Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC, V8Z 1M5, Canada.
USDA Forest Service, Rocky Mountain Research Station, 240 West Prospect Road, Fort Collins, CO, 80526, USA.
BMC Genomics. 2021 Jul 23;22(1):567. doi: 10.1186/s12864-021-07885-8.
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.
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.
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.
具有核苷酸结合位点（NBS）和富含亮氨酸重复（LRR）结构域的蛋白（NLR）是植物抵抗各种病原体和害虫侵袭的最重要抗性（R）家族之一。已有的白皮松（Pinus flexilis）转录组使我们能够对 NLR 基因和与宿主对白松疱锈病菌（Cronartium ribicola）抗性相关的抗性基因类似物（RGA）进行特征描述，白松疱锈病菌是全世界五针松上的白松疱锈病的致病真菌病原体。我们之前曾在白皮松主要基因座（Cr4）上定位了一个赋予白皮松对白松疱锈病菌完全抗性的基因座，该基因座位于 Pinus 共识连锁群 8（LG-8）上。然而，NLR 基因的遗传分布及其在抗性和敏感等位基因之间的分化仍不清楚。
为了在 Cr4 基因座上鉴定 NLR 基因，本研究使用靶向测序对一个 Cr4 分离的种子家系中的 480 个 RGA 进行了重新测序。在 SNP 调用和遗传图谱构建后，在 12 个 LG 上共定位了来自 155 个基因的 541 个 SNP。在 Cr4 区域中鉴定到了三个新的假定 NLR 基因，其中一个与 Cr4 共分离。通过对极端表型全基因组关联研究（XP-GWAS）进行显著检验的混合分离分析（BSA），进一步证实了 NLR 与 Cr4 控制的表型的紧密连锁关系。糖松基因组序列的共线性分析进一步支持了 Cr4 区域的局部串联重复。在 NLR 家族中观察到了显著的基因分化，表明在局部重复基因中，多样化选择压力相对较高。大多数基因表现出相似的低水平表达模式，但有些基因受到与抗病性相关的遗传背景的影响。精细遗传剖析、进化分析和表达谱分析的证据表明，两个 NLR 基因是 Cr4 对抗 WPBR 的最有希望的候选基因。
本研究为 Cr4 基因座的遗传结构以及白皮松标记辅助选择的一组 NLR 变体提供了基础见解。在 Cr4 基因座上鉴定到了新的 NLR 基因，这些 Cr4 候选基因将有助于在白皮松的育种计划中与其他主要/次要基因一起部署该 R 基因。
