Chromosome level assembly and annotation of Cuscuta campestris Yunck. ("field dodder"), a model parasitic plant

Abstract

We present the first chromosome-level genome assembly and annotation for the genus Cuscuta, a twining and leafless parasitic plant of the morning glory family (Convolvulaceae). C. campestris, the study species, is a widely studied model parasite, due in part to its worldwide occurrence as a weed of agricultural and natural plant communities. The species has served as a model parasite for studies of parasite biology, haustorium development, growth responses to chemical and light stimuli, gene content and expression, horizontal gene transfer, and interspecies RNA movement and has a recently developed transformation system. The 505 Mb (1C) genome is assembled into 31 chromosomes and supports annotation of 47,199 protein-coding genes, 214 small RNA loci (including 146 haustoria-specific miRNAs), and 3,238 interspecies mobile mRNA loci. C. campestris is a recent tetraploid with a high retention of duplicated genes and chromosomes, with less than 8% nucleotide divergence between homoeologous chromosomes. We also show that transformation of C. campestris with the RUBY marker system allows visualization of transformed Cuscuta-derived fluorescent mobile molecules that have entered the host stem. This genome, with an associated genome browser and BLAST server, will be of value for scientists performing fundamental research in a wide range of molecular, developmental, population, and evolutionary biology, as well as serve as a research tool for studying interspecies mobile molecules, generating genetic markers for species and genotype identification, and developing highly specific herbicides.

Keywords: Cuscuta campestris; RUBY transformation; comparative genomics; genome annotation; genome assembly; genome browser; genome sequence; mobile molecules; parasitic plants; plant genomics; polyploidy; reference genome.

Fig. 1.

Images of C. campestris in habitat. a) C. campestris growing on Impatiens sp. in Central Pennsylvania, United States. b) Close-up of the plant showing flowers and fruits around its host. Photos by Juan D. Cerda-Herrera.

Fig. 2.

Genome assembly of C. campestris. a) Contact map showing 31 distinct chromosomes. b) Visualization of genome, (1) chromosomes, (2) telomere repeat sequence distribution (window size = 30,000 bp), (3) gene content distribution (window size = 10,000 bp), (4) LTR content distribution (window size = 10,000 bp), (5) distribution of mobile mRNAs, (6) distribution of miRNAs, and (7) syntenic blocks connecting the chromosomes.

Fig. 3.

Repeat analysis and comparison between the parasitic plant C. campestris (this study, Convolvulaceae), the closely related, nonparasitic species I. cairica (Jiang et al. 2022; Convolvulaceae) and a more distantly related nonparasitic species S. lycopersicum (Fernandez-Pozo et al. 2015; Hosmani et al. 2019; Solanaceae). a), c), and e), showing the repeat landscape for the 3 species: X axis showing the Kimura substitution level (KSL) for among-copy divergence, and Y axis showing the percent of the genome occupied by each repeat at each KSL. b), d), and f), showing the percentage of genome occupied by any given type of repeat. Black = nonrepeat regions.

Fig. 4.

CoRe OrthoGroups (CROGs) analysis (Wafula et al. 2023) showing clustering of species by Z-score based on the number of annotated genes within given OGs. Redder and yellower OGs are expanded in gene number relative to other taxa, while Bluer OGs are reduced in gene number relative to other taxa. CuAu = C. australis (Sun et al. 2018), CuCa = C. campestris (this study), CuCaV = C. campestris (Vogel et al. 2018), IpNi = I. nil (Hoshino et al. 2016), IpCa = I. cairica (Jiang et al. 2022), IpPu = I. purpurea (Lyons et al. 2008; Lyons and Freeling 2008; Gupta et al. 2023), CaAn = C. annuum (Hulse-Kemp et al. 2018), SoLy = S. lycopersicum (Fernandez-Pozo et al. 2015; Hosmani et al. 2019), and SoTu = S. tuberosum (Hardigan et al. 2016).

Fig. 5.

Gene enrichment analysis of “lost” genes in Cuscuta: Molecular Functions, Cellular Components, Biological Processes. X axis shows the count of genes in each biological term; Y axis shows the primary biological terms. Color of the graphs represents the FDR-adjusted P-value for each term.

Fig. 6.

Species tree generated by OrthoFinder's STAG algorithm. Bar shows inferred substitution rate per site. Branches show STAG support value, and branches with no values have a support of 1.

Fig. 7.

Fluorescence of RUBY observed in C. campestris and within the host stem. Apical tissues of RUBY Cuscuta (35S:RUBY) and WT Cuscuta were harvested and observed with the bright-field light a) and eGFP filter b) using a widefield microscope. Vibratome-sectioned Cuscuta haustoria were imaged under the widefield microscope c). Zoomed-in image of the white box in (c) was analyzed using a confocal microscope with UV d) and YFP e) filters. The obtained images from UV and YFP filters were merged to produce f). The white dashed box indicates betalain accumulations inside the host stem. Abbreviations: cor, Arabidopsis cortex; end, Arabidopsis endodermis; hau, Cuscuta haustoria.