Gapless Genome Assembly of Puccinia triticina Provides Insights into Chromosome Evolution in Pucciniales

Abstract

Chromosome evolution drives species evolution, speciation, and adaptive radiation. Accurate genome assembly is crucial to understanding chromosome evolution of species, such as dikaryotic fungi. Rust fungi (Pucciniales) in dikaryons represent the largest group of plant pathogens, but the evolutionary process of adaptive radiation in Pucciniales remains poorly understood. Here, we report a gapless genome for the wheat leaf rust fungus Puccinia triticina determined using PacBio high-fidelity (HiFi) sequencing. This gapless assembly contains two sets of chromosomes, showing that one contig represents one chromosome. Comparisons of homologous chromosomes between the phased haplotypes revealed that highly frequent small-scale sequence divergence shapes haplotypic variation. Genome analyses of Puccinia triticina along with other rusts revealed that recent transposable element bursts and extensive segmental gene duplications synergistically highlight the evolution of chromosome structures. Comparative analysis of chromosomes indicated that frequent chromosomal rearrangements may act as a major contributor to rapid radiation of Pucciniales. This study presents the first gapless, phased assembly for a dikaryotic rust fungus and provides insights into adaptive evolution and species radiation in Pucciniales. IMPORTANCE Rust fungi (Pucciniales) are the largest group of plant pathogens. Adaptive radiation is a predominant feature in Pucciniales evolution. Chromosome evolution plays an important role in adaptive evolution. Accurate chromosome-scale assembly is required to understand the role of chromosome evolution in Pucciniales. We took advantage of HiFi sequencing to construct a gapless, phased genome for Puccinia triticina. Further analyses revealed that the evolution of chromosome structures in rust lineage is shaped by the combination of transposable element bursts and segmental gene duplications. Chromosome comparisons of Puccinia triticina and other rusts suggested that frequent chromosomal arrangements may make remarkable contributions to high species diversity of rust fungi. Our results present the first gapless genome for Pucciniales and shed light on the feature of chromosome evolution in Pucciniales.

Keywords: HiFi sequencing; chromosomal rearrangement; evolutionary radiation; gapless genome; rust fungi.

FIG 1

Assessment of genome assembly. (A) Genome completeness of the two haplotypes was assessed using conserved fungal BUSCO genes. (B) Read depth histogram plotted by mapping HiFi reads to the phased assembly, showing full phasing of the assembled genome.

FIG 2

Haplotypic variation. (A) Size distribution of structural variations between homologous chromosomes. Inv, inversions; Trans, translocations; InvTrans, invert translocations; Dup, duplications; InvDup, invert duplications. (B) K_a and K_s distribution for allelic genes. (C) Distribution of K_a/K_s ratios in conserved fungal BUSCO genes and secreted protein genes.

FIG 3

Analysis of TEs in rust species. (A) Comparison of different TEs in P. triticina (Pt), P. graminis f. sp. tritici (Pgt), P. coronata f. sp. avenae (Pca), P. striiformis f. sp. tritici (Pst), M. larici-populina (Mlp), and A. psidii (Ap). (B) Insertion times of LTR-RTs in P. triticina. The red curve represents the trend of the accumulation of TEs in selected rust species. Ma, 1 million years.

FIG 4

Gene duplication and evolution. (A) Dot plots showing gene duplicates in P. triticina. Chromosome numbers are shown. Best hits, secondary hits, and other hits are shown in red, blue, and gray, respectively. (B) K_s distribution for paralogues in P. triticina and orthologues in P. triticina and other rust species. (C) Bar plot of KEGG pathway enrichment for P. triticina genes after P. triticina-P. graminis divergence. TCA, tricarboxylic acid. (D) Distribution of K_a/K_s ratios for paralogues in P. triticina. (Left) After P. triticina-P. graminis divergence; (right) before P. triticina-P. graminis divergence. Positive selection, relaxed purifying selection, and purifying selection represent K_a/K_s > 1, 0.8 < K_a/K_s < 1, and K_a/K_s ≤ 0.8, respectively.

FIG 5

(A) Dot plots showing homologous genes in P. triticina-P. graminis, P. triticina-P. striiformis f. sp. tritici, and P. triticina-P. coronata f. sp. avenae. Best hits, secondary hits, and other hits are shown in red, blue, and gray, respectively. (B) Gene synteny in P. triticina-P. graminis, P. triticina-P. striiformis f. sp. tritici, and P. triticina-P. coronata f. sp. avenae. Syntenic blocks containing at least 10 genes are shown. Regular chromosomes are in blue and reversed chromosomes are in red. (C) K_s distribution of gene pairs between syntenic blocks. The median value is shown to represent the K_s distribution of the blocks containing at least 10 gene pairs. Chromosome numbers are shown for the four species.

FIG 6

Dot plots showing homologous genes in P. triticina-M. larici-populina (A) and P. triticina-A. psidii (B). Chromosome numbers are shown for P. triticina and M. larici-populina. Sequence numbers are shown for A. psidii.