Richard A. Sniezko

Proactive intervention to sustain high-elevation pine ecosystems threatened by white pine blister rust

Only recently have efforts begun to address how management might prepare currently healthy forests to affect the outcome of invasion by established non-native pests. Cronartium ribicola, the fungus that causes the disease white pine blister rust (WPBR), is among the introductions into North America where containment and eradication have failed; the disease continues to spread. Ecosystem function is impaired by high rust-caused mortality in mature five-needle white pine forests. This paper evaluates five proactive management options to mitigate the development of impacts caused by white pine blister rust in threatened remote high-elevation five-needle pine ecosystems of western North America. They are: reducing pest populations; managing forest composition; improving host vigor; introducing resistant stock with artificial regeneration; and diversifying age class structure to affect the natural selection process for resistance. Proactive intervention to manage and facilitate evolutionary change in the host species may sustain host populations and ecosystem function during pathogen naturalization.

Resistance breeding against nonnative pathogens in forest trees — current successes in North America

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.

Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions

The pollination droplet is a highly conservative pollination mechanism that is observed in all major gymnosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-β-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like protein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa β-d-glucan exohydrolase, a 47.5-kDa glucan 1,3-β-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen development. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success.

Western white pine SNP discovery and high-throughput genotyping for breeding and conservation applications

BackgroundWestern 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.ResultsA 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.ConclusionsThe 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.

Genetic Diversity and Population Structure of Whitebark Pine (Pinus albicaulis Engelm.) in Western North America.

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.

Genetic mapping of Pinus flexilis major gene (Cr4) for resistance to white pine blister rust using transcriptome-based SNP genotyping

BackgroundLinkage 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.ResultsNeedle 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.ConclusionsWe 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.

Pinus monticola pathogenesis-related gene PmPR10-2 alleles as defense candidates for stem quantitative disease resistance against white pine blister rust (Cronartium ribicola)

White pine blister rust, caused by the invasive fungus Cronartium ribicola, has been responsible for extremely high mortality of native western white pine (Pinus monticola) and other five-needle pines in natural stands throughout western North America. The presence of this non-native fungus has also led to greatly restricted use of western white pine for reforestation. A few families of defense proteins have been found as functional candidates involved in tree resistance to rust infection. Here we report genetic variation of a gene encoding a family 10 pathogenesis-related (PR) protein (PmPR10-2) in open-pollinated seed families with different levels of stem quantitative disease resistance (QDR). Six novel alleles and five common genotypes were identified inside the PmPR10-2 locus: these genetic variations included 33 single nucleotide polymorphisms (SNPs) throughout the gene regions and copy variation of a rare octanucleotide simple sequence repeat (SSR), 5′-AATTATTT-3′, in the gene intron. PmPR10-2 exhibited a moderate level (average r2 = 0.42) of linkage disequilibrium. Two-thirds of the identified SNPs and the SSR marker were significantly associated with stem QDR levels. The PmPR10-2 genotype (H3:H3) exhibited the highest level of stem QDR (P < 0.01). Cost-effective and co-dominant SSR markers were developed and used for genotyping the PmPR10-2 locus using simple PCR, providing a potential molecular tool for accelerating screening efforts of white pine resistance against C. ribicola.

The role of hybridization during ecological divergence of southwestern white pine (Pinus strobiformis) and limber pine (P. flexilis)

Interactions between extrinsic factors, such as disruptive selection and intrinsic factors, such as genetic incompatibilities among loci, often contribute to the maintenance of species boundaries. The relative roles of these factors in the establishment of reproductive isolation can be examined using species pairs characterized by gene flow throughout their divergence history. We investigated the process of speciation and the maintenance of species boundaries between Pinus strobiformis and Pinus flexilis. Utilizing ecological niche modelling, demographic modelling and genomic cline analyses, we illustrated a divergence history with continuous gene flow. Our results supported an abundance of advanced generation hybrids and a lack of loci exhibiting steep transition in allele frequency across the hybrid zone. Additionally, we found evidence for climate‐associated variation in the hybrid index and niche divergence between parental species and the hybrid zone. These results are consistent with extrinsic factors, such as climate, being an important isolating mechanism. A build‐up of intrinsic incompatibilities and of coadapted gene complexes is also apparent, although these appear to be in the earliest stages of development. This supports previous work in coniferous species demonstrating the importance of extrinsic factors in facilitating speciation. Overall, our findings lend support to the hypothesis that varying strength and direction of selection pressures across the long lifespans of conifers, in combination with their other life history traits, delays the evolution of strong intrinsic incompatibilities.

Saturated genic SNP mapping identified functional candidates and selection tools for the Pinus monticola Cr2 locus controlling resistance to white pine blister rust

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.

Characterization of Five Novel Mitoviruses in the White Pine Blister Rust Fungus Cronartium ribicola.

The white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.) is an exotic invasive forest pathogen causing severe stem canker disease of native white pine trees (subgenus Strobus) in North America. The present study reports discovery of five novel mitoviruses in C. ribicola by deep RNA sequencing. The complete genome of each mitovirus was determined by rapid amplification of cDNA ends (RACE) and reverse transcriptase-polymerase chain reaction (RT-PCR). A single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) was detected in each of the viral genomes using mitochondrial genetic codes. Phylogenetic analysis indicated that the C. ribicola mitoviruses (CrMV1 to CrMV5) are new putative species of the genus Mitovirus. qRT-PCR and RNA-Seq analyses revealed that viral RNAs were significantly increased in fungal mycelia in cankered pine stems compared to expression during two different stages of spore development, suggesting that viral genome replication and transcription benefit from active growth of the host fungus. CrMVs were widespread with relatively high levels of minor allele frequency (MAF) in western North America. As the first report of mitoviruses in the Class Pucciniomycetes, this work allows further investigation of the dynamics of a viral community in the WPBR pathosystem, including potential impacts that may affect pathogenicity and virulence of the host fungus.