Safflower is an important oilseed crop that has been used in traditional Chinese medicine for thousands of years because of the clinically valuable flavonoid glycosides in its flower petals. However, the biosynthesis and molecular regulation of these compounds are still elusive due to the lack of a high-quality reference genome and scarce identification of key biosynthetic pathway genes in a medicinal safflower variety. Here we leveraged an integrative multi-omics strategy by combining genomic, comparative genomics, and tissue-specific transcriptome profiling with biochemical analysis to identify uridine diphosphate glycosyltransferases (UGTs) for flavonoid glycoside biosynthesis in safflower. We assembled and annotated a high-quality reference genome of a medicinal safflower variety, ‘Yunhong3’. A comprehensive comparative genomic analysis indicated that an evolutionary whole-genome triplication event occurring in safflower contributed to gene amplification of the flavonoid biosynthetic pathway. By combining comparative transcriptome profiling with enzymatic reactions, we identified 11 novel UGTs that could catalyze the conversion of naringenin chalcone and phloretin to the corresponding O-glycosides. Moreover, we outlined the molecular pathway of hydroxysafflor yellow A (HSYA) biosynthesis featured by 17 newly identified UGTs with promising catalytic activity, laying the foundation for the synthetic production of HSYA. Our study reports systemic genome and gene expression information for flavonoid glycoside biosynthesis in medicinal safflower and provides insights into mechanisms regulating HSYA biosynthesis, which would facilitate the genetic improvement and synthetic bioengineering design for producing clinically valuable flavonoid glycosides in safflower.

Root-knot nematodes (RKNs) are the most widespread soil-borne obligate endoparasites. They can infect the roots of many crops and cause significant yield losses. The only commercially available RKN-resistant gene in tomatoes, Mi-1.2, fails at soil temperatures above 28°C. We cloned the heat-stable RKN-resistant gene, Mi-9, from a gene cluster composed of seven nucleotide-binding sites and leucine-rich repeat (NBS-LRR) type resistant genes in Solanum arcunum accession LA2157. Screening nematode infections in individual and combinatorial knockouts of five NBS-LRR genes showed that Mi-9 Candidate 4 (MiC-4) alone is sufficient to confer heat-stable RKN resistance. Our study identifies a new source of heat-stable resistance to RKN in tomatoes for challenging environmental conditions. We also showcase a roadmap for rapid characterization of resistance genes by combining comparative genomics and genome editing, with the potential to be utilized in other crops.