Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits.

Highlights d Long-read sequencing of 100 tomato genomes uncovered 238,490 structural variants d Transposons underlie many SVs, and SV hotspots revealed large introgressions d SVs associated with genes are predictive of population-scale changes in expression d New genome assemblies resolved complex breeding QTLs caused by SVs

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We present RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes. After the pseudomolecules are constructed, RaGOO identifies structural variants, including those spanning sequencing gaps. We show that RaGOO accurately orders and orients 3 de novo tomato genome assemblies, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open source at https://github.com/malonge/RaGOO.

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Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.

Plants evolved so that their flowering is triggered by seasonal changes in day length. However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication. Here we show that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). SP5G expression is induced to high levels during long days in wild species, but not in cultivated tomato because of cis-regulatory variation. CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. Our findings suggest that pre-existing variation in SP5G facilitated the expansion of cultivated tomato beyond its origin near the equator in South America, and they provide a compelling demonstration of the power of gene editing to rapidly improve yield traits in crop breeding.

Precise control of plant stem cell proliferation is necessary for the continuous and reproducible development of plant organs 1,2 . The peptide ligand CLAVATA3 (CLV3) and its receptor CLV1 maintain stem cell homeostasis within a deeply conserved negative feedback circuit 1,2 . In Arabidopsis, CLV1 paralogs also contribute to homeostasis, by compensating for the loss of CLV1 through transcriptional upregulation 3 . Here we show that compensation 4,5 operates in diverse