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

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.

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.

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.

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.

Transcriptome analysis of the white pine blister rust pathogen Cronartium ribicola: de novo assembly, expression profiling, and identification of candidate effectors.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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 SNP discovery and high-throughput genotyping for breeding and conservation applications.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Gene expression profiling of candidate virulence factors in the laminated root rot pathogen Phellinus sulphurascens.

BACKGROUND: Phellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease. RESULTS: We generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058. CONCLUSIONS: This first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.

Transcriptome analysis of Pinus monticola primary needles by RNA-seq provides novel insight into host resistance to Cronartium ribicola.

BACKGROUND: Five-needle pines are important forest species that have been devastated by white pine blister rust (WPBR, caused by Cronartium ribicola) across North America. Currently little transcriptomic and genomic data are available to understand molecular interactions in the WPBR pathosystem. RESULTS: We report here RNA-seq analysis results using Illumina deep sequencing of primary needles of western white pine (Pinus monticola) infected with WPBR. De novo gene assembly was used to generate the first P. monticola consensus transcriptome, which contained 39,439 unique transcripts with an average length of 1,303 bp and a total length of 51.4 Mb. About 23,000 P. monticola unigenes produced orthologous hits in the Pinus gene index (PGI) database (BLASTn with E values < e-100) and 6,300 genes were expressed actively (at RPKM ≥ 10) in the healthy tissues. Comparison of transcriptomes from WPBR-susceptible and -resistant genotypes revealed a total of 979 differentially expressed genes (DEGs) with a significant fold change > 1.5 during P. monticola- C. ribicola interactions. Three hundred and ten DEGs were regulated similarly in both susceptible and resistant seedlings and 275 DEGs showed regulatory differences between susceptible and resistant seedlings post infection by C. ribicola. The DEGs up-regulated in resistant seedlings included a set of putative signal receptor genes encoding disease resistance protein homologs, calcineurin B-like (CBL)-interacting protein kinases (CIPK), F-box family proteins (FBP), and abscisic acid (ABA) receptor; transcriptional factor (TF) genes of multiple families; genes homologous to apoptosis-inducing factor (AIF), flowering locus T-like protein (FT), and subtilisin-like protease. DEGs up-regulated in resistant seedlings also included a wide diversity of down-stream genes (encoding enzymes involved in different metabolic pathways, pathogenesis-related -PR proteins of multiple families, and anti-microbial proteins). A large proportion of the down-regulated DEGs were related to photosystems, the metabolic pathways of carbon fixation and flavonoid biosynthesis. CONCLUSIONS: The novel P. monticola transcriptome data provide a basis for future studies of genetic resistance in a non-model, coniferous species. Our global gene expression profiling presents a comprehensive view of transcriptomic regulation in the WPBR pathosystem and yields novel insights on molecular and biochemical mechanisms of disease resistance in conifers.

Gene expression profiling of a compatible interaction between Douglas-Fir and the root rot fungal pathogen Phellinus sulphurascens.

Douglas-fir (DF) (Pseudotsuga menziesii) is one of the largest and most economically important coniferous species in western North America. Its productivity is greatly affected by the root rot fungus Phellinus sulphurascens Pilát. Evidence of resistance by DF to fungal root pathogens such as P. sulphurascens has been reported but mechanisms of resistance in this compatible pathosystem are not yet known. To better understand the DF-P. sulphurascens interaction, especially at the molecular level, we selected 12 diverse plant genes already identified as defense-related from a cDNA library constructed using root tissues from P. sulphurascens-infected DF seedlings. Using quantitative reverse-transcriptase polymerase chain reaction on infected DF root samples collected at five different time points after inoculation, we found that P. sulphurascens infection significantly elevated expression of the 12 selected genes. In most cases the highest expression level was recorded within 2 to 3 days after inoculation. The constructed cDNA library, which is enriched with defense-related host genes and a number of fungal genes, will continue to serve as a useful resource for future larger-scale gene discovery and functional research on the P. sulphurascens and DF pathosystem.

A proteomics approach to identify proteins differentially expressed in Douglas-fir seedlings infected by Phellinus sulphurascens.

We carried out a comparative proteomic study to explore the molecular mechanisms that underlie the defense response of Douglas-fir (DF, Pseudotsuga menziesii) to laminated root rot, a disease caused by Phellinus sulphurascens. 2-DE was conducted on proteins extracted from roots of laboratory-grown, young DF seedlings inoculated with P. sulphurascens. A total of 1303 proteins was detected in 7 dpi infected and uninfected root samples. Among these 1303 proteins, 277 showed differential expression that was statistically significant (p<0.05). Of these 277 proteins, 74 upregulated and 85 downregulated proteins showed at least a two-fold change from controls. Forty seven upregulated and 23 downregulated proteins were selected to be excised and analyzed using LC-MS/MS followed by peptide matching. Our results indicate that the major proteins differentially expressed in P. sulphurascens-infected DF seedlings include those in the following functional groups: disease/defense (27%), metabolism (16%), transcription factors (11%), signal transduction (10%), secondary metabolism (7%) and energy (4%). A number of additional proteins involved in cell structure (3%) and protein synthesis (3%) were also identified. By providing an initial database of candidate pathogenesis-related proteins for the DF-Phellinus sulphurascens pathosystem the results of this study will enable future detailed investigation of gene expression and function.

Changes in anatomy and terpene chemistry in roots of Douglas-fir seedlings following treatment with methyl jasmonate.

Replicated trials were conducted on two full-sibling families of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings. In response to the application of a 0.01% solution of methyl jasmonate (MeJA) to the soil of potted seedlings, numerous anatomical and chemical changes were observed in the roots, stem and foliage. These changes were, for the most part, similar for both sib groups. Methyl jasmonate induced traumatic resin duct formation in roots and stems. Chemical differences between MeJA-treated and control seedlings were mainly limited to the roots and stem, though some changes also occurred in the foliage. A total of 35 terpenoids were observed in the P. menziesii seedlings. In response to MeJA treatment, several of the 22 detected monoterpenoids (linalool, bornyl acetate, camphene, myrcene, alpha- and beta-pinene, tricyclene and beta-phellandrene) increased significantly in roots and stems, whereas (E)-beta-ocimene decreased significantly in the foliage. Four of the five detected sesquiterpenoids (alpha-humulene, germacrene D, longifolene and (E)-caryophyllene) increased significantly following MeJA application, mainly in the root and stem. Four of the eight detected diterpenoids (abietate, levopimarate, palustrate and sandaracopimarate) increased in response to MeJA treatment, but only in root and stem tissue. This study provides the first description of the effects of MeJA applied to roots through the soil on the anatomy and terpene chemistry of a gymnosperm. This comprehensive inventory of terpenoids in P. menziesii, with and without MeJA treatment, may facilitate identification of terpenoid-related resistance traits. Potential practical applications of MeJA treatment of conifer roots as a pest management strategy are discussed.

Characterization of four terpene synthase cDNAs from methyl jasmonate-induced Douglas-fir, Pseudotsuga menziesii.

Numerous terpenoid compounds are present in copious amounts in the oleoresin produced by conifers, especially following exposure to insect or fungal pests. CDNA clones for many terpene synthases responsible for the biosynthesis of these defense compounds have been recovered from several conifer species. Here, the use of three terpene synthase sequences as heterologous probes for the discovery of related terpene synthase genes in Douglas-fir, Pseudotsuga menziesii (Mirbel) Franco (Pinaceae), is reported. Four full-length terpene synthase cDNAs were recovered from a methyl jasmonate-induced Douglas-fir bark and shoot cDNA library. These clones encode two multi-product monoterpene synthases [a (-)-alpha-pinene/(-)-camphene synthase and a terpinolene synthase] and two single-product sesquiterpene synthases [an (E)-beta-farnesene synthase and a (E)-gamma-bisabolene synthase].

Gene Cloning and Tissue Expression Analysis of a PR-5 Thaumatin-Like Protein in Phellinus weirii-Infected Douglas-Fir.

ABSTRACT In western North America, Douglas-fir (Pseudotsuga menziesii) is the most economically important conifer species susceptible to laminated root rot caused by Phellinus weirii. While attempting to internally sequence an endochitinase found to be up-regulated in P. weirii-infected Douglas-fir roots, we obtained overlapping peptide fragments showing 28% similarity with a PR-5 thaumatin-like protein (TLP) designated PmTLP (Pm for Pseudotsuga menziesi). A rabbit polyclonal antibody was reared against a synthetic peptide composed of a 29-amino-acid-long, conserved, internal sequence of PmTLP and purified by immunoaffinity. Western immunoblot analysis of infected roots of 24-year-old coastalfir showed significantly higher amounts of PmTLP (P < 0.01) closest to the point of P. weirii inoculation and infection than in uninfected regions of the same root. The antibody was also used to screen for PmTLP in roots of 25-year-old interior Douglas-firs naturally infected with a related pathogen, Armillaria ostoyae, and results showed significantly higher levels of PmTLP in bark tissues adjacent to infection (P < 0.05) than in uninfected tissue. Using polymerase chain reaction (PCR)-based cloning, the cDNA of PmTLP was shown to have a 702-bp open reading frame with a signal peptide cleavage site at 155 bp corresponding to a 29-amino-acid-long residue prior to the start of the N-terminal. Based on the deduced amino acid sequence, the molecular mass of the putative PmTLP was calculated to be 21.0 kDa with an isoelectric point of 3.71. Alignment analysis of PmTLP cDNA with a representative genomic DNA PCR sequence showed presence of one intron of variable size, within the coding region. The induction of PmTLP at the site of root infection and its presence in needle tissue suggests a general role for this protein in adaptation to stress and may be part of an integrated defense response initiated by the host to impede further pathogen spread.

Detection of a chitinase-like protein in the roots of Douglas-fir trees infected with Armillaria ostoyae and Phellinus weirii.

Protein was extracted from root bark of 11- and 25-year-old interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees that were naturally infected with Armillaria ostoyae (Romagnesi) Herink. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Root bark tissue adjacent to infected areas had a significantly higher protein concentration than healthy tissue (P < 0.05), whereas the protein concentration of infected tissue was consistently lower (P < 0.05) than that of healthy tissue. The SDS-PAGE profiles of healthy, infected, and adjacent-to-infected root bark tissues revealed significant differences in concentrations of a 29.3-kDa protein. The N-terminal amino acid sequence of the 29.3-kDa protein displayed significant homology (P = 0.013) to a basic endochitinase. Use of a polyclonal antibody raised against the 29.3-kDa putative endochitinase-like protein (ECP) indicated differences in the quantities of ECP in healthy roots compared with roots infected with A. ostoyae in 11- and 25-year-old interior Douglas-fir trees. The antibody was also used to screen for the presence of the 29.3-kDa protein in roots of 24-year-old coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) trees that were artificially inoculated with and colonized by Phellinus weirii (Murr.) Gilbn. The amount of ECP was elevated in root bark of coastal Douglas-fir in response to P. weirii infection, although in lower quantities relative to those found in the A. ostoyae-interior Douglas-fir pathosystem. The sequence homology of the ECP with a basic chitinase, together with its increased synthesis in response to two fungal pathogens, indicate a possible role for this protein in the defense of Douglas-fir against fungal pathogens.