A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop

Abstract

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress.

Keywords: Thlaspi arvense; comparative genomics; de novo assembly; field pennycress; whole genome sequencing.

Figure 1.

Comparative genomics of pennycress and other Brassicaceae species. (A) Syntenic path assembly dot plots comparing pennycress scaffolds >75 kilobases long to the seven Eutrema salsugineum pseudochromosomes fromYang et al. (B) BLASTp analysis of the 27,390 predicted pennycress peptides against predicted peptide sets from Capsella rubella, Brassica rapa, Arabidopsis thaliana (Bevan and Initiative, 2000), Arabidopsis lyrata, and Eutrema salsugineum. Highly similar is defined as pennycress predicted peptide having at least one BLASTp hit e < 1 × 10⁻⁵ and positive sequence similarity >70%. (C) BLASTp analysis of predicted pennycress peptides against a protein database containing the predicted peptides of the five Brassicaceae species listed. Predicted peptides with top hits (e ≤ 1 × 10⁻⁵ and >70% hit length) to a predicted protein from the corresponding species are shown, with pennycress peptides with hits falling below this threshold shown in the lower right half of the pie chart.

Figure 2.

CAPS analysis of Thlaspi arvense line MN106 (A) Schematic of the four PCR fragments produced by the primer sets listed in Supplementary Table S4. The largest fragments used to distinguish between individuals containing the SNP (MN106 A genotype fragment—top, and MN106 B genotype fragment—bottom). (B) DNA was isolated from progeny of each of the nine plants used to produce the draft genome assembly, and analysed using four CAPS markers. PCR products for each plant are shown side-by-side undigested (uncut) and post-digestion (cut) with the corresponding restriction endonucleases. In all cases, samples 3, 5, and 7 share restriction digest patterns, corresponding to the MN106-B genotype. A negative control for the PCR reaction is shown in the last lane. (C) Morphology of developing T. arvense flowers. The top panel (1–3) shows the morphology of the unaltered flowers, while the bottom panel (4–6) shows the same series of flowers with sepals and petals either removed or rearranged to reveal the status of the stamens with regard to filament elongation and the shedding of pollen. (4) Neither filament elongation nor pollen shedding has commenced in (1). (5) Filaments have elongated, and pollen is being shed inside of the closed flower shown in (2). (5) Pollen densely covers the stigmatic surface by the time the flower is fully open in (6). All scale bars equal 1 mm.

Figure 3.

Analysis of genes involved in glucosinolate metabolism and transport. (A) Overview of glucosinolate biosynthesis core structure (top) via methionine and tryptophan and breakdown (bottom) and corresponding orthologues in the pennycress genome pathway derived from Liu et al. Expression values (RPKM, in parentheses) are shown for each putative orthologues derived from the global RNAseq reads previously described. (B–G) Neighbour joining trees of TGG1/TGG2, MVP1, ESP, ESM1, GTR1, and GTR2-like predicted peptides (100 bootstrap replicates) from pennycress (identified in this study), Brassica rapa, and Brassica oleraceae.