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Societal impact statement Forests world‐wide are being negatively affected by non‐native, invasive pathogens and pests, and some tree species face uncertain futures. To retain these species as components of future forests, the rare genetic resistance that exists needs to be identified and harnessed. The applied tree improvement program for whitebark pine (Pinus albicaulis), a threatened (in the United States) and endangered (in Canada) keystone species in many forests in western North America, provides an example of what can be accomplished in a relatively short timeframe. The level and frequency of resistance vary by location, and this information will be used to implement the national restoration plan. Summary Forest trees face serious threats from non‐native diseases and pests, often causing high mortality of both the existing trees and regeneration. Developing populations with genetic resistance can help restore forests and retain affected species. Resistance programs have historically focused on species of high economic importance; however, the threats to species of little direct economic value that provide other important ecosystem services are also great. We examined the frequency, level, and geographic variation in genetic resistance to white pine blister rust in the threatened Pinus albicaulis (whitebark pine), a keystone species in high‐elevation ecosystems in western North America. In the two trials reported here, 2‐year‐old seedling progeny of 225 whitebark pine parent trees were inoculated with two geographic sources of the fungal pathogen Cronartium ribicola and evaluated over 5 years for an array of resistance traits. The trials focused primarily on parent trees from the Oregon and Washington populations. We found unexpectedly high levels of quantitative resistance in some seedling families and populations, in stark contrast to levels observed in similar resistance programs with other North American white pine species such as Pinus monticola and Pinus lambertiana. The level of resistance found in some whitebark pine populations provides optimism about potential recovery efforts for this species. Restoration efforts are underway by government agencies, tribes, and non‐government organizations in both the United States and Canada. These efforts may help boost support for applied genetic resistance programs in other forest tree species severely affected by non‐native pathogens or pests. Forests world‐wide are being negatively affected by non‐native, invasive pathogens and pests, and some tree species face uncertain futures. To retain these species as components of future forests, the rare genetic resistance that exists needs to be identified and harnessed. The applied tree improvement program for whitebark pine (Pinus albicaulis), a threatened (in the United States) and endangered (in Canada) keystone species in many forests in western North America, provides an example of what can be accomplished in a relatively short timeframe. The level and frequency of resistance vary by location, and this information will be used to implement the national restoration plan.

Whitebark pine (WBP, Pinus albicaulis Engelm.) is an endangered conifer species due to heavy mortality from white pine blister rust (WPBR, caused by Cronartium ribicola) and mountain pine beetle (Dendroctonus ponderosae). Information about genetic diversity and population structure is of fundamental importance for its conservation and restoration. However, current knowledge on the genetic constitution and genomic variation is still limited for WBP. In this study, an integrated genomics approach was applied to characterize seed collections from WBP breeding programs in western North America. RNA-seq analysis was used for de novo assembly of the WBP needle transcriptome, which contains 97,447 protein-coding transcripts. Within the transcriptome, single nucleotide polymorphisms (SNPs) were discovered, and more than 22,000 of them were non-synonymous SNPs (ns-SNPs). Following the annotation of genes with ns-SNPs, 216 ns-SNPs within candidate genes with putative functions in disease resistance and plant defense were selected to design SNP arrays for high-throughput genotyping. Among these SNP loci, 71 were highly polymorphic, with sufficient variation to identify a unique genotype for each of the 371 individuals originating from British Columbia (Canada), Oregon and Washington (USA). A clear genetic differentiation was evident among seed families. Analyses of genetic spatial patterns revealed varying degrees of diversity and the existence of several genetic subgroups in the WBP breeding populations. Genetic components were associated with geographic variables and phenotypic rating of WPBR disease severity across landscapes, which may facilitate further identification of WBP genotypes and gene alleles contributing to local adaptation and quantitative resistance to WPBR. The WBP genomic resources developed here provide an invaluable tool for further studies and for exploitation and utilization of the genetic diversity preserved within this endangered conifer and other five-needle pines.

Since its introduction to North America in the early 1900s, white pine blister rust (WPBR) caused by the fungal pathogen has resulted in substantial economic losses and ecological damage to native North American five-needle pine species. The high susceptibility and mortality of these species, including limber pine ( ), creates an urgent need for the development and deployment of resistant germplasm to support recovery of impacted populations. Extensive screening for genetic resistance to WPBR has been underway for decades in some species but has only started recently in limber pine using seed families collected from wild parental trees in the USA and Canada. This study was conducted to characterize Alberta limber pine seed families for WPBR resistance and to develop reliable molecular tools for marker-assisted selection (MAS). Open-pollinated seed families were evaluated for host reaction following controlled infection using basidiospores. Phenotypic segregation for presence/absence of stem symptoms was observed in four seed families. The segregation ratios of these families were consistent with expression of major gene resistance (MGR) controlled by a dominant R locus. Based on linkage disequilibrium (LD)-based association mapping used to detect single nucleotide polymorphism (SNP) markers associated with MGR against , MGR in these seed families appears to be controlled by or other R genes in very close proximity to . These associated SNPs were located in genes involved in multiple molecular mechanisms potentially underlying limber pine MGR to , including NBS-LRR genes for recognition of effectors, signaling components, and a large set of defense-responsive genes with potential functions in plant effector-triggered immunity (ETI). Interactions of associated loci were identified for MGR selection in trees with complex genetic backgrounds. SNPs with tight Cr4-linkage were further converted to TaqMan assays to confirm their effectiveness as MAS tools. This work demonstrates the successful translation and deployment of molecular genetic knowledge into specific MAS tools that can be easily applied in a selection or breeding program to efficiently screen MGR against WPBR in Alberta limber pine populations.

Societal Impact Statement Non‐native pathogens and pests cause high mortality to tree species globally and may imperil the future viability of associated forest ecosystems. Phytophthora lateralis, an oomycete, causes Port‐Orford‐cedar root disease and is a major cause of mortality in the ecologically and economically important conifer species Chamaecyparis lawsoniana (Port‐Orford‐cedar). The P. lateralis resistance program shows promise to help stabilize C. lawsoniana in its native range of northwestern California and southwestern Oregon, USA, and serves as a leading example of disease resistance breeding in forest trees Summary A non‐native, invasive pathogen, Phytophthora lateralis, has caused extensive mortality within the native range, northern California and southern Oregon USA, of Chamaecyparis lawsoniana (Port‐Orford‐cedar), as well as in horticultural and amenity plantings in the USA and Europe. Restoration of affected sites is contingent upon development of populations with genetic resistance. Naturally occurring genetic resistance has been identified in C. lawsoniana, and an active selective breeding program seeks to characterize and increase resistance levels. Two seedling root dip inoculation trials, assessed for mortality for nearly three years each, are used to examine the types and levels of genetic resistance in C. lawsoniana. Most seedlings utilized in these trials are progeny of crosses from parent trees that exhibited apparent resistance to the disease in earlier trials. Seedling trials suggest that both qualitative major gene and quantitative disease resistance occurs in C. lawsoniana. Both types of resistance to P. lateralis appear to be present at levels high enough to be immediately useful for restoration and reforestation. The data suggest that the qualitative resistance is conditioned by a single major gene (designated here as Pla), but nothing is known about the number of genes involved in quantitative disease resistance. Seedling progeny from resistant parent trees in containerized seed orchards are now being used for restoration and reforestation. Resistant seedlings or clones could also be used to re‐establish C. lawsoniana in urban forests. Non‐native pathogens and pests cause high mortality to tree species globally and may imperil the future viability of associated forest ecosystems. Phytophthora lateralis, an oomycete, causes Port‐Orford‐cedar root disease and is a major cause of mortality in the ecologically and economically important conifer species Chamaecyparis lawsoniana (Port‐Orford‐cedar). The P. lateralis resistance program shows promise to help stabilize C. lawsoniana in its native range of northwestern California and southwestern Oregon, USA, and serves as a leading example of disease resistance breeding in forest trees.

Pathogen perception generates the activation of signal transduction cascades to host defense. White pine blister rust (WPBR) is caused by Cronartium ribicola J.C. Fisch and affects a number of species of Pinus. One of the most severely affected species is Pinus albicaulis Engelm (whitebark pine). WPBR resistance in the species is a polygenic and complex trait that requires an optimized immune response. We identified early responses in 2-year-old seedlings after four days of fungal inoculation and compared the underlying transcriptomic response with that of healthy non-inoculated individuals. A de novo transcriptome assembly was constructed with 56,796 high quality-annotations derived from the needles of susceptible and resistant individuals in a resistant half-sib family. Differential expression analysis identified 599 differentially expressed transcripts, from which 375 were upregulated and 224 were downregulated in the inoculated seedlings. These included components of the initial phase of active responses to abiotic factors and stress regulators, such as those involved in the first steps of flavonoid biosynthesis. Four days after the inoculation, infected individuals showed an overexpression of chitinases, reactive oxygen species (ROS) regulation signaling, and flavonoid intermediates. Our research sheds light on the first stage of infection and emergence of disease symptoms among whitebark pine seedlings. RNA sequencing (RNA-seq) data encoding hypersensitive response, cell wall modification, oxidative regulation signaling, programmed cell death, and plant innate immunity were differentially expressed during the defense response against C. ribicola.

Summary Molecular breeding incorporates efficient tools to increase rust resistance in five‐needle pines. Susceptibility of native five‐needle pines to white pine blister rust (WPBR), caused by the non‐native invasive fungus Cronartium ribicola (J.C. Fisch.), has significantly reduced wild populations of these conifers in North America. Major resistance (R) genes against specific avirulent pathotypes have been found in several five‐needle pine species. In this study, we screened genic SNP markers by comparative transcriptome and genetic association analyses and constructed saturated linkage maps for the western white pine (Pinus monticola) R locus (Cr2). Phenotypic segregation was measured by a hypersensitive reaction (HR)‐like response on the needles and disease symptoms of cankered stems post inoculation by the C. ribicola avcr2 race. SNP genotypes were determined by HRM‐ and TaqMan‐based SNP genotyping. Saturated maps of the Cr2‐linkage group (LG) were constructed in three seed families using a total of 34 SNP markers within 21 unique genes. Cr2 was consistently flanked by contig_2142 (encoding a ruvb‐like protein) and contig_3772 (encoding a delta‐fatty acid desaturase) across the three seed families. Cr2 was anchored to the Pinus consensus LG‐1, which differs from LGs where other R loci of Pinus species were mapped. GO annotation identified a set of NBS‐LRR and other resistance‐related genes as R candidates in the Cr2 region. Association of one nonsynonymous SNP locus of an NBS‐LRR gene with Cr2‐mediated phenotypes provides a valuable tool for marker‐assisted selection (MAS), which will shorten the breeding cycle of resistance screening and aid in the restoration of WPBR‐disturbed forest ecosystems.

Pathogen perception generates the activation of signal transduction cascades to host defense. White pine blister rust (WPBR) is caused by Cronartium ribicola J.C. Fisch and affects a number of species of Pinus. One of the most severely affected species is Pinus albicaulis Engelm (whitebark pine). WPBR resistance in the species is a polygenic and complex trait that requires an optimized immune response. We identified early responses in 2-year-old seedlings after four days of fungal inoculation and compared the underlying transcriptomic response with that of healthy non-inoculated individuals. A de novo transcriptome assembly was constructed with 56,796 high quality-annotations derived from the needles of susceptible and resistant individuals in a resistant half-sib family. Differential expression analysis identified 599 differentially expressed transcripts, from which 375 were upregulated and 224 were downregulated in the inoculated seedlings. These included components of the initial phase of active responses to abiotic factors and stress regulators, such as those involved in the first steps of flavonoid biosynthesis. Four days after the inoculation, infected individuals showed an overexpression of chitinases, reactive oxygen species (ROS) regulation signaling, and flavonoid intermediates. Our research sheds light on the first stage of infection and emergence of disease symptoms among whitebark pine seedlings. RNA sequencing (RNA-seq) data encoding hypersensitive response, cell wall modification, oxidative regulation signaling, programmed cell death, and plant innate immunity were differentially expressed during the defense response against C. ribicola.

Background 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 . Results 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. Conclusions 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.

Genetic variation in a plant species is a key to its ability to survive and evolve in the face of changing environmental pressures. Due to insect and disease impacts, changes in fire regimes, and a changing climate, many populations of high elevation white pine species continue to experience high mortality levels and potentially worrisome decreases in genetic variation. In recent years, some trees rated highly for resistance to the non-native white pine blister rust have been killed by fire or mountain pine beetle. Ex situ genetic conservation offers the possibility to conserve the genetic variation within a species before much of it is lost. For many conifer species, freezer storage of seed offers a relatively inexpensive, long-term method of storing germplasm for future use. However, there is uncertainty concerning how long seed of some conifers can be stored and retain viability. We report here on results of germination testing of the oldest known seedlots of whitebark pine ( Pinus albicaulis Engelm.) and foxtail pine ( P. balfouriana Grev. & Balf.), some of which had been in storage for several decades. The 52 whitebark pine seedlots averaged 47.7% germination (average seed age of 19.2 years), while the four foxtail pine seedlots had an average germination of 71.3% (average seed age of 15.3 years). Some seedlots of both species had greater than 90% germination. Refinements to the stratification procedure have since been developed which should enhance germination further. A follow-up study examining seedling vigor of long-stored whitebark pine seed is planned.

Whitebark pine (WBP, Pinus albicaulis Engelm.) is an endangered conifer species due to heavy mortality from white pine blister rust (WPBR, caused by Cronartium ribicola) and mountain pine beetle (Dendroctonus ponderosae). Information about genetic diversity and population structure is of fundamental importance for its conservation and restoration. However, current knowledge on the genetic constitution and genomic variation is still limited for WBP. In this study, an integrated genomics approach was applied to characterize seed collections from WBP breeding programs in western North America. RNA-seq analysis was used for de novo assembly of the WBP needle transcriptome, which contains 97,447 protein-coding transcripts. Within the transcriptome, single nucleotide polymorphisms (SNPs) were discovered, and more than 22,000 of them were non-synonymous SNPs (ns-SNPs). Following the annotation of genes with ns-SNPs, 216 ns-SNPs within candidate genes with putative functions in disease resistance and plant defense were selected to design SNP arrays for high-throughput genotyping. Among these SNP loci, 71 were highly polymorphic, with sufficient variation to identify a unique genotype for each of the 371 individuals originating from British Columbia (Canada), Oregon and Washington (USA). A clear genetic differentiation was evident among seed families. Analyses of genetic spatial patterns revealed varying degrees of diversity and the existence of several genetic subgroups in the WBP breeding populations. Genetic components were associated with geographic variables and phenotypic rating of WPBR disease severity across landscapes, which may facilitate further identification of WBP genotypes and gene alleles contributing to local adaptation and quantitative resistance to WPBR. The WBP genomic resources developed here provide an invaluable tool for further studies and for exploitation and utilization of the genetic diversity preserved within this endangered conifer and other five-needle pines.