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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3079-2) contains supplementary material, which is available to authorized users.

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Limber pine, Pinus flexilis E. James, a wide-ranging tree species in western North America, is highly susceptible to white pine blister rust (WPBR), caused by the non-native fungal pathogen Cronartium ribicola J.C. Fisch. The Canadian populations in particular have been heavily impacted, and in 2014, limber pine was designated endangered in Canada by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). Little is known about genetic resistance to WPBR in limber pine, but major gene resistance (MGR) has been characterized in some populations in the United States. This study examines resistance in seedling families from 13 parent trees from British Columbia, Alberta, and Oregon, representing the northern- and northwestern-most populations. Most families were susceptible, with 100% of the seedlings cankered, but one family from Alberta segregated 1:1 for cankered and canker free. This is the first report of (a) MGR in Canada of any of the four species of five-needle pines native to Canada and (b) any resistance in limber pine in Canadian populations and is the northernmost known incidence of putative R-gene resistance in a natural stand of any five-needle pine species. Many of the Canadian selections were from stands with high incidence of WPBR infection, and their high susceptibility in this trial suggests that further infection and mortality is likely in the Canadian populations.

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.

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SummaryMolecular 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.

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