A genomic map enriched for markers linked to Avr1 in Cronartium quercuum f.sp. fusiforme.

A novel approach is presented to map avirulence gene Avr1 in the basidiomycete Cronartium quercuum f.sp. fusiforme, the causal agent of fusiform rust disease in pines. DNA markers tightly linked to resistance gene Fr1 in loblolly pine tree 10-5 were used to classify 10-5 seedling progeny as either resistant or susceptible. A single dikaryotic isolate (P2) heterozygous at the corresponding Avr1 gene was developed by crossing Fr1 avirulent isolate SC20-21 with Fr1 virulent isolate NC2-40. Bulk basidiospore inoculum derived from isolate P2 was used to challenge the pine progeny. The ability to unambiguously marker classify 10-5 progeny as resistant (selecting for virulence) or susceptible (non-selecting) permitted the genetic mapping of the corresponding Avr1 gene by bulked segregant analysis. Using this approach, 14 genetic markers significantly linked to Avr1 were identified and placed within the context of a genome-wide linkage map produced for isolate P2 using samples from susceptible seedlings.

Genome size variation in the pine fusiform rust pathogen Cronartium quercuum f.sp. fusiforme as determined by flow cytometry.

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.

Evolution of genome size and complexity in Pinus.

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.

Reference karyotype and cytomolecular map for loblolly pine (Pinus taeda L.).

A reference karyotype is presented for loblolly pine (Pinus taeda L., subgenus Pinus, section Pinus, subsection Australes), based on fluorescent in situ hybridization (FISH), using 18S-28S rDNA, 5S rDNA, and an Arabidopsis-type telomere repeat sequence (A-type TRS). Well separated somatic chromosomes were prepared from colchicine-treated root meristems, using an enzymatic digestion technique. Statistical analyses performed on chromosome-arm lengths, centromeric indices, and interstitial rDNA and telomeric positions were based on observations from 6 well-separated metaphase cells from each of 3 unrelated trees. Statistically, 7 of the 12 loblolly pine chromosomes could be distinguished by their relative lengths. Centromeric indices were unable to distinguish additional chromosomes. However, the position and relative strength of the rDNA and telomeric sites made it possible to uniquely identify all of the chromosomes, providing a reference karyotype for use in comparative genome analyses. A dichotomous key was developed to aid in the identification of loblolly pine chromosomes and their comparison to chromosomes of other Pinus spp. A cytomolecular map was developed using the interstitial 18S-28S rDNA and A-type TRS signals. A total of 54 bins were assigned, ranging from 3 to 5 bins per chromosome. This is the first report of a chromosome-anchored physical map for a conifer that includes a dichotomous key for accurate and consistent identification of the P. taeda chromosomes.

Molecular evidence for an Asian origin and a unique westward migration of species in the genus Castanea via Europe to North America.

The genus Castanea (Fagaceae) is widely distributed in the deciduous forests of the Northern Hemisphere. The striking similarity between the floras of eastern Asia and those of eastern North America and the difference in chestnut blight resistance among species has been of interest to botanists for a century. To infer the biogeographical history of the genus, the phylogeny of Castanea was estimated using DNA sequence data from different regions of the chloroplast genome. Sequencing results support the genus Castanea as a monophyletic group with Castanea crenata as basal. 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 the European/North American species during the middle Eocene and a split between the European and the North American species in the late Eocene. The differentiation within North America and China might have occurred in early or late Miocene. 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 Castanea dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.

Markers linked to vegetative incompatibility (vic) genes and a region of high heterogeneity and reduced recombination near the mating type locus (MAT) in Cryphonectria parasitica.

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.

A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers.

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 x 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.

Genetic interaction of the fusiform rust fungus with resistance gene fr1 in loblolly pine.

ABSTRACT We propose a method for defining DNA markers linked to Cronartium quercuum f. sp. fusiforme avirulence (Avr) genes. However, before this method can be successfully employed, a spore competition study was needed to determine the genetic composition of single pycnial drops and multiple drops on single galls when using the standard inoculation procedure, whether virulent (avr1) basidiospores ever predispose some resistant (Fr1/fr1) trees to infection by avirulent (Avr1) basidiospores, and whether avr1 and Avr1 basidiospores equally infect susceptible (fr1/fr1) trees. Results of this study suggest that multiple infections within a single gall are common using the concentrated basidiospore system, resulting on average in >4 infection events per tree. Due to multiple infections within a single gall, an individual pycnial drop cannot be assumed to consist of spores from only a single haploid pycnium. Roughly 57% of the drops harvested were found to consist of more than one haploid genotype, most likely due to the physical mixing of spores from genetically different pycnia. Most importantly, although multiple infections do occur in the formation of a single gall, there is no evidence to suggest that the genetics of the proposed gene-for-gene interaction are compromised. Only avr1 basidiospores were observed to cause infection on Fr1/fr1 trees, whereas both avr1 and Avr1 basidiospores were observed to cause infection on fr1/fr1 trees, albeit not at equal frequencies.