleotide microsatellite markers for the green‐eyed tree frog (Litoria genimaculata)

We have isolated 16 polymorphic microsatellite markers for the green‐eyed tree frog, Litoria genimaculata, from genomic libraries enriched for (AAGG)_n and (AAAG)_n repetitive elements. The number of alleles ranges from four to 14 per locus with the observed heterozygosity ranging from 0.36 to 1.00. These markers will be useful for analysis of questions concerning population genetic structure and speciation.     

Isolation and characterization of polymorphic microsatellite DNA markers in the Japanese eight‐barbel loach,Lefua echigonia

We isolated and characterized 17 polymorphic microsatellite loci for the Japanese eight‐barbel loach, Lefua echigonia, an endangered freshwater species in streams including agricultural canals in Japan. The number of observed alleles for each locus ranged from two to nine, and the values of observed and expected heterozygosities ranged from 0.125 to 0.844 and from 0.148 to 0.876, respectively. All loci conformed to Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Ten polymorphic microsatellite markers for Scaphoideus titanus,the vector of flavescence dorée phytoplasma

The leafhopper Scaphoideus titanus is a vector of flavescence dorée phytoplasma, the causal agent of the most important grapevine yellow disease in European vineyards. Ten polymorphic microsatellite loci were developed from a genomic library enriched for AC and AG repeats. Levels of polymorphism were evaluated in 106 individuals from S. titanus European and American populations. An average of 16 alleles per locus was detected and the observed heterozygosity ranged from 0.141 to 0.813. Cross‐species amplification was successful in four other Cicadellidae species. These 10 microsatellites are valuable markers for population genetic and phylogeographical studies of S. titanus.     

Solution structure of LC5, the CCR5‐ derived peptide for HIV‐1 inhibition

The synthetic peptide fragment (LC5: LRCRNEKKRHRAVRLIFTI) inhibits human immunodeficiency virus type 1 (HIV‐1) infection of MT‐4 cells. In this study, the solution structure of LC5 in SDS micelles was elucidated by using the standard ¹H two‐dimensional NMR spectroscopic method along with circular dichroism and fluorescence quenching. The peptide adopts a helical structure in the C‐terminal region (residues 13–16), whereas the N‐terminal part remains unstructured. The importance of Phe17 in maintaining the structure of LC5 was demonstrated by replacing Phe17 with Ala, which resulted in the dramatic conformational change of LC5. The solution structure of LC5 elucidated in the present work provides a basis for further study of the mechanism of the inhibition of HIV‐1 infection. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation of polymorphic microsatellite markers in the sub‐Saharan tree,Acacia (Senegalia) mellifera (Fabaceae: Mimosoideae)

We isolated 11 polymorphic microsatellite markers from Acacia mellifera, a savannah woodland tree in sub‐Saharan Africa and southern Arabia. The loci were screened for polymorphism using 48 Kenyan individuals. Allelic diversity ranged from three to 19 per locus and the polymorphic information content varied from 0.287 to 0.893. These loci will be useful in studies of genetic structure, gene flow and breeding systems.     

Isolation and characterization of 11 polymorphic microsatellite markers for the marine gastropod Concholepas concholepas (Brugière, 1789)

Because of its long‐lived planktonic stage, the marine gastropod Concholepas concholepas is expected to exchange larvae over large distances. However, discrepancies between expected and realized dispersal have been documented in marine invertebrates. To investigate relationships between potential and effective (i.e. gene flow) dispersal, we developed 11 microsatellite markers and investigate their usefulness by analysing two populations distant by c. 4000 km. The 11 loci were found to be highly polymorphic in both populations, with 12–51 alleles according to the locus. This polymorphism is strong enough to allow fine‐scale population analyses including larval studies and paternity analyses.     

Development and characterization of six new microsatellite markers for the silver‐ or gold‐lipped pearl oyster,Pinctada maxima (Pteriidae)

Six di‐, tri‐ and tetranucleotide microsatellite loci were developed for the silver‐ or gold‐lipped pearl oyster Pinctada maxima using a linker‐ligated, magnetic bead enrichment protocol. Based on a minimum of 134 Indonesian pearl oyster samples, number of alleles and observed heterozygosity at each locus ranged from six to 17 alleles and from 0.172 to 0.813 (mean = 0.448), respectively. Mean polymorphic information content for the six loci was 0.562. These loci should be very useful in DNA parentage analyses and population differentiation of P. maxima in Australia and Indonesia.