Conifers Concentrate Large Numbers of NLR Immune Receptor Genes on One Chromosome

Abstract

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

Keywords: NBS-LRR; comparative genomics; gene clusters; gene family evolution; genomic architecture; resistance genes.

Fig. 1.

Overview of main gymnosperm clades, with total number of NLR genes and their corresponding categories indicated as bar charts, as found in this study (Table 1). Cladogram is based on the phylogeny presented in Leslie et al. (2018). Genome sizes are indicated beneath each bar chart and are based on the chromosome-level assemblies used in this study (see “Genomic Distribution of NLR Genes in Pinaceae and Other Conifer Families” section) or, in the case of Pinaceae (P. flexilis), obtained from the Kew Plant DNA C-values database (release 7.1, Pellicer and Leitch 2020). Gymnosperms invariably have large genomes (∼10 Gb), but display large variations in NLR gene numbers. Despite the 3-fold increase in genome size observed in Pinaceae, the number of discovered NLR genes remained within the average range of conifers. Pictures were obtained from the Wikimedia Commons repository (https://commons.wikimedia.org) and correspond to the broader taxonomic clades: A—Cycas rumphii Miq., Andy King; B—Ginkgo biloba L., Susanna Giaccai; C—Taxus baccata L., Mykola Swarnyk; D—Sequoiadendron giganteum, W. Bulach; E—Pinus flexilis, Greg Woodhouse.

Fig. 2.

Chromosomal distribution of NLR genes across gymnosperms. A) Scatter plot indicating the observed number of NLR genes versus the expected number of NLR genes (calculated based on the length of the chromosome and the total number of NLR genes discovered in the genome; see “Genomic Distribution of NLR Genes in Pinaceae and Other Conifer Families” section) for each chromosome in each taxon analyzed in this study. The black line follows the function y = x, indicating a perfectly homogeneous distribution of NLR genes over the chromosomes. Deviations from this line therefore indicate a nonhomogeneous distribution. Highly deviant chromosomes of four taxa are highlighted and have their intrachromosomal NLR distribution displayed in histograms (B–E). Bin width equals ±1% of the length of the largest chromosome in the genome of the respective taxon (see “Genomic Distribution of NLR Genes in Pinaceae and Other Conifer Families” section). The colors indicate NLR class as determined with the NLR Annotator (Steuernagel et al. 2020) and manual BLASTs (see “NLR Classification” section). Ultra-dense NLR clusters are indicated for each taxon.

Fig. 3.

Histogram plots of NLR genes on each chromosome within the genome of five gymnosperm lineages. Chromosomes are ordered based on the ordering in the respective assembly and do not reflect synteny. Red arrows indicate particularly dense clusters.

Fig. 4.

Intragenomic phylogenetic relationships of NLR genes based on the conserved central NB-ARC domain, calculated with maximum likelihood algorithms using IQTree v1.6.12 (Nguyen et al. 2015; (see “NLR Phylogenies” section) and annotated using the iTOL web server (Letunic and Bork 2021). The color strips around the circular trees indicate NLR class as determined with the NLR Annotator (Steuernagel et al. 2020) and manual BLASTs (see “NLR Classification” section). Main gymnosperm lineages represented by Cycadales (A), Ginkgoales (B), Cuppressaceae (C), Taxaceae (D), and Pinaceae: Pinus (E), and Picea (F).

Fig. 5.

Chromosomal structuring in phylogenetic relationships within a conifer species (T. chinensis) displayed in an intragenomic framework. The branch colors correspond to the NLR gene subfamily, as indicated in the same colors next to the phylogeny. The colored squares at the tips of branches represent the chromosome on which the respective NLR genes are located. The NLR genes found on scaffolds that were not assembled into chromosomes are indicated with empty color squares (“Unclassified”).