Genome assembly of primitive cultivated potato Solanum stenotomum provides insights into potato evolution.

Genetic diversity is the raw material for germplasm enhancement. Landraces and wild species relatives of potato, which contain a rich gene pool of valuable agronomic traits, can provide insights into the genetic diversity behind the adaptability of the common potato. The diploid plant, Solanum stenotomum (Sst), is believed to have an ancestral relationship with modern potato cultivars and be a potential source of resistance against disease. Sequencing of the Sst genome generated an assembly of 852.85 Mb (N50 scaffold size, 3.7 Mb). Pseudomolecule construction anchored 788.75 Mb of the assembly onto 12 pseudochromosomes, with an anchor rate of 92.4%. Genome annotation yielded 41,914 high-confidence protein-coding gene models and comparative analyses with closely related Solanaceae species identified 358 Sst-specific gene families, 885 gene families with expansion along the Sst lineage, and 149 genes experiencing accelerated rates of protein sequence evolution in Sst, the functions of which were mainly associated with defense responses, particularly against bacterial and fungal infection. Insights into the Sst genome and the genomic variation of cultivated potato taxa are valuable in elaborating the impact of potato evolution in early landrace diploid and facilitate modern potato breeding.

Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice.

CRISPR-Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non-coding RNAs in plants is still at its infancy. Despite being a major class of non-coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR-Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR-Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs' participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA-miRNA-mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.

Multiplex QTL editing of grain-related genes improves yield in elite rice varieties.

KEY MESSAGE: Significant yield increase has been achieved by simultaneous introduction of three trait-related QTLs in three rice varieties with multiplex editing by CRISPR-Cas9. Using traditional breeding approaches to develop new elite rice varieties with high yield and superior quality is challenging. It usually requires introduction of multiple trait-related quantitative trait loci (QTLs) into an elite background through multiple rounds of crossing and selection. CRISPR-Cas9-based multiplex editing of QTLs represents a new breeding strategy that is straightforward and cost effective. To test this approach, we simultaneously targeted three yield-related QTLs for editing in three elite rice varieties, namely J809, L237 and CNXJ. The chosen yield-related QTL genes are OsGS3, OsGW2 and OsGn1a, which have been identified to negatively regulate the grain size, width and weight, and number, respectively. Our approach rapidly generated all seven combinations of single, double and triple mutants for the target genes in elite backgrounds. Detailed analysis of these mutants revealed differential contributions of QTL mutations to yield performance such as grain length, width, number and 1000-grain weight. Overall, the contributions are additive, resulting in 68 and 30% yield per panicle increase in triple mutants of J809 and L237, respectively. Our data hence demonstrates a promising genome editing approach for rapid breeding of QTLs in elite crop varieties.