Nonnative invasive pathogens have decimated North American forests for over 100 years, and additional pathogens continue to arrive. These pathogens are destined to be permanent fixtures in the ecosystem. In many cases, management activities have been unsuccessful in slowing the spread of these pathogens or in restoring forests. Genetic resistance potentially provides an invaluable management tool for restoring these species or using them in plantations. Although native tree species are highly susceptible to some nonnative pathogens, a low frequency of resistance is present in even those North American host species most affected. Classical breeding methods can produce genetically diverse and resistant populations for reforestation or restoration of natural forests. However, any operational program for developing resistant populations of forest trees must contend with relatively long generation times, as well as the long-lived nature of trees and the potential of the pathogen to evolve. Western white pine (Pinus monticola), Port-Orford-cedar (Chamaecyparis lawsoniana), American chestnut (Castanea dentata), and American elm (Ulmus americana) are examples of species for which there are successful breeding programs for disease resistance. Examples from these species, with a particular focus on western white pine and white pine blister rust [Cronartium ribicola] resistance, will be used to illustrate some of the successes in operational programs, as well as to discuss some of the research needs and current unknowns in developing durable resistance. Some of these programs have been ongoing for 50 years, while the Port-Orford-cedar program began only in the last decade. Resistant seedlings from several programs are now being used in reforestation and restoration.

Damage or mortality from pathogens can reduce productivity of forest plantations, as well as significantly harm natural forest ecosystems. Genetic resistance within the host species is the first line of defense for tree species. Resistance breeding programs for the native fusiform rust and exotic (to North America) white pine blister rust diseases are two of the longest concerted efforts in forest trees, spanning more than 50 years. Advances in developing greater genetic resistance have been made in both pathosystems, but unique challenges and opportunities in each system translate to different approaches. Fusiform rust resistance programs have mainly emphasized complete resistance, while partial resistance plays a prominent role in white pine blister rust resistance programs. Advances in the development of molecular genetic tools now permit investigations in conifers and their associated rust pathogens. Good progress has been made in identifying resistant populations and understanding resistance in these pathosystems, and resistant stock is now being used extensively for reforestation and restoration. These programs represent great success stories brought to fruition by the long-term efforts. However, continued support will be needed to enhance the level and fully realize the potential of durable genetic resistance in these invaluable North American conifer species.

The fungus Cronartium ribicola (Cri) is an economically and ecologically important forest pathogen that causes white pine blister rust (WPBR) disease on five-needle pines. To cause stem cankers and kill white pine trees the fungus elaborates a life cycle with five stages of spore development on five-needle pines and the alternate host Ribes plants. To increase our understanding of molecular WP-BR interactions, here we report genome-wide transcriptional profile analysis of C. ribicola using RNA-seq. cDNA libraries were constructed from aeciospore, urediniospore, and western white pine (Pinus monticola) tissues post Cri infection. Over 200 million RNA-seq 100-bp paired-end (PE) reads from rust fungal spores were de novo assembled and a reference transcriptome was generated with 17,880 transcripts that were expressed from 13,629 unigenes. A total of 734 unique proteins were predicted as a part of the Cri secretome from complete open reading frames (ORFs), and 41 % of them were Cronartium-specific. This study further identified a repertoire of candidate effectors and other pathogenicity determinants. Differentially expressed genes (DEGs) were identified to gain an understanding of molecular events important during the WPBR fungus life cycle by comparing Cri transcriptomes at different infection stages. Large-scale changes of in planta gene expression profiles were observed, revealing that multiple fungal biosynthetic pathways were enhanced during mycelium growth inside infected pine stem tissues. Conversely, many fungal genes that were up-regulated at the urediniospore stage appeared to be signalling components and transporters. The secreted fungal protein genes that were up-regulated in pine needle tissues during early infection were primarily associated with cell wall modifications, possibly to mask the rust pathogen from plant defenses. This comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions. The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.

Western white pine (WWP, Pinus monticola Douglas ex D. Don) is of high interest in forest breeding and conservation because of its high susceptibility to the invasive disease white pine blister rust (WPBR, caused by the fungus Cronartium ribicola J. C. Fisch). However, WWP lacks genomic resource development and is evolutionarily far away from plants with available draft genome sequences. Here we report a single nucleotide polymorphism (SNP) study by bulked segregation-based RNA-Seq analysis. A collection of resistance germplasm was used for construction of cDNA libraries and SNP genotyping. Approximately 36–89 million 2 × 100-bp reads were obtained per library and de-novo assembly generated the first shoot-tip reference transcriptome containing a total of 54,661 unique transcripts. Bioinformatic SNP detection identified >100,000 high quality SNPs in three expressed candidate gene groups: Pinus highly conserved genes (HCGs), differential expressed genes (DEGs) in plant defense response, and resistance gene analogs (RGAs). To estimate efficiency of in-silico SNP discovery, genotyping assay was developed by using Sequenom iPlex and it unveiled SNP success rates from 40.1% to 61.1%. SNP clustering analyses consistently revealed distinct populations, each composed of multiple full-sib seed families by parentage assignment in the WWP germplasm collection. Linkage disequilibrium (LD) analysis identified six genes in significant association with major gene (Cr2) resistance, including three RGAs (two NBS-LRR genes and one receptor-like protein kinase -RLK gene), two HCGs, and one DEG. At least one SNP locus provided an excellent marker for Cr2 selection across P. monticola populations. The WWP shoot tip transcriptome and those validated SNP markers provide novel genomic resources for genetic, evolutionary and ecological studies. SNP loci of those candidate genes associated with resistant phenotypes can be used as positional and functional variation sites for further characterization of WWP major gene resistance against C. ribicola. Our results demonstrate that integration of RNA-seq-based transcriptome analysis and high-throughput genotyping is an effective approach for discovery of a large number of nucleotide variations and for identification of functional gene variants associated with adaptive traits in a non-model species.

Linkage of DNA markers with phenotypic traits provides essential information to dissect clustered genes with potential phenotypic contributions in a target genome region. Pinus flexilis E. James (limber pine) is a keystone five-needle pine species in mountain-top ecosystems of North America. White pine blister rust (WPBR), caused by a non-native fungal pathogen Cronartium ribicola (J.C. Fisch.), has resulted in mortality in this conifer species and is still spreading through the distribution. The objective of this research was to develop P. flexilis transcriptome-wide single nucleotide polymorphism (SNP) markers using RNA-seq analysis for genetic mapping of the major gene (Cr4) that confers complete resistance to C. ribicola. Needle tissues of one resistant and two susceptible seedling families were subjected to RNA-seq analysis. In silico SNP markers were uncovered by mapping the RNA-seq reads back to the de novo assembled transcriptomes. A total of 110,573 in silico SNPs and 2,870 indels were identified with an average of 3.7 SNPs per Kb. These SNPs were distributed in 17,041 unigenes. Of these polymorphic P. flexilis unigenes, 6,584 were highly conserved as compared to the genome sequence of P. taeda L (loblolly pine). High-throughput genotyping arrays were designed and were used to search for Cr4-linked genic SNPs in megagametophyte populations of four maternal trees by haploid-segregation analysis. A total of 32 SNP markers in 25 genes were localized on the Cr4 linkage group (LG). Syntenic relationships of this Cr4-LG map with the model conifer species P. taeda anchored Cr4 on Pinus consensus LG8, indicating that R genes against C. ribicola have evolved independently in different five-needle pines. Functional genes close to Cr4 were annotated and their potential roles in Cr4-mediated resistance were further discussed. We demonstrated a very effective, low-cost strategy for developing a SNP genetic map of a phenotypic trait of interest. SNP discovery through transcriptome comparison was integrated with high-throughput genotyping of a small set of in silico SNPs. This strategy may be applied to mapping any trait in non-model plant species that have complex genomes. Whole transcriptome sequencing provides a powerful tool for SNP discovery in conifers and other species with complex genomes, for which sequencing and annotation of complex genomes is still challenging. The genic SNP map for the consensus Cr4-LG may help future molecular breeding efforts by enabling both Cr4 positional characterization and selection of this gene against WPBR.