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Due to increasing resistance of pathogenic fungi to antifungal treatments, new types of drugs are needed. For this purpose, active substances with antifungal properties occurring in natural compounds should be considered. The herb Psephellus bellus shows strong antifungal activity and is characterized by unique guaianolides, which have an ester on C-2. Thus, a specialized method of isolation and testing was applied to assess the pharmacological effects of these guaianolides. After phytochemical analysis (chromatography and spectral methods), selected lipophilic compounds and the herb extract of this species containing 26 sesquiterpene lactones were tested. The antifungal effect of the herbal compounds was determined on clinical strains of fungi Candida, Rhodotorula, Trichophyton, Microsporum, and Scopulariopsis using a diffusion test. The MTT assay was employed to study the cytotoxic effects of the extract against human fibroblasts. Statistical analysis was performed. All analyzed compounds exhibited antifungal activity in cultivations suitable for assessment. Most lipophilic cebellins from Psephellus bellus prevent the growth of most fungal strains.

Single-urediniospore cultures of the fusiform rust fungus were used to inoculate seedlings from 10 full-sib families of a five-parent slash pine diallel at two different times in 1994. The presence or absence of fusiform rust galls was recorded for each inoculated seedling at 9 months postinoculation, and percent infection levels for each family-inoculum-time combination were used for detecting differences among host families and fungal cultures and for identifying differential interactions. The existence of differential interactions between two or more fungal cultures and two or more host families verifies that complementary gene action does exist in this pathosystem. Some host families may be excluded from more detailed interaction studies on the basis of their redundancy and lack of participation in differential interactions.

We report a case of a 34-year-old Polish Caucasian male who was diagnosed with tinea manuum caused by Trichophyton rubrum var. raubitschekii. It would be the first described case of a dermatophytosis caused by this fungus in Poland and one of a few cases in Central Europe described so far. Admittedly, it would be the first case in Central Europe with no evidence pointing to African origin. The clinical condition improved after administering itraconazole (daily dose 100 mg orally) supplemented with a topical treatment, while the patient was totally cured after 2 months. The histopathological examination turned out to be highly useful in the diagnostic process. The genetic analysis of the urease gene pointed to a urease-positive T. rubrum rather than T. rubrum var. raubitschekii.

The Chinese chestnut (Castanea mollissima) carries resistance to Cryphonectria parasitica, the fungal pathogen inciting chestnut blight. The pathogen, introduced from Asia, devastated the American chestnut (Castanea dentata) through- out its native range early in the twentieth century. A highly informative genetic map of Chinese chestnut was constructed to extend genomic studies in the Fagaceae and to aid the intro- gression of Chinese chestnut blight resistance genes into Amer- ican chestnut. Two mapping populations were established with three Chinese chestnut parents, 'Mahogany', 'Nanking' ,a nd 'Vanuxem', totaling 337 progeny. The transcriptome-based ge- netic map was created with 329 simple sequence repeat and 1,064 single nucleotide polymorphism markers all derived from expressed sequence tag sequences. Genetic maps for each parent were developed and combined to establish 12 consensus linkage groups spanning 742 cM, providing the the most com- prehensive genetic map for a Fagaceae species to date. Over 75 % of the mapped markers from the Chinese chestnut con- sensus genetic map were placed on the physical map using overgo hybridization, providing a fully integrated genetic and physical map resource for Castanea spp. About half (57 %) of the Chinese chestnut genetic map could be assigned to regions of segmental homology with 58 % of the peach (Prunus per- sica) genome assembly. A three quantitative trait loci (QTL) model for blight resistance was verified using the new genetic markers and an existing interspecies (C. mollissima × C. den- tata)F2mapping population. Two of the blight resistance QTLs in chestnut shared synteny with two QTLs for powdery mildew resistance in peach, indicating the potential conservation of disease resistance genes at these loci.

The genome size of the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme (Cqf) was determined by flow cytometric analysis of propidium iodide-stained, intact haploid pycniospores with haploid spores of two genetically well characterized fungal species, Sclerotinia sclerotiorum and Puccinia graminis f.sp. tritici, as size standards. The Cqf haploid genome was estimated at ~90 Mb, similar to other Pucciniales species for which reference genome sequences are available. Twenty-three Cqf pycniospore samples were compared that comprised three samples obtained from naturally occurring pine galls and 20 samples obtained after artificial inoculation with parental isolates and their progeny. Significant variation in genome size (>10% of mean) was detected among unrelated as well as sibling Cqf samples. The unexpected plasticity in Cqf genome size observed among sibling samples is likely to be driven by meiosis between parental genomes that differ in size.

The National Science Foundation of the United States has funded a four-year project to develop genomic tools for the Fagaceae, the beech family, which includes chestnut (Castanea), oak (Quercus), and beech (Fagus). A large amount of cDNA sequence of Castanea, Quercus, and Fagus is being generated by pyrosequencing, a new technique that can produce large amounts of DNA sequence at a lower cost per nucleotide than conventional Sanger-Coulson sequencing. New molecular markers, primarily Single-Nucleotide Polymorphisms, are being discovered, and three Bacterial Artificial Chromosome Libraries of Castanea mollissima cv. 'Vanuxem' have been generated and are available for purchase at the Clemson University Genomics Institute. All sequence information generated by the project will be available on-line at www.fagaceae.org.

The American chestnut (Castanea dentata), once known as ‘The King of the Forest’ in the Appalachian Mountains of the eastern United States and southeastern Canada, has been all but extirpated by chestnut blight disease caused by an Asiatic bark fungus, Cryphonectria parasitica. A group of scientists at The American Chestnut Foundation has been working since 1983 to develop blight resistant American chestnut by inter-species backcross breeding with the relatively resistant Chinese chestnut (Castanea mollissima). In an effort to facilitate this breeding program we have initiated cytogenetic studies of these two species and their F1 and backcross hybrids. Here we report our successes in preparing somatic chromosomes using enzymatic digestion of American chestnut seedling root tips and demonstrate the quality of the chromosome spreads using fluorescent in situ hybridization with ribosomal DNA probes. Our results show two 18S-28S rDNA sites, one major and one minor, and one 5S rDNA site in American chestnut. This is the first such report for American chestnut and our preliminary results appear similar for Chinese chestnut.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

The genus Castanea (Fagaceae), which contains three sections and seven species, is widely distributed in the deciduous forests of the Northern Hemisphere. The phylogeny of Castanea was estimated using DNA sequence data from five different regions of the chloroplast genome. Sequencing results support the genus Castanea as a paraphyletic group with C. crenata, the Japanese chestnut, representing an early divergence in the genus. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene, followed by intercontinental dispersion and divergence between the Chinese and European/North American species during the Oligocene and a split between the European and North American species in the early Miocene. The differentiation within North America and China might have occurred in late Miocene or early Pliocence. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and C. dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.

We report on 53 microsatellite loci for use in population genetic or linkage mapping studies in Cryphonectria parasitica . In 40 isolates collected from throughout the Northern Hemisphere, the number of alleles per locus ranged from two to 14 (mean 5.17) with gene diversity values ranging from 0.049 to 0.859 (mean 0.437). Samples from Asia were more diverse than those from Europe and North America. Most of the markers (48 of 53) were developed from an expressed sequence tag library, and hence, offer the opportunity to examine population structure or provide genome location information for specific expressed genes vs. anonymous genomic regions.

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.

Cronartium quercuum f.sp. fusiforme is the pathogen that incites fusiform rust disease of southern pine species. To date, a number of host resistance genes have been mapped. Although genomic mapping studies have provided valuable information on the genetic basis of disease interactions in this pine-rust pathosystem, the interaction at the molecular level is poorly understood. To further our understanding of this interaction, we implemented a microarray study to examine the differential expression of genes in pathogenchallenged progeny of a full-sib loblolly pine family known to be segregating at a single dominant resistance gene (Fr1). Statistical analyses revealed shifts in gene expression that may reflect discrete stages of gall development.

Summary Aggressive chemotherapy and immunosuppressive treatment may prolong patients’ life, but influence the risk of severe, life-threatening infections. Here, we report the case of a 21-year-old caucasian female who developed a disseminated infection of Scedosporium prolificans after allogenic stem cell transplantation performed for treatment of relapsed acute lymphoblastic leukaemia. The pathogen was isolated from the blood and identified on the basis of its macroscopic and microscopic morphological features. The empirical treatment with amphotericin B provided no improvement. However, introduction of intravenous voriconazole resulted in amelioration of fever. Unfortunately, the patient died due to progression of underlying disease and multiorgan failure. However, this case report indicates a possible relevance of voriconazole-based treatment regimens in invasive S. prolificans infections.

A total of 1,110 decamer primers were screened for RAPD markers linked to a dominant allele in hazelnut ( Corylus avellana) that confers resistance to eastern filbert blight caused by Anisogramma anomala. Twenty RAPD markers linked in coupling, and five markers linked in repulsion, were found. A seedling population was used to construct a linkage map of the region flanking the resistance locus. The map spans 46.6 cM, with 14 markers on one side of the resistance locus and eight on the other side. Eleven markers showed less than 3% recombination with resistance, including three that showed no recombination. Seven of these 11 markers are sufficiently robust to allow their use in marker-assisted selection. These include AA12(850) which shows no recombination, and six markers on one side of the resistance locus: 173(500), 152(800), 122(825), 275(1130), H19(650) and O16(1250). Marker 268(580), which flanks the resistance locus on the other side, is also suitable for use in marker-assisted selection, but shows 5.8% recombination with resistance. Other markers are less suitable for marker-assisted selection because of sensitivity to changes in primer or MgCl(2) concentration, or the long time required for electrophoresis to separate bands of similar size. The 16 markers closest to the resistance locus were cloned and sequenced. The W07(365) marker, which showed no recombination with the resistance locus but is difficult to score, includes a CT microsatellite repeat. The sequence information will allow the design of SCAR primers and eventual map-based cloning of the resistance allele.

Phylogenetic relationship in Actinidia was investigated using RAPD and PCR-RFLPs of mtDNA. Forty taxa of 31 species, encompassing all four sections and six series of the genus, and 2 species of related genus were analyzed with 22 pre-screened random decamers out of a total of 144 primers. A total of 220 DNA scorable bands were amplified across all samples, and 204 DNA scorable bands were amplified within Actinidia, of which 188 (92.16%) were polymorphic. Meanwhile, Thirty-four taxa of Actinidia were investigated through PCR-RFLP analysis of two mtDNA fragments, including nad1/B-C gene (approximately 1,565 bp) and nad4/1-2 gene (approximately 2,061 bp). Digestion of these two fragments by seven restriction endonucleases yielded twenty polymorphic restiction fragments. The phylogenetic analysis revealed a clear indication that section Leiocarpae was a monophyletic group, but subdividing other three traditional sections was poorly supported by RAPDs; however, none of four sections was a monophyletic group in PCR-RFLPs of mtDNA. Both UPGMA phenogram and the strict consensus tree showed that majority of the species were clustered into geographic subgroups of Yangtzi River, southeastern, southern and southwestern region of China in accordance with their natural distribution. The intrageneric subdivisions of Actinidia appeared to be difficult, but could be more appropriate by taking into account the geographic distribution of species, particularly for species of Liang’s sections of Maculatae and Stellatae. The results of phylogenetic analysis with RAPD data suggest to divide the genus into two sections of Leicarpae and Maculatae and to divide the latter section into four series of Yangtzi River, southeastern, southern and southwestern China. The phylogenetic relationships among several species with taxonomic uncertainty have been also discussed on the basis of the results of RAPD and mtDNA data. The results obtained in the present study supplements our understandings of the Actinidia taxonomy obtained from the morphological characters.

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely identified by as few as 14 loci of eight primers. Genetic diversity of the existing gene pool of cultivated pawpaws, as estimated by Nei's gene diversity (He), was similar to that of wild pawpaw populations. The genetic relatedness among the cultivated pawpaws examined by UPGMA cluster analysis separated 34 cultivars and selections into two distinct clusters, a cluster of PPF (The PawPaw Foundation) selections and a cluster including a majority of the extant cultivars selected from the wild and their derived selections. The results are in general agreement with the known selection history and pedigree information available. The consensus fingerprint profile using the genetically defined RAPD markers is a useful and reliable method for establishing the genetic identities of the pawpaw cultivars and advanced selections. This also proved to be an improved discriminating tool over isozyme markers for the assessment of genetic diversity and relatedness. RAPD profiling of data presented in this study provides a useful reference for germplasm curators engaged in making decisions of sampling strategies, germplasm management and for breeders deciding which parents to select for future breeding efforts.

Phylogenetic relationships within the Actinidia were investigated using randomly amplified polymorphic DNA (RAPD) markers. DNAs from 40 taxa, including 31 species encompassing all four sections and four series of the traditional subdivisions within the genus, were amplified using 22 preselected 10-mer oligonucleotide primers. A total 204 DNA bands were scored across the 40 taxa, of which 188 (92%) were polymorphic. A wide range of genetic similarity was observed among the taxa (0.13 to 0.61). The average similarity between varieties of the same species was 0.54, and between different species was 0.28, respectively. Although the phylogenetic analysis revealed a clear indication that section Leiocarpae was a monophyletic group, subdivisions of the other three traditional sections were poorly supported. The UPGMA phenogram showed that the majority of the species clustered into geographic subgroups in accordance with their natural distribution (the Yangtzi River, southeastern China, southern China and southwestern China). The intrageneric subdivisions of Actinidia appeared to be difficult, but some subdivisions could be explained by the geographic distribution of the species, particularly for species of Liang's sections of Maculatae and Stellatae. The phylogenetic relationships among several species with previous taxonomic uncertainty are also discussed on the basis of the RAPD data. The results of this study supplement our previous understanding of the Actinidia taxonomy based solely on morphological characters.

Random amplified polymorphic DNA (RAPD) markers were employed to map the genome and quantitative trait loci controlling the early growth of a pine hybrid F(1) tree ( Pinus palustris Mill. x P. elliottii Engl.) and a recurrent slash pine tree ( P. elliottii Engl.) in a (longleaf pine x slash pine) x slash pine BC(1) family consisting of 258 progeny. Of the 150 hybrid F(1) parent-specific RAPD markers, 133 were mapped into 17 linkage groups covering a genetic distance of 1,338.2 cM. Of the 116 slash pine parent-specific RAPD markers, 83 were mapped into 19 linkage groups covering a genetic distance of 994.6 cM. A total of 11 different marker intervals were found to be significantly associated with 13 of the 20 traits on height and diameter growth using MAPMAKER/QTL. Nine of the eleven marker intervals were unique to the hybrid parent 488 genome, and two were unique to the recurrent parent 18-27 genome. The amount of phenotypic variance explained by the putative QTLs ranged from 3.6% to 11.0%. Different QTLs were detected at different ages. Two marker intervals from the hybrid parent 488 were found to have QTL by environment interactions.

The loss of genetic diversity in fragmented populations ofself-incompatible plant species may result in sexual reproductive failure andlocal extinctions. Florida ziziphus (Ziziphus celata) is aself-incompatible clonal shrub known only from five genetically depauperatepopulations on the Lake Wales Ridge, Florida, USA. Recovery of this speciesrequires identification of cross-compatible genotypes that can be used to createviable (i.e., sexually reproducing) populations. To further development of arecovery program for this highly imperiled species, we investigated its geneticstructure and sexual reproductive viability. We used random amplifiedpolymorphic DNAs (RAPDs) to investigate genetic variability within remnantpopulations and we conducted experimental compatibility trials to determine thecross-compatibility of remnant genotypes. One hundred and ninety-nine uniquestem samples collected from one ex situ and fivein situ populations were assayed for the presence orabsence of a band for 32 RAPD markers. Based on unweighted pair-group meancluster analysis (UPGMA), only 11 multi-locus genotypes (MLGs) were identified.Eight of these MLGs correspond to MLGs identified in an earlier allozyme study.In addition, we identified three new RAPD-based MLGs. Three of the five naturalpopulations consisted of only one MLG, while the largest and most geneticallydiverse population comprised only four MLGs. Coefficients of similarity rangedfrom 96.6% for the most closely related MLGs to 20.7% for the most distantlyrelated. The compatibility trials demonstrated that most MLGs arecross-incompatible. With 69% of all possible one-way crosses tested (38/55), wehave identified only eight compatible crosses via germination trials. Based onthe results of the compatibility trials, we assigned MLGs toself-incompatibility (SI) mating types. On present evidence, the currentbreeding population of Florida ziziphus may comprise as few as two SI matingtypes. These SI mating types will be used to guide future breeding efforts andan experimental introduction.

Morphological traits were examined in an F3 generation derived from a cross between C. lanatus var. lanatus [(Thunb.) Matsum. & Nakai] and C. lanatus var. citroides. At least three genes, C (yellow) vs. c (red), i (inhibitory to C) vs. I (non-inhibitory to C), and y (yellow) vs. yw (white), with epistatic and inhibitory actions were found to govern the inheritance of fruit flesh color. The high frequency of yellow-fleshed fruit and low frequencies of white and red fruits can be explained by the presence of a new allele (yw recessive to y) in the multiple allele series at the Y locus. The low frequency of tan colored seeds in segregating populations could be explained by at least three genes governing inheritance of seed-coat color. Single factor analysis of variance was conducted for each pairwise combination of random amplified polymorphic DNA (RAPD) locus and fruit or seed characteristics. Several RAPD loci were identified to be loosely linked to morphological characteristics.

We tested the constancy of linkage relationships of randomly amplified polymorphic DNA (RAPD) marker loci used to construct a population-based consensus map in material from an Italian stand of Picea abies (L.) Karst. in 29 individuals from three Norwegian populations. Thirteen marker loci linked in the Italian stand did show a consistent locus ordering in the Norwegian population. The remaining 16 unlinked marker loci were spread over different linkage groups and (or) too far apart both in the population map and in this study. The limited validity of RAPD markers as genomic "hallmarks" resilient across populations is discussed. We also investigated the reliability of RAPD markers; only 58% of the RAPD markers previously used to construct the consensus map in the Italian population were repeat- able in the same material. Of the repeatable ones 76.3% were amplified and found polymorphic in 29 megagametophyte sibships from three Norwegian populations. R&urn6 : Les auteurs ont voulu verifier, chez 29 individus provenant de trois populations norvegiennes de Pica abies (L.) Karst., la stabilite des relations de liaison entre les loci de marqueurs d'amplification au hasard des polymorphis- mes de I'ADN (RAPD) utiiises pour construire une carte consensus de population a partir de materiel qui origine d'un peuplement italien de Ia m&me espece. Un groupe de 13 loci de marqueurs du peuplement italien a demontre un ordre similaire des loci chez les populations norvegiennes. Les autres I6 loci de marqueurs non lies Ctaient repartis entre dif

Isozyme, randomly amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers were used to generate a linkage map in an F2 and F3 watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai] population derived from a cross between the fusarium wilt (Fusarium oxysporum f. sp. niveum) susceptible `New Hampshire Midget' and resistant PI 296341-FR. A 112.9 cM RAPD-based map consisting of 26 markers spanning two linkage groups was generated with F2 data. With F3 data, a 139 cM RAPD-based map consisting of 13 markers covering five linkage groups was constructed. Isozyme and SSR markers were unlinked. About 40% to 48% of the RAPD markers were significantly skewed from expected Mendelian segregation ratios in both generations. Bulked segregant analysis and single-factor analysis of variance were employed to identify RAPD markers linked to fusarium wilt caused by races 1 and 2 of F. oxysporum f. sp. niveum. Current linkage estimates between the resistance trait and the marker loci were too large for effective use in a marker-assisted selection program.

ADDITIONAL INDEX WORDS. Annonaceae, dominant inheritance, allele frequency, Nei's genetic identity and distance, cluster analysis ABSTRACT. Twelve, 10-base primers amplified a total of 20 intense and easily scorable polymorphic bands in an interspecific cross of PPF1-5 pawpaw (Asimina triloba (L.) Dunal.) x RET (Asimina reticulata Shuttlew.). In this cross, all bands scored were present in, and inherited from, the A. triloba parent PPF1-5. Nineteen of the 20 bands were found to segregate as expected (1:1 or 3:1) based on chi-square goodness-of-fit tests, and were subsequently used to evaluate genetic diversity in populations of A. triloba collected from six states (Georgia, Illinois, Indiana, Maryland, New York, and West Virginia) within its natural range. Analysis of genetic diversity of the populations revealed that the mean number of alleles per locus was A = 1.64, percent polymorphic loci was P = 64, and expected heterozygosity was He = 0.25. No significant differences were found among populations for any of the polymorphic indices. Partitioning of the population genetic diversity showed that the average genetic diversity within populations was Hs = 0.26, accounting for 72% of the total genetic diversity. Genetic diversity among populations was Dst = 0.10, accounting for 28% of the total genetic diversity. Nei's genetic identity and distance showed a high mean identity of 0.86 between populations. Genetic relationships among the populations examined by unweighted pair-group mean clustering analysis separated the six populations into two primary clusters: one composed of Georgia, Maryland, and New York, and the other composed of Illinois, Indiana, and West Virginia. The Georgia and Indiana populations were further separated from the other populations within each group. This study provides additional evidence that marginal populations within the natural range of A. triloba should be included in future collection efforts to capture most of the rare and local alleles responsible for this differentiation. The genus Asimina Adans. is the only temperate representa- tive of the tropical Annonaceae, the largest primitive family of flowering plants, containing ≈130 genera and 2000 species (Cronquist, 1981). The genus Asimina includes nine species, which are indigenous to the United States (Kral, 1960). The best- known species is Asimina triloba, the North American pawpaw, which is the largest tree fruit native to the United States. Pawpaw fruit have an oblong shape with green skin and a taste that can be described as a very sweet blend of banana ( Musa ×paradisiaca L.), mango (Mangifera indica L.), and pineapple (Ananas comosus (L.) Merr.) flavors with a mild pleasant fruit aroma. Its fruit quality, nutritional value, natural defense compounds (annonaceous acetogenins) and landscaping appeal, make paw- paw of great potential for commercial development as a new alternative fruit tree crop and ornamental in the United States and elsewhere (Johnson et al., 1996; Jones and Layne, 1997; Layne, 1996). The natural range of A. triloba covers most of eastern North America, ranging from northern Florida to southern Ontario

(1999). Genetic Linkage Mapping of Genomic Regions Conferring Tolerance to High Aluminum in Slash Pine. Journal of Sustainable Forestry: Vol. 10, No. 1-2, pp. 69-78.

Microsatellite markers were developed for conservation genetic studies of Lindera melissifolia (pondberry), a federally endangered shrub of southern bottomland ecosystems. Microsatellite sequences were obtained from DNA libraries that were enriched for the (AC) n simple sequence repeat motif. From 35 clone sequences, 20 primer pairs were designed and evaluated. Eleven primer pairs amplified polymorphic marker loci in pondberry while two did so in Lindera benzoin (spicebush). In 46 samples from a single pondberry site the number of microsatellite alleles ranged from two to 11 per locus with observed heterozygosity values of 0.07–0.91.

A three-generation American chestnut × Chinese chestnut pedigree was used to construct a genetic linkage map for chestnut and to investigate the control of resistance to Endothia parasitica (chestnut blight fungus). DNA genotypes for 241 polymorphic markers (eight isozymes, 17 restriction fragment length polymorphisms [RFLPs], and 216 random amplified polymorphic DNAs [RAPDs]) were assayed on an F2 family consisting of 102 individuals. Of these markers, 196 were segregating as expected and, subsequently, used for primary linkage mapping. Two isozymes, 12 RFLPs, and 170 RAPDs were mapped to 12 linkage groups spanning a total genetic distance of 530.1 Kosambi centimorgans. F2 plants were evaluated for a response to E. parasitica infection by directly inoculating them with two unique fungal isolates and measuring canker expansion over a period of 3.5 months. Results were compared with the marker genotype data, thereby identifying genomic regions significantly associated with a resistance response. Single-marker or nonsimultaneous analyses of variance identified seven genomic regions that appear to have an effect on host response. Multiple-marker or simultaneous models suggest that three of these regions have a significant effect on host response, together explaining as much as 42.2% of the total variation for canker size. At each of the three putative resistance loci, alleles derived from the Chinese chestnut grandparent were associated with smaller canker size, or higher levels of resistance.

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we describe the development of 11 codominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were cloned and sequenced, and polymerase chain reaction (PCR) primers were designed flanking insertions/deletions. Primers labelled with fluorescent dyes were combined in multiplex reactions to assay five or six loci simultaneously in a capillary sequencing system. These codominant markers have the potential to complement RFLP methods for studying C. parasitica.

Zinc is required for the folding and function of many proteins. In Saccharomyces cerevisiae, homeostatic and adaptive responses to zinc deficiency are regulated by the Zap1 transcription factor. One Zap1 target gene encodes the Tsa1 peroxiredoxin, a protein with both peroxidase and protein chaperone activities. Consistent with its regulation, Tsa1 is critical for growth under low zinc conditions. We previously showed that Tsa1's peroxidase function decreases the oxidative stress that occurs in zinc deficiency. In this report, we show that Tsa1 chaperone, and not peroxidase, activity is the more critical function in zinc-deficient cells. Mutations restoring growth to zinc-deficient tsa1 cells inactivated TRR1, encoding thioredoxin reductase. Because Trr1 is required for oxidative stress tolerance, this result implicated the Tsa1 chaperone function in tolerance to zinc deficiency. Consistent with this hypothesis, the tsa1Δ zinc requirement was complemented by a Tsa1 mutant allele that retained only chaperone function. Additionally, growth of tsa1Δ was also restored by overexpression of holdase chaperones Hsp26 and Hsp42, which lack peroxidase activity, and the Tsa1 paralog Tsa2 contributed to suppression by trr1Δ, even though trr1Δ inactivates Tsa2 peroxidase activity. The essentiality of the Tsa1 chaperone suggested that zinc-deficient cells experience a crisis of disrupted protein folding. Consistent with this model, assays of protein homeostasis suggested that zinc-limited tsa1Δ mutants accumulated unfolded proteins and induced a corresponding stress response. These observations demonstrate a clear physiological role for a peroxiredoxin chaperone and reveal a novel and unexpected role for protein homeostasis in tolerating metal deficiency.

The European chestnut, Castanea sativa Mill, covers a total area of 2.53 million hectares two million of which are chestnut forests, i.e. forests where chestnut is the dominant tree species, being the remaining 0.53 million hectares devoted to fruit production (20.9% of the total chestnut-growing area). Chestnut fruit production in Europe declined considerably during the XX century to the current 200,000 t (almost 300 million euros). This decline arose mainly due to serious diseases and changes in the structure of society. Towards the end of the century, there was a sharp increase in chestnut demand which triggered new planting and the restoration of old orchards throughout Europe, although ink disease (Phytophthora spp.), canker blight (Cryphonectria parasitica (Murrill) M.E. Barr.) and more recently Dryocosmus kuriphilus Yasumatsu, still represent major threats to the species. In Europe, chestnut breeding has been focused on the improvement of cultivars and rootstocks through selection and hybridisation with Asian species resistant to ink disease. Considerable success was obtained using this approach, however little information was acquired until now on the genetic basis of resistance in chestnut. A set of interspecific crosses were established between European chestnut and Asian species and two separate full-sib pedigrees have been produced, SC (C. sativa x C. crenata) and SM (C.sativa x C. mollissima). The goal is to perform DNA marker:trait association analysis to identify QTLs related to ink disease metrics and also to identify putative resistance genes to P cinnamomi using a transcriptomic approach. An overview of the study will be presented including the genotyping of the mapping population (parents and progenies) with nuclear SSR markers designed for Castanea mollissima and provided by the American team of Fagaceae Genomics Project, for the construction of the first genetic linkage map. The methodologies implemented for the determination of the metrics of resistance of mapping population, for further identification of QTLs, will also be presented.

Molecular Ecology Resources Primer Development Consortium: et al., This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

In the north central United States, leaf rust caused by Melampsora medusae is a major disease problem on Populus deltoides. In this study we identified molecular markers linked to a M. medusae resistance locus (Lrd1) that was segregating 1:1 within an intraspecific P. deltoides family (C9425DD). Previous field results were confirmed in the controlled environment of a growth chamber through an excised whole-leaf inoculation method. Using bulked segregant analysis we identified two random amplified polymorphic DNA (RAPD) markers (OPG10340 and OPZ191800) that are linked to Lrd1. Based on segregation in a total of 116 progeny, the genetic distances between OPG10340 and OPZ191800 and the resistance locus were estimated as 2.6 and 7.4 Haldane centimorgans (cM), respectively. Multipoint linkage analyses strongly suggest the most likely order for these loci is Lrd1, OPG10340, and OPZ191800. These markers may prove to be instrumental in the eventual cloning of Lrd1, as well as for marker-assisted selection of leaf-rust resistant genotypes.

A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 × OSU 414.062 was used. RAPD markers in testcross configuration, segregating 1:1, were used to construct separate maps for each parent. Fifty additional RAPD loci were assigned to linkage groups as accessory markers whose exact location could not be determined. Markers in intercross configuration, segregating 3:1, were used to pair groups in one parent with their homologues in the other. Eleven groups were identified for each parent, corresponding to the haploid chromosome number of hazelnut (n = x = 11). Thirty of the 31 SSR loci were able to be assigned to a linkage group. The maternal map included 249 RAPD and 20 SSR markers and spanned a distance of 661 cM. The paternal map included 271 RAPD and 28 SSR markers and spanned a distance of 812 cM. The maps are quite dense, with an average of 2.6 cM between adjacent markers. The S-locus, which controls pollen?stigma incompatibility, was placed on chromosome 5S where 6 markers linked within a distance of 10 cM were identified. A locus for resistance to eastern filbert blight, caused by Anisogramma anomala, was placed on chromosome 6R for which two additional markers tightly linked to the dominant allele were identified and sequenced. These maps will serve as a starting point for future studies of the hazelnut genome, including map-based cloning of important genes. The inclusion of SSR loci on the map will make it useful in other populations. Fil: Mehlenbacher, Shawn A. M. State University of Oregon; Estados Unidos Fil: Brown, Rebecca N.. State University of Oregon; Estados Unidos Fil: Nouhra, Eduardo Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Gokirmak, Tufan. State University of Oregon; Estados Unidos Fil: Bassil, Nahla V.. State University of Oregon; Estados Unidos Fil: Kubisiak, Thomas L.. Southern Institute of Forest Genetics; Estados Unidos

To find markers linked to vegetative incompatibility (vic) genes in the chestnut blight fungus, Cryphonectria parasitica, we constructed a preliminary linkage map. In general, this map is characterized by low levels of polymorphism, as evident from the more than 24 linkage groups observed, compared to seven expected from electrophoretic karyotyping. Nonetheless, we found markers closely linked to two vic genes (vic1 and vic2) making them candidates for positional cloning. Two markers were found to be linked to vic2: one cosegregated with vic2, i.e., it is 0.0 cM from vic2, the other was at a distance of 4.5 cM; a single marker was found 4.0 cM from vic1. The closest markers linked to three other vic genes (vic4, vic6, and vic7) were >15 cM away; additional markers are needed before efficient positional cloning of these three vic genes can be realized. In contrast to the low levels of polymorphism observed across most of the C. parasitica genome, the linkage group containing the MAT locus appears to harbor an extremely high level of RAPD heterogeneity and reduced recombination. Markers within this highly heterogeneous region are in linkage disequilibrium in some natural populations; however, recombination is clearly evident between this region and the MAT locus.

Random amplified polymorphic DNAs (RAPDs) were used to construct linkage maps of the parent of a longleaf pine (Pinus palustris Mill.) slash pine (Pinus elliottii Englm.) F1 family. A total of 247 segregating loci [233 (1∶1), 14 (3∶1)] and 87 polymorphic (between parents), but non-segregating, loci were identified. The 233 loci segregating 1∶1 (testcross configuration) were used to construct parent-specific linkage maps, 132 for the longleaf-pine parent and 101 for the slash-pine parent. The resulting linkage maps consisted of 122 marker loci in 18 groups (three or more loci) and three pairs (1367.5 cM) for longleaf pine, and 91 marker loci in 13 groups and six pairs for slash pine (952.9 cM). Genome size estimates based on two-point linkage data ranged from 2348 to 2392 cM for longleaf pine, and from 2292 to 2372 cM for slash pine. Linkage of 3∶1 loci to testcross loci in each of the parental maps was used to infer further linkages within maps, as well as potentially homologous counterparts between maps. Three of the longleaf-pine linkage groups appear to be potentially homologous counterparts to four different slash-pine linkage groups. The number of heterozygous loci (previously testcross in parents) per F1 individual, ranged from 96 to 130. With the 87 polymorphic, but non-segregating, loci that should also be heterozygous in the F1 progeny, a maximum of 183-217 heterozygous loci could be available for mapping early height growth (EHG) loci and for applying genomic selection in backcross populations.

Background: Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. Methodology/Principal Findings: Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. Conclusions/Significance: Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f.sp.niveum(FON), is one of the most economically important diseases of watermelon (Citrullus lanatus) worldwide. FON has been divided into three races (races 0, 1, and 2) determined by differential pathogenicity assays. Most commercial cultivars are resistant to races 0 and 1; whereas none are resistant to race 2. Few morphological and isozyme variations are seen in the cultivated watermelon, but molecular markers, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs), have been shown to generate several polymorphisms. Segregating populations, derived from a cross between watermelon cultivar New Hampshire Midget, susceptible to all three races of FON and the resistant C. lanatus plant introduction PI 296341, were used to detect molecular markers linked to Fusarium wilt resistance. In a preliminary experiment, more than 180 10-mer primers were screened against the parents and the F1. Informative RAPD primers were identified and used in conjunction with AFLP and SSR markers to generate a genome map.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f.sp. niveum (FON), is a serious disease of the watermelon (Citrullus lanatus). Three races of this pathogen (races 0, 1, and 2) have been identified based on differential pathogenicity assays. Most commercially available cultivars are resistant to races 0 and 1. Inheritance for resistance to these races is thought to be controlled by a single dominant gene. No cultivars are resistant to race 2 and resistance is thought to be a quantitative trait. F2 lines derived from a cross between the Fusarium-resistant Citrullus lanatus PI296341, and the Fusarium-susceptible watermelon cultivar `New Hampshire Midget' were used to generate a RAPD-based map of the Citrullus genome. F2:3 families were assayed in the greenhouse for resistance to races 1 and 2. Those families that were either highly resistant or highly susceptible were used in identifying markers linked to Fusarium wilt resistance. A preliminary map of the Citrullus genome based on random amplified polymorphic DNA (RAPD) markers has been expanded with the inclusion of simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), and isozymes.

Kentucky State Univ. (KSU) is the national clonal germplasm repository for Asimina species. Previous evaluation of the KSU pawpaw collection using 24 isozyme markers demonstrated that pawpaw has a relatively higher genetic diversity than that noted for other plant species with similar species characteristics (long-lived, woody, perennial, out-crossing, temperate, widespread, etc.). Current evaluation using RAPD markers will provide us with a more-accurate insight into pawpaw genetic diversity and population structure. In a preliminary experiment, one hundred 10-mer primers (OA1-20 through OE1-20, Operon Technologies Inc.) were screened against 32 commercial cultivars or advanced selections. A subset of 24 primers that amplify only the most-informative markers were used for germplasm evaluation. Sixty-eight RAPD markers were identified and used for determining genetic parameters. One-hundred-twenty pawpaw accessions were sampled from the KSU repository for RAPD analysis. These accessions represented nine widely distributed states within pawpaw's native range. RAPD data were subjected to various analyses using the NTSYS-PC computer program (ver. 1.8). Information generated from isozyme and RAPD markers will be used to formulate future germplasm collection strategies from wild populations within the native range. The implications of such information to the genetic enhancement of our repository and establishment of a core collection will be discussed.

Successful restoration of wild turkeys (Meleagris gallopavo) to Wisconsin has stimulated increased interest in hunting and a need for specific management information. We determined survival and causes of mortality for 224 radiomarked hens during 1988-94 in Vernon County, Wisconsin under a relatively liberal fall harvest regime. Annual survival ranged from 43.1 to 66.0 and averaged 52.7% (SE = 4.8). Seasonal survival averaged 72.2 (SE = 2.9), 81.3 (SE = 3.3), and 89.3% (SE = 2.5) during recruitment (16 Mar to 14 Jul), post-recruitment (15 Jul to 21 Nov), and winter (22 Nov to 15 Mar), and was lower (P = 0.001) during recruitment than during winter. Predation accounted for 71.2% of all deaths and was highest during recruitment. Localized starvation occurred during a winter with 49 consecutive days of deep snow and cold temperatures. Cumulative fall hunter densities were about 3.5/km 2 of woodland and the fall harvest rate of radioed hens averaged 7.3%. Turkey densities declined during the study and more conservative fall harvests may be necessary.