Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of Spirodela

Abstract

Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation.

Keywords: aquatic adaptation; disease resistance; long reads; root evolution; tandem duplication.

Fig. 1.

Comparison of genome assembly from short reads and long reads. From outside to inside, the circles represent karyotype (a), sequence gaps (b), GC content (c), full-length LTRs (d), gene density (e), and syntenous connections (f). The metrics are calculated in 1-Mb sliding windows. The right half circle represents genome assembly from long reads (Sp7498V3). The left half circle represents genome assembly from short reads (Sp7498V2). Every blue vertical bar indicates one gap in layer b. There are 270 gaps in Sp7498V3 and 13,459 gaps in Sp7498V2. The inner lines denote the synteny of two versions of genomes. Chr, chromosome.

Fig. 2.

A benchmark of the nested LTRs in Spirodela. Scaffold 15 with contiguous sequence length of 539 kb exhibits regions of TE islands and a gene cluster. Zone I and IV are nested with LTR regions. Red numbers next to LTRs represent insertion ages in million years ago (MYA). Retrotransposons are indicated by filled triangles of different colors. Zone II and Zone V are simple repeat regions. Simple repeats are represented by blue vertical lines. Zone III contains 23 genes without any transposon interruption. The genes are shown as green pentagons or triangles. Genes and LTRs have been drawn to scale. Hap, Doc, Sne, Sle, Dop, and Bas indicate different types of LTR retrotransposons.

Fig. 3.

Anatomy of the Spirodela root. (A). A dorsal overview of a Spirodela plant, showing the location of cross-sections. (Scale bar: 500 μm.) The cross-sections were sampled at 8 (c), 1.5 (d), 0.5 (e), and 0.2 mm (f) from the root tip, corresponding to Fig. 3 C–F, respectively. (B). A ventral overview of a Spirodela plant exhibits as many as 12 adventitious roots. (Scale bar: 500 μm.) (C–F) Cross-sections of Spirodela roots, illustrating the structures of the epidermis (Ep), cortex (C), endodermis (En), and vascular tissue (V). A close investigation shows vascular tissues. The central cell (X), located in the middle, is a tracheary element, which is surrounded by a ring of phloem tissue (P). The endodermis (En) is between the vascular tissue and the cortex parenchyma cells (Pc). (Scale bars: 50 μm.) (G) The genetic pathways involved in adventitious root (AR), lateral root (LR), and root hair (RH) development. The homologous genes in Spirodela are defined by using the rice protein sequences.

Fig. 4.

Genomic distribution and transcriptomic expression of tandemly duplicated disease-resistant genes. (A) Disease-resistant gene copies at each locus in the genome are illustrated as yellow arrowheads. The yellow arrowheads indicate the gene coding direction. The sign of * next to the yellow arrowhead indicates that the gene is supported by full-length cDNA evidence. Contig 44 (Ctg44) is one of contigs that could not be incorporated into chromosomes with current sequence data. (B) Gene expression level is represented in the y axis with the value of fragments per kilobase of transcript per million mapped reads (FPKM) analyzed from RNA-Seq. The horizontal dotted line shows the average gene expression of total expressed genes. The bars are labeled in different colors based on chromosomes.