Plant Evolution and Environmental Adaptation Unveiled by Long-Read Whole-Genome Sequencing of Spirodela
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Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of Spirodela Dong Ana,1, Yong Zhoua,1, Changsheng Lib, Qiao Xiaob, Tao Wangb, Yating Zhanga, Yongrui Wub, Yubin Lic, Dai-Yin Chaob, Joachim Messingd,2, and Wenqin Wanga,2 aSchool of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China; bNational Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China; cBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081 Beijing, China; and dWaksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854 Contributed by Joachim Messing, August 5, 2019 (sent for review June 24, 2019; reviewed by Yves Van de Peer and Rod A. Wing) Aquatic plants have to adapt to the environments distinct from against diseases. The previous assembly of the Spirodela genome where land plants grow. A critical aspect of adaptation is the was based on a read length of ∼350 base pairs (bp) (7), too short to dynamics of sequence repeats, not resolved in older sequencing assess critical properties of plant aquatic adaptation. Indeed, the platforms due to incomplete and fragmented genome assemblies assembly included more than 16,055 small contigs and 11.8% of from short reads. Therefore, we used PacBio long-read sequencing missing sequences, limiting access to total gene content. Here, we of the Spirodela polyrhiza genome, reaching a 44-fold increase of were able to extend read length to more than 10 kilobases (kb), contiguity with an N50 (a median of contig lengths) of 831 kb and using PacBio long-read sequencing. We filled 95.4% of preexisting filling 95.4% of gaps left from the previous version. Reconstruction gaps, and, with PacBio sequencing of full-length complementary of repeat regions indicates that sequentially nested long terminal DNA (cDNA), we could identify 18,708 protein-coding genes, repeat (LTR) retrotranspositions occur early in monocot evolution, many of which were larger than previously thought. In particular, featured with both prokaryote-like gene-rich regions and eukaryotic the expansion of disease-resistant gene families by tandem dupli- repeat islands. Protein-coding genes are reduced to 18,708 gene cations can explain the innate immunity and worldwide distribution models supported by 492,435 high-quality full-length PacBio comple- SCIENCES of the species. Furthermore, new sequence information gives us mentary DNA (cDNA) sequences. Different from land plants, the AGRICULTURAL primitive architecture of Spirodela’s adventitious roots and lack of new insights into the minimal morphological requirements of a lateral roots and root hairs are consistent with dispensable functions higher plant and its environmental adaptation. of nutrient absorption. Disease-resistant genes encoding antimicro- Results and Discussion bial peptides and dirigent proteins are expanded by tandem dupli- cations. Remarkably, disease-resistant genes are not only amplified, Genome Sequencing, Assembly, and Annotation. AcloneofS. polyrhiza but also highly expressed, consistent with low levels of 24-nucleotide 7498 from North Carolina, United States, had been subjected to (nt) small interfering RNA (siRNA) that silence the immune system of physical mapping and whole-genome shotgun sequencing using the land plants, thereby protecting Spirodela against a wide spectrum of “454” platform (7). Because of the short reads (∼350 bp) of the pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, Significance but also facilitates applications in bioenergy and phytoremediation. Constant exposure of aquatic plants to freely exchangeable nu- long reads | root evolution | disease resistance | tandem duplication | trients and pathogenic microbes requires regulation of gene aquatic adaptation expression different to land plants. However, short-read se- quencing platforms fail to provide vital information that com- reater duckweeds, i.e., Spirodela polyrhiza, are small and prises genes involved in the response to such a challenge. Here, Gfast-growing aquatic plants, belonging to the early-diverging we applied long-read sequencing to retrieve missing sequences monocot order of the Alismatales (1) (SI Appendix,Fig.S1). As to the duckweed species of Spirodela polyrhiza. Evolution of the one of the most yielding biomass species, they can be found from genetic network and root morphology show that roots play a temperate to tropical regions, surviving in freshwater ponds and function as sea anchors rather than nutrient uptake. Moreover, animal waste lagoons. The plant is composed of a tiny frond and a disease-resistance gene clusters are constitutively active whereas few adventitious roots (ARs), providing unique material to study they are silenced by phasiRNA in land plants. root evolution at early angiosperm lineages. Two other genera of Author contributions: J.M. and W.W. designed research; D.A., Y. Zhou, Q.X., T.W., and Lemnaceae (Wolffiella and Wolffia) appear to lack roots, based on D.C. performed research; D.A., Y. Zhou, C.L., Y. Zhang, Y.W., Y.L., and J.M. analyzed data; close morphological and microscopic inspections (2). Because of and D.A., Y. Zhou, J.M., and W.W. wrote the paper. larger fronds of the genus Spirodela, sticky roots perhaps help Reviewers: Y.V.d.P., Ghent University; and R.A.W., University of Arizona. maintain their upright position in the water and promote vegetative The authors declare no conflict of interest. dispersal with the attachment to animals, rather than act primarily Published under the PNAS license. as an organ for the uptake of water and nutrients (3). On the other Data deposition: Genome assembly and consensus sequences have been deposited in the hand, the presence of water brings the fronds into immediate National Center for Biotechnology Information (NCBI) GenBank database, https:// contact with a diverse population of microbes. Interestingly, the www.ncbi.nlm.nih.gov (accession no. SWLF00000000). Full-length cDNA sequences have duckweed crude extract exhibited antimicrobial resistance toward been uploaded to the NCBI Genbank database (accession no. SRX5321175). RNA-Seq data have been deposited in the NCBI GenBank BioProject database (accession no. waterborne fungi and bacteria (4, 5), which could be a potential PRJNA557001). source for medicinal herbs and new antimicrobial therapeutics. To 1D.A. and Y. Zhou contributed equally to this work. understand the molecular nature of these properties, a compre- 2To whom correspondence may be addressed. Email: [email protected] or hensive repertoire of genes is essential, not available with older [email protected]. sequencing platforms. Due to larger gaps from short-read (6) se- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. quence assembly, missing information could be critical for under- 1073/pnas.1910401116/-/DCSupplemental. standing the molecular mechanism of root formation and defenses www.pnas.org/cgi/doi/10.1073/pnas.1910401116 PNAS Latest Articles | 1of7 Downloaded by guest on September 25, 2021 “454” system, the genome of Spirodela (Sp7498V2) was highly and Spo000617 in Sp7498V3 were concatenated from 4, 3, and 2 fragmented and could not resolve complex regions, leading to ge- genes in Sp7498V2, respectively (SI Appendix,Fig.S5). The average nome misassembly or misannotations. Although shotgun DNA se- gene length was significantly increased by 24.5% from 3,458 to 4,342 quencing (8) facilitated the assembly of a complete genome (9), the bp, with longer and higher exon numbers for each gene (Table 1). presence of tandemly repeated sequences, like gene copies, trans- BUSCO (Benchmarking Universal Single-Copy Orthologs) evalu- posable elements, or satellite repeats of large genomes, prevents ation revealed that Sp7498V3 had a higher rate (86%) of complete earlier developed sequencing platforms from delivering contiguous homologous proteins, compared with 79% of Sp7498V2 (SI Ap- sequence information. PacBio with long reads (>10 kb) can cover pendix,TableS6), indicating a high-quality assembly and annotation sequence junctions between single repeats, such as retrotransposons, of Sp7498V3. and can overcome assembly problems in terms of gap filling and Repetitive elements play important roles in shaping genome repeat reconstruction in many plants, such as maize (10, 11), architecture and gene regulation. Most repeats were incomplete, Oropetium (12), quinoa (13), and broomcorn millet (14). To this unassembled, or highly collapsed in Sp7498V2 due to short-read end, we resequenced S. polyrhiza 7498 with ∼126-fold coverage assembly. The improved genome of Sp7498V3 allowed us to ac- of PacBio long reads, resulting in 411 contigs and an N50 length curately survey its completeness of repetitive features. We found (a median of contig lengths) of 831 kb (15) (SI Appendix, Figs. S2 that repetitive sequences account for 30.91% of the genome (SI and S3 and Tables S1 and S2), which improved contiguous se- Appendix, Fig. S6 and Table S7), 18.61% of which were long quence information 44 times compared with the previous version terminal repeat (LTR) retrotransposons that arose before 2 mil- (Table 1) (7). The new genome assembly version, named lion years ago (MYA) and were species-specific (SI