The first high-altitude autotetraploid haplotype-resolved genome assembled (Rhododendron nivale subsp. boreale) provides new insights into mountaintop adaptation

Abstract

Background: Rhododendron nivale subsp. boreale Philipson et M. N. Philipson is an alpine woody species with ornamental qualities that serve as the predominant species in mountainous scrub habitats found at an altitude of ∼4,200 m. As a high-altitude woody polyploid, this species may serve as a model to understand how plants adapt to alpine environments. Despite its ecological significance, the lack of genomic resources has hindered a comprehensive understanding of its evolutionary and adaptive characteristics in high-altitude mountainous environments.

Findings: We sequenced and assembled the genome of R. nivale subsp. boreale, an assembly of the first subgenus Rhododendron and the first high-altitude woody flowering tetraploid, contributing an important genomic resource for alpine woody flora. The assembly included 52 pseudochromosomes (scaffold N50 = 42.93 Mb; BUSCO = 98.8%; QV = 45.51; S-AQI = 98.69), which belonged to 4 haplotypes, harboring 127,810 predicted protein-coding genes. Conjoint k-mer analysis, collinearity assessment, and phylogenetic investigation corroborated autotetraploid identity. Comparative genomic analysis revealed that R. nivale subsp. boreale originated as a neopolyploid of R. nivale and underwent 2 rounds of ancient polyploidy events. Transcriptional expression analysis showed that differences in expression between alleles were common and randomly distributed in the genome. We identified extended gene families and signatures of positive selection that are involved not only in adaptation to the mountaintop ecosystem (response to stress and developmental regulation) but also in autotetraploid reproduction (meiotic stabilization). Additionally, the expression levels of the (group VII ethylene response factor transcription factors) ERF VIIs were significantly higher than the mean global gene expression. We suspect that these changes have enabled the success of this species at high altitudes.

Conclusions: We assembled the first high-altitude autopolyploid genome and achieved chromosome-level assembly within the subgenus Rhododendron. In addition, a high-altitude adaptation strategy of R. nivale subsp. boreale was reasonably speculated. This study provides valuable data for the exploration of alpine mountaintop adaptations and the correlation between extreme environments and species polyploidization.

Keywords: Rhododendron; autotetraploid; evolutionary history; harsh environment; mountaintop adaptation.

Figure 1:

Habitat and genomic characteristics of R. nivale subsp. boreale. (A) Habitat. (B) Habit. (C) Genome landscape. a, 52 pseudochromosomes, which belong to 13 homologous groups, and the length of the pseudochromosome; b, gene density; c, GC density; d, transposon element density; e, copia density; f, gypsy density; g, tandem repeat density. Curved lines inside the circles link syntenic genes between different pseudochromosomes, the synteny between haplotype 1 and haplotype 2 is indicated in red, the synteny between haplotype 1 and haplotype 3 is indicated in green, and the synteny between haplotype 1 and haplotype 4 is indicated in yellow. (D) Hi-C heatmap for assembled pseudochromosomes. (E) Smudgeplot analysis based on 21 k-mers.

Figure 2:

Phylogenetic and comparative analysis between related species and haplotypes. (A) Dot plot between R. nivale subsp. boreale and R. ovatum. (B) Syntenic blocks between 4 haplotypes. (C) Phylogenetic relationships of subsect. Lapponica based on the maximum likelihood (ML) analysis; yellow and green blocks show R. nivale and the sister clade of R. nivale, respectively; red block represents the data generated in this study (n1, n2, n3, n4, and R. nivale subsp. boreale represent the 4 haplotypes and transcriptome of R. nivale subsp. boreale, respectively), and the blue block represents downloaded species data. (D) Gene family characteristics between 4 haplotypes.

Figure 3:

Comparative genomic analysis. (A) ML phylogenetic tree showing the relationship between R. nivale subsp. boreale and 18 other species. Estimated divergence times (Mya) are labeled at nodes in black. Bootstrap values are displayed on the nodes in circles (100%) and squares (≥95%). Expansion (orange) and contraction (blue) of gene families are shown on the branch, contraction and expansion of ancestors are represented by a pie chart, and extant species are indicated by numbers. WGD and WGT events are marked with D and T, respectively. (B) A number of other orthologs, unique paralogs, multicopy orthologs, and single-copy orthologs in 19 species. (C) Ks of paralog frequency distribution chart of 7 species, namely, 6 Ericales (Actinidia, Vaccinium and subg. Hymenanthes, subg. Furthermore, subg. Rhododendron, subg. Tsutsusi, 1 species each) and 1 V. vinifera; polyploidization events are represented by dotted lines. (D) Homologous gene dot plots between R. nivale subsp. boreale and V. vinifera. The red box exemplifies the orthologous ratio of 1:2 between V. vinifera and R. nivale subsp. boreale.

Figure 4:

KEGG and GO enrichment and gene duplication analysis of R. nivale subsp. boreale. (A) KEGG (left) and GO (right) enrichment of genes in significantly expanded gene families. (B) Venn diagram showing the number of shared and specific gene duplications between the significantly expanded genes (SEGs) and 5 categories of duplications (DSD, dispersed duplications; PD, proximal duplications; TD, tandem duplications; TRD, transposed duplications; WGD, whole-genome duplications). (C) Ka/Ks ratios of the 5 types of duplications. (D) KEGG pathway enrichment analysis of the 5 duplication types.

Figure 5:

Single-matched allelic expression analysis. (A) The total amount of single-match allelic expression of 52 pseudochromosomes; the colors represent 13 homologous groups (HGs). (B) Heatmap clustering analysis of single-match alleles in screening the position of DELs in R. nivale subsp. boreale. Each row represents a set of differentially expressed alleles, and each column represents a chromosome. The heatmap shows a homologous group.

Figure 6:

Identification and evolution of key family and genes for adaptation to low mountaintop temperature and hypoxia. (A) Rootless ML phylogenetic tree based on ultra-fast 1,000 bootstrap samplings showed diversified AP2/ERF superfamily in 13 species, including 10 Rhododendron species and A. thaliana, kiwifruit, and V. darrowii. The color of the clades indicates 5 subfamilies of the AP2/ERF superfamily. The labels are differently colored according to species. (B) Schematic diagram of the gain and loss of key genes in 12 species of Ericales; numbers in pink and blue depict ERF VII and CBF gene family turnover. The numbers in the rectangles and circles represent the number of genes in ancestral and existing species. The + and − signs represent the gain and loss of genes, respectively. (C) Expression levels of ERF VIIs and CBFs. The numbers in parentheses indicate the number of genes that are expressed. P represents the adjusted P value.

Figure 7:

Characteristics of Ericales cytochrome P450 (CYP). (A) ML phylogenetic tree showing the relationship between 10 CYP clans (higher-order groupings of CYP families). (B) Heatmap showing the number of clan members for each species. (C) Phylogenetic tree of the CYP members of 13 species based on GTR (generalized time-reversible). Different clans are represented by different colors. (D) Number of CYP genes produced by duplication events in 10 species of Rhododendron.