Abstract
BackgroundProteins 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.ResultsTo 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.ConclusionThis 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.
Highlights
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
Targeted sequencing and Single nucleotide polymorphism (SNP) calling Fluidigm custom access arrays were designed for 480 RGASs, which were selected from a limber pine transcriptome shotgun assembly
Following adapter trimming and quality control, Illumina MiSeq generated a total 14.9 million 250-bp PE reads with high-quality, averaging 155 ± 22 thousand (K) reads per sample, with a range of 73 K ~ 206 K PE reads for individual samples (Table S2)
Summary
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. Because most plant disease resistance (R) genes encode proteins containing nucleotidebinding site (NBS) and leucine-rich repeat (LRR) domains (NLRs) or leucine-rich repeat receptor-like protein kinases (LRR-RLKs) [4], plant genomic regions encoding NLR proteins are attractive targets of TS. As one TS approach, resistance gene enrichment sequencing (RenSeq) has been used for improving genome annotations and genetic mapping of plant NLR genes [5, 6], the prioritization of novel NLR genes [7, 8], and identification of candidate R genes [9, 10]
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