Characterization of a Lolium multiflorum diploid assembly

Grasses of the genera Lolium and Festuca are the main feed sources for a sustainable livestock production due to their high palatability and biomass production. Since decades, their importance for the agriculture of temperate regions led to the development of new varieties through traditional breeding programs. However, newer crop improvement methods such as genomic selection could benefit from a high quality reference genome assembly. In the past, attempts in delivering such a dataset have struggled due to the complexity of the genome and the high heterozygosity of individual genotypes. We sequenced an individual of the L. multiflorum (Italian ryegrass) cv. Rabiosa, producing a highly contiguous (N50 of 3 Mb) and complete assembly (97% of the BUSCO gene models). Due to the high heterozygosity of the line, the assembly (4.5 Gb) resulted to be as large as the diploid genome, and presented the sequence of both alleles in separate scaffolds. About ~70,000 gene models were identified, and the repeat content approached 80%. The comparison of a representative allelic region showed an extensive amount of intergenic sequence variation, supporting the high dynamicity of grass genomes. Compared to the available Lolium genome assemblies, the Rabiosa assembly improves contiguity by >40-fold, contains both haplotypes of the diploid parent, and assigns most of the sequence to chromosomes. The availability of a complete and highly-contiguous genome assembly of Italian ryegrass paves the road to the exploitation of the forage crops genetic resources by means of modern genomic platforms

A ~phased diploid genome assembly of Italian ryegrass

Grasses of the genera Lolium and Festuca are the main feed sources for a sustainable livestock production due to their high palatability and biomass production. Since decades, their importance for the agriculture of temperate regions led to the development of new varieties through traditional breeding programs. However, newer crop improvement methods such as genomic selection could benefit from a high-quality reference genome assembly. In the past, attempts at producing genomic resources have struggled due to the complexity of the genome and the outcrossing nature of the species. We sequenced an individual of the L. multiflorum (Italian ryegrass) cv. Rabiosa, producing a highly-contiguous and complete assembly. Due to the high heterozygosity of the genotype, the resulting assembly was as large as the diploid genome, thus presenting the sequence of both alleles in separate collinear scaffolds. The generation of large-scale scaffolding datasets (i.e. chromosome conformation capture data and optical maps) allowed to phase sequences, reaching chromosome-level contiguity. The comparison of the two allelic sequences for a region showed an extensive amount of intergenic sequence variation, confirming that ryegrass genomes are highly dynamic. The high-quality genome assembly of the cv. Rabiosa is the first phased diploid assembly of a plant genome and provides a high-quality reference for expediting ryegrass breeding and studying the genome biology of outcrossing species

Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots

Aim 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. Background 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. Results 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. Conclusions 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.ISSN:1746-481

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch

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

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch

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.status: publishe

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch

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.ISSN:2375-254