Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)–mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering—an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

Mapping-by-sequencing has emerged as a powerful technique for genetic mapping in several plant and animal species. As this resequencing-based method requires a reference genome, its application to complex plant genomes with incomplete and fragmented sequence resources remains challenging. We perform exome sequencing of phenotypic bulks of a mapping population of barley segregating for a mutant phenotype that increases the rate of leaf initiation. Read depth analysis identifies a candidate gene, which is confirmed by the analysis of independent mutant alleles. Our method illustrates how the genomic resources of barley together with exome resequencing can underpin mapping-by-sequencing.

Background: Cassava is an important food crop in tropical and sub-tropical regions worldwide. In Africa, cassava production is widely affected by cassava mosaic disease (CMD), which is caused by the African cassava mosaic geminivirus that is transmitted by whiteflies. Cassava breeders often use a single locus, CMD2, for introducing CMD resistance into susceptible cultivars. The CMD2 locus has been genetically mapped to a 10-Mbp region, but its organization and genes as well as their functions are unknown. Results: We report haplotype-resolved de novo assemblies and annotations of the genomes for the African cassava cultivar TME (tropical Manihot esculenta), which is the origin of CMD2, and the CMD-susceptible cultivar 60444. The assemblies provide phased haplotype information for over 80% of the genomes. Haplotype comparison identified novel features previously hidden in collapsed and fragmented cassava genomes, including thousands of allelic variants, inter-haplotype diversity in coding regions, and patterns of diversification through allele-specific expression. Reconstruction of the CMD2 locus revealed a highly complex region with nearly identical gene sets but limited microsynteny between the two cultivars. Conclusions: The genome maps of the CMD2 locus in both 60444 and TME3, together with the newly annotated genes, will help the identification of the causal genetic basis of CMD2 resistance to geminiviruses. Our de novo cassava genome assemblies will also facilitate genetic mapping approaches to narrow the large CMD2 region to a few candidate genes for better informed strategies to develop robust geminivirus resistance in susceptible cassava cultivars.

We report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots. VIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function. We developed an African cassava mosaic virus—based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots. The African cassava mosaic virus—based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.