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.This research was supported in part by Cooperative Agreement 09-CA-11330126-058 between the USDA-Forest Service Southern Research Station (Southern Institute of Forest Genetics) and the University of Florida

On the Origin of Fusiform Rust Resistance in Loblolly Pine

Studies of geographic variation in loblolly pine (Pinus taeda L.) have shown that seed sources from the western (generally west of the Mississippi River) and the northeastern part of the natural distribution are relatively resistant to fusiform rust, and those from elsewhere are susceptible. The greatest incidence of infection, on the other hand, is in the center of the distribution, exactly where the frequency of resistant genotypes is low. One might expect that the frequency of resistant genotypes would be higher where the disease is more prevalent, due to natural selection. It has been proposed that (1) fusiform rust resistance in loblolly pine in the west originates from hybridization with shortleaf pine. It is well known that shortleaf is resistant to fusiform rust, and it is also known that natural hybrids between the two species exist, and they seem to be more common in the west. (2) In the northeastern loblolly, it has been proposed that hybridization with pond pine is the source of resistance. Once again, natural hybridization between loblolly and pond pine is known to exist in the northeast, but not much is known about the relative resistance of pond pine to fusiform rust. Allozyme data was used to refute hypothesis (1) and cortical monoterpene data was used to refute hypothesis (2). A hypothesis is proposed involving selection during the Pleistocene to explain the present pattern of resistance and the development of a gene-for-gene pathosystem.Papers and abstracts from the 27th Southern Forest Tree Improvement Conference held at Oklahoma State University in Stillwater, Oklahoma on June 24-27, 2003

Top Grafting Longleaf x Slash Pine F1 Hybrids on Mature Longleaf and Slash Pine Interstocks

Top grafting is used to accelerate the breeding cycle of loblolly pine (Pinus taeda L). Scions, collected from seedlings as young as 1 year from seed, are grafted onto mature interstocks of the same or a related species. Male and female strobili production often begins 1 or 2 years after grafting, thus potentially decreasing the generation time by several years over conventional accelerated breeding methods. We are interested in applying top grafting to our breeding program involving interspecific hybrids, in particular longleaf x slash pine (Pinus palustris Mill. x Pinus elliottii Englem. var. elliottii) hybrids and their backcross generations. Towards this end, we grafted scions from 16 longleaf x slash pine F1 selections onto two longleaf and two slash pine interstock trees of different genotype. The F1 selections were 6 years old and none showed any signs of strobili development prior to, or during the experiment. A total of 100 grafts were made on to each interstock species. Scion survival after the second year was significantly higher on slash (72%) pine interstocks than on longleaf pine (18%). However there were no differences in male or female strobili production per living scion between the interstock species. Scions grew longer and produced more branches on slash pine interstocks than they did on longleaf. Given the relatively poor survival of the scions grafted onto longleaf interstocks and the reasonably good strobili production of scions grafted onto slash, we recommend using slash pine as the interstock species for top grafting longleaf x slash F1 hybrids.Papers and abstracts from the 27th Southern Forest Tree Improvement Conference held at Oklahoma State University in Stillwater, Oklahoma on June 24-27, 2003

Development of Differential Screening Panels for Slash Pine-Fusiform Rust Reaction Types

Understanding the genetic basis for disease resistance is crucial to ensure a successful breeding and deployment strategy for slash pine. A complementary genetic interaction model has been hypothesized for the pine-fusiform rust pathosystem. In such a model, resistance is due to a major gene effect that confers resistance to some strains of pathogens, but not others. If this model for the pine-fusiform rust pathosystem is correct, it will be necessary to identify the genes for resistance in order to develop successful strategies. Some resistance genes are dispersed throughout the genome and can be identified by genetic mapping. However, resistance genes may be clustered in the host and it may be possible to identify them only by empirically determining their differential interaction with different pathogen strains. In the latter case, it will be necessary to develop a panel of slash pine parents that can be used to screen for presence/absence of avirulence among collections of the pathogen and a panel of pathogen strains that can be used to screen for presence/absence of resistance genes in the host. The feasibility of developing such screening panels was tested by evaluating 43 slash pine (Pinus elliottii, Engelm. var elliottii) parents with 8 single-urediniospore cultures of Cronartium quercuum f. sp. fusiforme. The slash pine parents were selected to include a range of resistance levels based on their performance in an earlier screening trial. Each parent was control-pollinated with a pollen mix collected from 10 trees known to be highly susceptible to bulk inocula. The 8 inocula were derived from eight single-urediniospore rust cultures obtained from galls from across the southeastern USA. Examination of the reaction data under the assumption of a gene-for-gene host:pathogen system allowed us to infer the presence of at least 8 putative genotypes for resistance in the host and pathogenicity in the fungus, suggesting that screening panels can be developed with current propagation technologies.Papers and abstracts from the 27th Southern Forest Tree Improvement Conference held at Oklahoma State University in Stillwater, Oklahoma on June 24-27, 2003

Loblolly Pine Karyotype Using FISH and DAPI Positive Banding

A loblolly pine (Pinus taeda L.) karyotype has been developed based on fluorescent insitu hybridization (FISH) using cyto-molecular landmarks including plant telomere repeat, 18S-28S rDNA and 5S rDNA probes and DAPI positive bands. Somatic chromosome spreads of loblolly pine root tips were prepared using a modified enzymatic digestion technique. We observed ten pairs of long metacentric, one pair of long submetacentric and one pair of short sub-metacentric chromosomes. All the chromosomes showed characteristic DAPI positive bands (A-T rich regions) near and/or around the centromeres. At least one DAPI positive band was also observed in intercalary positions on all chromosome arms. Plant telomere FISH signals were observed towards the end of each chromosomal arm as expected. In addition, most of the chromosomes showed telomeric sites near and/or around the centromeres except for one or possibly two chromosomes. A total of seventeen 18S-28S rDNA sites were identified per haploid genome. Eight of these were located near and/or around the centromeres and seven were at intercalary positions. One major 5S rDNA site was observed in an intercalary region of a metacentric chromosome that lacked 18S-28S rDNA sites. One or possibly two minor 5S rDNA sites were observed near the ends of two different chromosomes. We are also developing a slash pine karyotype for direct comparison with loblolly as well as a comparison with a previously published slash karyotype (Doudrick et al. 1995, Journal of Heredity 86:289-296). Finally, we will provide an update on our progress toward using BAC clones as FISH probes on pine chromosomes.Papers and abstracts from the 27th Southern Forest Tree Improvement Conference held at Oklahoma State University in Stillwater, Oklahoma on June 24-27, 2003

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC(1)F(1) plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs

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

Finding Single Copy Genes Out of Sequenced Genomes for Multilocus Phylogenetics in Non-Model Fungi

Historically, fungal multigene phylogenies have been reconstructed based on a small number of commonly used genes. The availability of complete fungal genomes has given rise to a new wave of model organisms that provide large number of genes potentially useful for building robust gene genealogies. Unfortunately, cross-utilization of these resources to study phylogenetic relationships in the vast majority of non-model fungi (i.e. “orphan” species) remains an unexamined question. To address this problem, we developed a method coupled with a program named “PHYLORPH” (PHYLogenetic markers for ORPHans). The method screens fungal genomic databases (107 fungal genomes fully sequenced) for single copy genes that might be easily transferable and well suited for studies at low taxonomic levels (for example, in species complexes) in non-model fungal species. To maximize the chance to target genes with informative regions, PHYLORPH displays a graphical evaluation system based on the estimation of nucleotide divergence relative to substitution type. The usefulness of this approach was tested by developing markers in four non-model groups of fungal pathogens. For each pathogen considered, 7 to 40% of the 10–15 best candidate genes proposed by PHYLORPH yielded sequencing success. Levels of polymorphism of these genes were compared with those obtained for some genes traditionally used to build fungal phylogenies (e.g. nuclear rDNA, β-tubulin, γ-actin, Elongation factor EF-1α). These genes were ranked among the best-performing ones and resolved accurately taxa relationships in each of the four non-model groups of fungi considered. We envision that PHYLORPH will constitute a useful tool for obtaining new and accurate phylogenetic markers to resolve relationships between closely related non-model fungal species