Canadian Forest Service Publications
Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family. 2021. Liu, J-J, Schoettle, A.W., Sniezko, R.A., Williams, H., Zamany, A., Rancourt, B. BMC Genomics 22, 567.
Issued by: Pacific Forestry Centre
Catalog ID: 40432
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Background Proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine (Pinus flexilis) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus (Cr4) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown.
Results To identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4-segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4. The tight linkage of NLRs with Cr4-controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR.
Conclusion This study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.
Plain Language Summary
Most plant resistance (R) proteins against pests and pathogens belong to the family containing nucleotide binding site and leucine-rich repeat domains (NLR). White pine blister rust (WPBR) is caused by Cronartium ribicola, causing high mortality and disruption to ecosystems of limber pine (Pinus flexilis) and other five-needle pines. To identify candidates of limber pine R gene (Cr4) against WPBR, we sequenced the limber pine NLR gene using one genetic mapping population. Through a call of single nucleotide polymorphisms (SNPs) and genetic mapping, we successfully mapped a total of 541 SNPs of 155 genes across 12 linkage groups (LGs), including six SNPs of three NLR genes in the Cr4 region on LG8. Bulked segregation analysis (BSA) and TaqMan array-based SNP genotyping further confirmed the tight linkages of NLRs to Cr4. A comparative and syntenic analysis using sugar pine genome sequence draft implied that limber pine NLRs might have evolved at the Cr4 region through local tandem duplication under diversifying selection pressures at relatively high levels. All lines of evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggest that Cr4-linked NLR genes are the most promising R candidates against WPBR in limber pine. Overall, this study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding.