A copy-number variation generates a long-noncoding RNA that enhances ClBige1b expression to suppress cell expansion and seed growth in watermelon.

Seed size is a key agronomic trait in watermelon: large seeds are favorable for sowing, whereas small-seeded watermelons are preferred for consumption. However, the causal genetic variations and underlying mechanisms that govern seed size in watermelon remain poorly understood. Here, we identified a copy-number variation (CNV) tightly linked to seed size; a single copy is present in large-seeded watermelons, whereas four copies are present in small-seeded lines. Compared with the single-copy CNV, the four-copy CNV contains two additional TATA boxes and activates the transcription of a downstream long-noncoding RNA (C. lanatus lncRNA seed size 1 [ClLNC-SS1]) on the antisense strand. Knockout of ClLNC-SS1 in the small-seeded line resulted in larger seeds and repressed the expression of ClBige1b, which is located downstream of the CNV on the sense strand. ClBige1b encodes a multidrug and toxic compound extrusion transporter, and its overexpression in a large-seeded line resulted in smaller seeds. By contrast, knockout of ClBige1b increased seed size in a small-seeded line, markedly increasing cell expansion in the exocarp, endocarp, and endosperm by reducing auxin contents in the developing seeds. Our findings demonstrate that the newly identified CNV regulates seed size via the ClLNC-SS1-ClBige1b module in watermelon, providing novel insight into the regulation of seed size and laying a foundation for its genetic improvement in watermelon and other crops.