Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

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 beta-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.

Microsatellite DNA suggests regional structure in the fusiform rust fungus Cronartium quercuum f. sp fusiforme.

This paper reports results obtained from microsatellite DNA analysis of genetic structure for populations of the native fungus Cronartium quercuum f. sp fusiforme infecting loblolly pine (Pinus taeda L.) over much of this host's natural range. Mostly all fusiform rust galls formed under field conditions are produced as a result of infection and colonization by haploid mycelium originating from a single basidiospore of C. quercuum fusiforme. If multiple infections do occur, then only a single haplotype must ultimately dominate and be responsible for gall formation. High levels of microsatellite variability exist in C. quercuum fusiforme and most of this variation occurs within local populations (average 88.4%). A statistically significant proportion, however, is found among populations, and the magnitude of this differentiation is closely associated with geographic distance between populations. Unweighted pair-group mean analysis and principal components analysis both indicate that at least four genetically distinct regional groups of C. quercuum fusiforme exist in the south Atlantic and Gulf coastal plains. In summary, the distribution of genetic variability in C. quercuum fusiforme is consistent with a hypothesis of at least four metapopulations with gene flow occurring less among regions than among populations within regions, and where overall levels of gene migration are related to geographic distance between populations.

RAPD markers linked to eastern filbert blight resistance in Corylus avellana.

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.

On the number of genes controlling the grass stage in longleaf pine.

The grass stage is an inherent and distinctive developmental trait of longleaf pine (Pinus palustris), in which height growth in the first few years after germination is suppressed. In operational forestry practice the grass stage extends for two to several years and often plays a role in planting failures and decisions to plant alternative species. Interspecies hybrids involving loblolly (P. taeda) and slash (P. elliottii var. elliottii) pines have been investigated as a means to produce planting stock with improved early height growth and to develop backcross populations for advanced generation breeding. We have reevaluated data from several interspecies populations, with the objective of estimating the number of genes contributing to the difference in first-year height growth between longleaf and loblolly pines. Estimates based on means and variances of parental and interspecies hybrid and backcross families suggest a minimum of 4 to 10 genes with standard errors less than half the estimates. These results suggest that the grass stage has evolved through the accumulation of alleles at several loci, each with small effects on various components of first-year height growth. Given the complexity of the grass-stage trait, tree breeders may need to combine genetic marker analysis with recurrent backcross breeding to efficiently develop longleaf pine planting stock for improved reforestation.

Bulked Segregant Analysis Identifies Molecular Markers Linked to Melampsora medusae Resistance in Populus deltoides.

ABSTRACT 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 (OPG10(340) and OPZ19(1800)) that are linked to Lrd1. Based on segregation in a total of 116 progeny, the genetic distances between OPG10(340) and OPZ19(1800) 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, OPG10(340), and OPZ19(1800). 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.

Prescreening Slash Pine and Cronartium Pedigrees for Evaluation of Complementary Gene Action in Fusiform Rust Disease.

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.

Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae).

Genetic variation among 12 populations of the American chestnut (Castanea dentata) was investigated. Population genetic parameters estimated from allozyme variation suggest that C. dentata at both the population and species level has narrow genetic diversity as compared to other species in the genus. Average expected heterozygosity was relatively low for the population collected in the Black Rock Mountain State Park, Georgia (He = 0.096 +/- 0.035), and high for the population in east central Alabama (He = 0.196 +/- 0.048). Partitioning of the genetic diversity based on 18 isozyme loci showed that ~10% of the allozyme diversity resided among populations. Cluster analysis using unweighted pair-group method using arithmetric averages of Rogers' genetic distance and principal components analysis based on allele frequencies of both isozyme and RAPD loci revealed four groups: the southernmost population, south-central Appalachian populations, north-central Appalachian populations, and northern Appalachian populations. Based on results presented in this study, a conservation strategy and several recommendations related to the backcross breeding aimed at restoring C. dentata are discussed.

Molecular mapping of resistance to blight in an interspecific cross in the genus castanea.

ABSTRACT A three-generation American chestnut x 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 F(2) 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. F(2) 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.

RAPD linkage mapping in a longleaf pine x slash pine F1 family.

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.