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Chestnut blight (caused by Cryphonectria parasitica ), together with Phytophthora root rot (caused by Phytophthora cinnamomi ), has nearly extirpated American chestnut ( Castanea dentata ) from its native range. In contrast to the susceptibility of American chestnut, many Chinese chestnut ( C. mollissima ) genotypes are resistant to blight. In this research, we performed a series of genome-wide association studies for blight resistance originating from three unrelated Chinese chestnut trees (Mahogany, Nanking and M16) and a Quantitative Trait Locus (QTL) study on a Mahogany-derived inter-species F2 family. We evaluated trees for resistance to blight after artificial inoculation with two fungal strains and scored nine morpho-phenological traits that are the hallmarks of species differentiation between American and Chinese chestnuts. Results support a moderately complex genetic architecture for blight resistance, as 31 QTLs were found on 12 chromosomes across all studies. Additionally, although most morpho-phenological trait QTLs overlap or are adjacent to blight resistance QTLs, they tend to aggregate in a few genomic regions. Finally, comparison between QTL intervals for blight resistance and those previously published for Phytophthora root rot resistance, revealed five common disease resistance regions on chromosomes 1, 5, and 11. Our results suggest that it will be difficult, but still possible to eliminate Chinese chestnut alleles for the morpho-phenological traits while achieving relatively high blight resistance in a backcross hybrid tree. We see potential for a breeding scheme that utilizes marker-assisted selection early for relatively large effect QTLs followed by genome selection in later generations for smaller effect genomic regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fan, Georgi, Hebard, Zhebentyayeva, Yu, Sisco, Fitzsimmons, Staton, Abbott and Nelson.)

The American chestnut (Castanea dentata, 2n = 2x = 24), once known as the "King of the Appalachian Forest", was decimated by chestnut blight during the first half of the twentieth century by an invasive fungus (Cryphonectria parasitica). The Chinese chestnut (C. mollissima, 2n = 2x = 24), in contrast to American chestnut, is resistant to this blight. Efforts are being made to transfer this resistance to American chestnut through backcross breeding and genetic engineering. Both chestnut genomes have been genetically mapped and recently sequenced to facilitate gene discovery efforts aimed at assisting molecular breeding and genetic engineering. To complement and extend this genomic work, we analyzed the distribution and organization of their ribosomal DNAs (35S and 5S rDNA), and the chromatin composition of the nucleolus organizing region (NOR)-associated satellites. Using fluorescent in situ hybridization (FISH), we have identified two 35S (one major and one minor) and one 5S rDNA sites. The major 35S rDNA sites are terminal and sub-terminal in American and Chinese chestnuts, respectively, originating at the end of the short arm of the chromosome, extending through the secondary constriction and into the satellites. An additional 5S locus was identified in certain Chinese chestnut accessions, and it was linked distally to the major 35S site. The NOR-associated satellite in Chinese chestnut was found to comprise a proximal region packed with 35S rDNA and a distinct distal heterochromatic region. In contrast, the American chestnut satellite was relatively small and devoid of the distal heterochromatic region., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The fields of tree breeding and genetic engineering can be perceived as being antagonistic towards each other-genetic engineers suggesting that tree breeding is too slow and expensive and tree breeders suggesting that genetic engineering is not practical and too expensive. We argue here that both fields have much to offer forestry and the success of each is intimately tied to the other. The major purposes of genetic engineering in forestry are described as well as the importance of evaluating tree engineering initiatives in the context of tree improvement and silviculture and integrating genetic engineering with tree breeding from start to finish. A generalized approach is developed that meets these requirements and demonstrates the interrelationships between the activities and phases of each program. In addition, a case study of the American chestnut ( Castanea dentata ) is provided to underscore the value of integrating genetic engineering and tree breeding programs to achieve a long-term conservation goal., Competing Interests: Conflict of interestCDN declares no conflicts of interest., (© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022.)

Resistance to fusiform rust disease in loblolly pine (Pinus taeda) is a classic gene-for-gene system. Early resistance gene mapping in the P. taeda family 10-5 identified RAPD markers for a major fusiform rust resistance gene, Fr1. More recently, single nucleotide polymorphism (SNP) markers associated with resistance were mapped to a full-length gene model in the loblolly pine genome encoding for a nucleotide-binding site leucine-rich repeat (NLR) protein. NLR genes are one of the most abundant gene families in plant genomes and are involved in effector-triggered immunity. Inter- and intraspecies studies of NLR gene diversity and expression have resulted in improved disease resistance. To characterize NLR gene diversity and discover potential resistance genes, we assembled de novo transcriptomes from 92 loblolly genotypes from across the natural range of the species. In these transcriptomes, we identified novel NLR transcripts that are not present in the loblolly pine reference genome and found significant geographic diversity of NLR genes providing evidence of gene family evolution. We designed capture probes for these NLRs to identify and map SNPs that stably cosegregate with resistance to the SC20-21 isolate of Cronartium quercuum f.sp. fusiforme (Cqf) in half-sib progeny of the 10-5 family. We identified 10 SNPs and 2 quantitative trait loci associated with resistance to SC20-21 Cqf. The geographic diversity of NLR genes provides evidence of NLR gene family evolution in loblolly pine. The SNPs associated with rust resistance provide a resource to enhance breeding and deployment of resistant pine seedlings., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The soilborne oomycete Phytophthora cinnamomi -which causes root rot, trunk cankers, and stem lesions on an estimated 5,000 plant species worldwide-is a lethal pathogen of American chestnut ( Castanea dentata ) as well as many other woody plant species. P. cinnamomi is particularly damaging to chestnut and chinquapin trees ( Castanea spp.) in the southern portion of its native range in the United States due to relatively mild climatic conditions that are conductive to disease development. Introduction of resistant genotypes is the most practical solution for disease management in forests because treatment with fungicides and eradication of the pathogen are neither practical nor economically feasible in natural ecosystems. Using backcross families derived from crosses of American chestnuts with two resistant Chinese chestnut cultivars Mahogany and Nanking, we constructed linkage maps and identified quantitative trait loci (QTLs) for resistance to P. cinnamomi that had been introgressed from these Chinese chestnut cultivars. In total, 957 plants representing five cohorts of three hybrid crosses were genotyped by sequencing and phenotyped by standardized inoculation and visual examination over a 6-year period from 2011 to 2016. Eight parental linkage maps comprising 7,715 markers were constructed, and 17 QTLs were identified on four linkage groups (LGs): LG_A, LG_C, LG_E, and LG_K. The most consistent QTLs were detected on LG_E in seedlings from crosses with both 'Mahogany' and 'Nanking' and LG_K in seedlings from 'Mahogany' crosses. Two consistent large and medium effect QTLs located ∼10 cM apart were present in the middle and at the lower end of LG_E; other QTLs were considered to have small effects. These results imply that the genetic architecture of resistance to P. cinnamomi in Chinese chestnut × American chestnut hybrid progeny may resemble the P. sojae -soybean pathosystem, with a few dominant QTLs along with quantitatively inherited partial resistance conferred by multiple small-effect QTLs.

Trees use many mechanisms to adapt and respond to stressful conditions. The phenylpropanoid pathway in particular is known to be associated with a diverse suite of plant stress responses. In this study, we explored the relationship between the phenylpropanoid pathway metabolite production, gene expression and adaptive trait variation associated with floral bud reactivation during and following dormancy in Prunus armeniaca L. (apricot). Concentrations of eight phenylpropanoid metabolites were measured during chill accumulation and at developmental stages corresponding to the emergence of sepals and petals in floral buds of varieties that differ phenotypically in bloom date (BD). A significant interaction effect of chill hours and BD phenotype on the concentration of each of the compounds was observed (mixed analysis of variance, P < 0.05), with the concentration of most phenylpropanoid metabolites dropping precipitously when sepals and petals emerged. While phenylpropanoid biosynthetic gene expression patterns were more variable in general, expression changed over time and was impacted, although to a lesser degree, by BD phenotype. Furthermore, separation of BD phenotypic groups was most pronounced when early and late BD varieties were at different developmental stages, i.e., 800 chill hours. Taken together, these results suggest that the phenylpropanoid pathway is associated with floral bud reactivation in apricot. Furthermore, we show that the phenylpropanoid pathway is also impacted by phenological trait variation associated with dormancy. A better understanding of how apricot and other perennial tree species respond and adapt to environmental perturbations will be critical for improvement programs aimed at identifying and breeding trees more suitable for rapidly changing environments., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To understand how diverse mutualisms coevolve and how species adapt to complex environments, a description of the underlying genetic basis of the traits involved must be provided. For example, in diverse coevolving mutualisms, such as the interaction of host plants with a suite of symbiotic mycorrhizal fungi, a key question is whether host plants can coevolve independently with multiple species of symbionts, which depends on whether those interactions are governed independently by separate genes or pleiotropically by shared genes. To provide insight into this question, we employed an association mapping approach in a clonally replicated field experiment of loblolly pine (Pinus taeda L.) to identify genetic components of host traits governing ectomycorrhizal (EM) symbioses (mycorrhizal traits). The relative abundances of different EM fungi as well as the total number of root tips per cm root colonized by EM fungi were analyzed as separate mycorrhizal traits of loblolly pine. Single-nucleotide polymorphisms (SNPs) within candidate genes of loblolly pine were associated with loblolly pine mycorrhizal traits, mapped to the loblolly pine genome, and their putative protein function obtained when available. The results support the hypothesis that ectomycorrhiza formation is governed by host genes of large effect that apparently have independent influences on host interactions with different symbiont species., (© 2019 John Wiley & Sons Ltd.)

Background: Loblolly pine (Pinus taeda L.) is one of the most widely planted and commercially important forest tree species in the USA and worldwide, and is an object of intense genomic research. However, whole genome resequencing in loblolly pine is hampered by its large size and complexity and a lack of a good reference. As a valid and more feasible alternative, entire exome sequencing was hence employed to identify the gene-associated single nucleotide polymorphisms (SNPs) and to genotype the sampled trees., Results: The exons were captured in the ADEPT2 association mapping population of 375 clonally-propagated loblolly pine trees using NimbleGen oligonucleotide hybridization probes, and then exome-enriched genomic DNA fragments were sequenced using the Illumina HiSeq 2500 platform. Oligonucleotide probes were designed based on 199,723 exons (≈49 Mbp) partitioned from the loblolly pine reference genome (PineRefSeq v. 1.01). The probes covered 90.2 % of the target regions. Capture efficiency was high; on average, 67 % of the sequence reads generated for each tree could be mapped to the capture target regions, and more than 70 % of the captured target bases had at least 10X sequencing depth per tree. A total of 972,720 high quality SNPs were identified after filtering. Among them, 53 % were located in coding regions (CDS), 5 % in 5' or 3' untranslated regions (UTRs) and 42 % in non-target and non-coding regions, such as introns and adjacent intergenic regions collaterally captured. We found that linkage disequilibrium (LD) decayed very rapidly, with the correlation coefficient (r (2)) between pairs of SNPs linked within single scaffolds decaying to half maximum (r (2) = 0.22) within 55 bp, to r (2) = 0.1 within 192 bp, and to r (2) = 0.05 within 451 bp. Population structure analysis using unlinked SNPs demonstrated the presence of two main distinct clusters representing western and eastern parts of the loblolly pine range included in our sample of trees., Conclusions: The obtained results demonstrated the efficiency of exome capture for genotyping species such as loblolly pine with a large and complex genome. The highly diverse genetic variation reported in this study will be a valuable resource for future genetic and genomic research in loblolly pine.

A consensus genetic map for Pinus taeda (loblolly pine) and Pinus elliottii (slash pine) was constructed by merging three previously published P. taeda maps with a map from a pseudo-backcross between P. elliottii and P. taeda. The consensus map positioned 3856 markers via genotyping of 1251 individuals from four pedigrees. It is the densest linkage map for a conifer to date. Average marker spacing was 0.6 cM and total map length was 2305 cM. Functional predictions of mapped genes were improved by aligning expressed sequence tags used for marker discovery to full-length P. taeda transcripts. Alignments to the P. taeda genome mapped 3305 scaffold sequences onto 12 linkage groups. The consensus genetic map was used to compare the genome-wide linkage disequilibrium in a population of distantly related P. taeda individuals (ADEPT2) used for association genetic studies and a multiple-family pedigree used for genomic selection (CCLONES). The prevalence and extent of LD was greater in CCLONES as compared to ADEPT2; however, extended LD with LGs or between LGs was rare in both populations. The average squared correlations, r(2), between SNP alleles less than 1 cM apart were less than 0.05 in both populations and r(2) did not decay substantially with genetic distance. The consensus map and analysis of linkage disequilibrium establish a foundation for comparative association mapping and genomic selection in P. taeda and P. elliottii., (Copyright © 2015 Westbrook et al.)

Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

Rapidly enhancing oleoresin production in conifer stems through genomic selection and genetic engineering may increase resistance to bark beetles and terpenoid yield for liquid biofuels. We integrated association genetic and genomic prediction analyses of oleoresin flow (g 24 h(-1)) using 4854 single nucleotide polymorphisms (SNPs) in expressed genes within a pedigreed population of loblolly pine (Pinus taeda) that was clonally replicated at three sites in the southeastern United States. Additive genetic variation in oleoresin flow (h(2) ≈ 0.12-0.30) was strongly correlated between years in which precipitation varied (r(a) ≈ 0.95), while the genetic correlation between sites declined from 0.8 to 0.37 with increasing differences in soil and climate among sites. A total of 231 SNPs were significantly associated with oleoresin flow, of which 81% were specific to individual sites. SNPs in sequences similar to ethylene signaling proteins, ABC transporters, and diterpenoid hydroxylases were associated with oleoresin flow across sites. Despite this complex genetic architecture, we developed a genomic prediction model to accelerate breeding for enhanced oleoresin flow that is robust to environmental variation. Results imply that breeding could increase oleoresin flow 1.5- to 2.4-fold in one generation., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

We propose a conceptual framework for restoration of threatened plant species that encourages integration of technological, ecological, and social spheres. A sphere encompasses ideas relevant to restoration and the people working within similar areas of influence or expertise. Increased capacity within a sphere and a higher degree of coalescing among spheres predict a greater probability of successful restoration. We illustrate this with Castanea dentata, a foundation forest tree in North America that was annihilated by an introduced pathogen; the species is a model that effectively merges biotechnology, reintroduction biology, and restoration ecology. Because of C. dentata's ecological and social importance, scientists have aggressively pursued blight resistance through various approaches. We summarize recent advancements in tree breeding and biotechnology that have emerged from C. dentata research, and describe their potential to bring new tools to bear on socio-ecological restoration problems. Successful reintroduction of C. dentata will also depend upon an enhanced understanding of its ecology within contemporary forests. We identify a critical need for a deeper understanding of societal influences that may affect setting and achieving realistic restoration goals. Castanea dentata may serve as an important model to inform reintroduction of threatened plant species in general and foundation forest trees in particular., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

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., (Published by Elsevier Inc.)

Background: Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map., Results: The updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi) across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs) and 149 were from non-transcribed genomic sequences (genomic-SSRs). Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO) terms. Duplicate (i.e., redundant accessory) and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He, among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at http://www.conifergdb.org/pinemap., Conclusions: Many polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped genes with UniGene clusters and GO terms allowed assessment of redundant and paralogous EST markers and further improved the quality and utility of the genetic map for P. taeda.

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

Simple sequence repeats (SSRs) have proven to be extremely valuable DNA markers for genetic mapping and population genetic analyses. However, data collected across laboratories or even within laboratories are difficult to combine due to challenges in standardizing allele names, especially for nonmodel systems. Here we provide a new approach for standardizing SSR allele names that combines several previously recognized components for standardization, including reference samples/alleles, cumulative binsets, static between-allele spacing, and interval allele naming.

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.

Natural populations of forest trees exhibit striking phenotypic adaptations to diverse environmental gradients, thereby making them appealing subjects for the study of genes underlying ecologically relevant phenotypes. Here, we use a genome-wide data set of single nucleotide polymorphisms genotyped across 3059 functional genes to study patterns of population structure and identify loci associated with aridity across the natural range of loblolly pine (Pinus taeda L.). Overall patterns of population structure, as inferred using principal components and Bayesian cluster analyses, were consistent with three genetic clusters likely resulting from expansions out of Pleistocene refugia located in Mexico and Florida. A novel application of association analysis, which removes the confounding effects of shared ancestry on correlations between genetic and environmental variation, identified five loci correlated with aridity. These loci were primarily involved with abiotic stress response to temperature and drought. A unique set of 24 loci was identified as F(ST) outliers on the basis of the genetic clusters identified previously and after accounting for expansions out of Pleistocene refugia. These loci were involved with a diversity of physiological processes. Identification of nonoverlapping sets of loci highlights the fundamental differences implicit in the use of either method and suggests a pluralistic, yet complementary, approach to the identification of genes underlying ecologically relevant phenotypes.

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.

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)

The recent completion of a draft sequence of the poplar (Populus trichocarpa Torr. & Gray ex Brayshaw) genome has advanced forest tree genetics to an unprecedented level. A "parts list" for a forest tree has been produced, opening up new opportunities for dissecting the interworkings of tree growth and development. In the relatively near future we can anticipate additional reference genome sequences, including the much larger Pinus genome. One goal is to use this information to define the genomic attributes that affect the phenotypic performances of trees growing in various environments. A first step is the definition of ideotypes that constitute optimal tree and stand-level performance. Following this, the genome can be systematically searched for genetic elements and their allelic variants that affect the specified traits. Knowledge of these alleles and their effects will facilitate the development of efficient tree improvement programs through genome-guided breeding and genetic engineering and further our mechanistic understanding of trait variation. Improved mechanistic understanding of tree growth and development is needed to develop process models that will allow us to anticipate and manage change in forest ecosystems. Here we consider the development of an ideotype for loblolly pine (Pinus taeda L.) and discuss genomic approaches for studying the component traits that will enable advances in process model development and the genetic improvement of this important conifer.

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.

Genetic association is a powerful method for dissecting complex adaptive traits due to (i) fine-scale mapping resulting from historical recombination, (ii) wide coverage of phenotypic and genotypic variation within a single experiment, and (iii) the simultaneous discovery of loci and alleles. In this article, genetic association among single nucleotide polymorphisms (58 SNPs) from 20 wood- and drought-related candidate genes and an array of wood property traits with evolutionary and commercial importance, namely, earlywood and latewood specific gravity, percentage of latewood, earlywood microfibril angle, and wood chemistry (lignin and cellulose content), was tested using mixed linear models (MLMs) that account for relatedness among individuals by using a pairwise kinship matrix. Population structure, a common systematic bias in association studies, was assessed using 22 nuclear microsatellites. Different phenotype:genotype associations were found, some of them confirming previous evidence from collocation of QTL and genes in linkage maps (for example, 4cl and percentage of latewood) and two that involve nonsynonymous polymorphisms (cad SNP M28 with earlywood specific gravity and 4cl SNP M7 with percentage of latewood). The strongest genetic association found in this study was between allelic variation in alpha-tubulin, a gene involved in the formation of cortical microtubules, and earlywood microfibril angle. Intragenic LD decays rapidly in conifers; thus SNPs showing genetic association are likely to be located in close proximity to the causative polymorphisms. This first multigene association genetic study in forest trees has shown the feasibility of candidate gene strategies for dissecting complex adaptive traits, provided that genes belonging to key pathways and appropriate statistical tools are used. This approach is of particular utility in species such as conifers, where genomewide strategies are limited by their large genomes.

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.

A targeted genomics approach was used to construct a cDNA array of potential pathogen-regulated genes for investigating host-pathogen interactions in pine trees (Pinus species). This array, containing a nonredundant set of 311 cDNAs, was assembled by combining smaller sets of cDNAs generated by differential display or suppression subtraction hybridization using a variety of pathogen treatments and elicitors. The array was probed to identify host genes regulated by Fusarium circinatum, a necrotrophic fungus that incited pitch canker disease on pine stems. A set of 29 cDNAs were induced during the disease state. Notably, cDNAs on the array that were derived from experiments with fusiform rust, incited by Cronartium quercuum f. sp. fusiforme (a biotrophic fungus) were unregulated by Fusarium. the results imply distinct genetic responses in pine to diseases incited by necrotrophs and biotrophs. This cDNA collection expands the genomics toolkit for understanding interactions between conifers and their microbial associates in forest ecosystems.