Both the fruit mesocarp and the seeds of winter squash can be used for consumption, although the focus of breeding efforts varies by cultivar. Cultivars bred for fruit consumption are selected for fruit mesocarp quality traits such as carotenoid content, percent dry matter, and percent soluble solids, while these traits are essentially ignored in oilseed pumpkins. To compare fruit development in these two types of squash, we sequenced the fruit transcriptome of two cultivars bred for different purposes: an acorn squash, ‘Sweet REBA’, and an oilseed pumpkin, ‘Lady Godiva’. Putative metabolic pathways were developed for carotenoid, starch, and sucrose synthesis in winter squash fruit and squash homologs were identified for each of the structural genes in the pathways. Gene expression, especially of known rate-limiting and branch point genes, corresponded with metabolite accumulation both across development and between the two cultivars. Thus, developmental regulation of metabolite genes is an important factor in winter squash fruit quality.

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