SummaryEffective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.

Summary Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype‐dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi‐C to resolve each haplotype of the genome of a farmer‐preferred cassava line, TME7 (Oko‐iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON‐Phase and Hi‐C read data. The ultralong‐range information from Hi‐C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype‐specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele‐specific expression was further explored. RNA‐sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy‐to‐use web‐based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype‐specific structural variation, gene hemizygosity, and allele‐specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Cassava (Manihot esculenta Crantz, 2n=36) is a global food security crop. Cassava has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultra-long-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed more than 5,000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele specific expression was further explored. RNA-seq data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Key message We demystify recent advances in genome assemblies for the heterozygous staple crop cassava (Manihot esculenta), and highlight key cassava genomic resources. Abstract Cassava, Manihot esculenta Crantz, is a crop of societal and agricultural importance in tropical regions around the world. Genomics provides a platform for accelerated improvement of cassava’s nutritional and agronomic traits, as well as for illuminating aspects of cassava’s history including its path towards domestication. The highly heterozygous nature of the cassava genome is widely recognized. However, the full extent and context of this heterozygosity has been difficult to reveal because of technological limitations within genome sequencing. Only recently, with several new long-read sequencing technologies coming online, has the genomics community been able to tackle some similarly difficult genomes. In light of these recent advances, we provide this review to document the current status of the cassava genome and genomic resources and provide a perspective on what to look forward to in the coming years.

The zig-zag model of host-pathogen interaction describes the relative strength of defense response across a spectrum of pathogen-induced plant phenotypes. A stronger defense response results in increased resistance. Here, we investigate the strength of pathogen virulence during disease and place these findings in the context of the zig-zag model. Xanthomonas vasicola pv. holcicola (Xvh) causes sorghum bacterial leaf streak. Despite being widespread, this disease has not been described in detail at the molecular level. We divided diverse sorghum genotypes into three groups based on disease symptoms: water-soaked lesions, red lesions, and resistance. Bacterial growth assays confirmed that these three phenotypes represent a range of resistance and susceptibility. To simultaneously reveal defense and virulence responses across the spectrum of disease phenotypes, we performed dual RNA-seq on Xvh-infected sorghum. Consistent with the zig-zag model, the expression of plant defense-related genes was strongest in the resistance interaction. Surprisingly, bacterial virulence genes related to the type III secretion system (T3SS) and type III effectors (T3Es) were also most highly expressed in the resistance interaction. This expression pattern was observed at multiple time points within the sorghum-Xvh pathosystem. Further, a similar expression pattern was observed in Arabidopsis infected with Pseudomonas syringae for effector-triggered immunity via AvrRps4 but not AvrRpt2. Specific metabolites were able to repress the Xvh virulence response in vitro and in planta suggesting a possible signaling mechanism. Taken together, these findings reveal multiple permutations of the continually evolving host-pathogen arms race from the perspective of host defense and pathogen virulence responses.

Cassava mosaic disease suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to the cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for loss of resistance, we performed whole genome sequencing and genetic variant analysis and fine-mapped the CMD2 locus to a 190 kilobase interval. Data suggest that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a Cassava mosaic disease-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional resistance alleles within MePOLD1. MePOLD1 resistance alleles represent important genetic resources for resistance breeding or genome editing, and elucidating mechanisms of resistance to geminiviruses.

A correction to this paper has been published: https://doi.org/10.1007/s11103-021-01139-7