2018 ASHS Annual Conference

Lettuce seed germination is promoted by light through activation of gibberellin (GA) biosynthesis. A quantitative trait locus for inhibition of lettuce seed germination by the darkness was mapped to chromosome 7 using a RIL population derived from a dark-germinating cultivar (Lactuca sativa cv. “Salinas”) and a light-requiring accession of L. serriola (US96UC23). The LsGA2ox2 gene encoding a GA-inactivation enzyme was located in this QTL region and exhibited distinct expression patterns in Salinas and US96UC23 seeds imbibed in the dark. Nine of twelve SNPs between Salinas and US96UC23 (UC) were within a 93 bp window in the promoter of LsGA2ox2, and the same 9 SNPs were conserved in light-requiring Grand Rapids (L. sativa) and dark-germinating W48 (L. serriola). Expression of ProUCGA2ox2::UCGA2ox2 in Salinas lines resulted in strong inhibition of seed germination in the dark. Site-directed mutagenesis of three SNPs associated with an abscisic acid-responsive motif in the UCGA2ox2 promoter resulted in failure to complement atga2ox2 mutants, suggesting that these SNPs are critical for upregulation of LsGA2ox2 in seeds imbibed in the dark. Knockout of LsGA2ox2 through CRISPR/Cas9 in Grand Rapids or RNAi silencing of LsGA2ox2 in US96UC23 resulted in stem elongation and enhanced seed germination in the dark that are associated GA upregulation. The combination of genetic mapping, mutant complementation, and conserved SNPs implicates degradation of GA by LsGA2ox2 in the inhibition of germination by darkness. Our results may facilitate breeding of lettuce varieties with more uniform germination of pelleted seeds and improve lettuce stand establishment and yield.