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Cucurbita moschata is a short-day plant that is sensitive to photoperiod and exhibits delayed female flowering or no female flowers under long days(LDs).Understanding the biological mechanisms underlying photoperiod-controlled female flowering is required to advance the breeding of photoperiod-insensitive cultivars and develop improved cultivation habits for existing cultivars.We studied two pumpkin germplasms,a photoperiod-insensitive(PPIS)germplasm normally capable of blossoming and producing fruits and a common photoperiod-sensitive(PPS)germplasm bearing almost no female flowers under LDs.These were studied using RNA-Seq,Gibberellin(GA)measurement,microRNA(miRNA),and hormone treatment analysis.RNA-Seq analysis of shoot apices from 3-week-old seedlings in PPS compared to PPIS under LDs disclosed 995 up-and 651 down-regulated differentially expressed genes(DEGs).GA mediated signaling pathway was one significantly different pathway among process ontology terms in which five DEGs,GAIP,GID1,HY5,SPINDLY and GAST1 were enriched.Transcriptional expression of the five DEGs and GA biosynthetic genes GA20ox2,GA3oxl,and KS was detected in DEGs before filtration and were validated by quantitative real time RT-PCR.The significantly different expression of these 8 genes revealed that a GA signaling pathway and GA biosynthesis may play key roles in photoperiod femaleness of PPIS.Femaleness of the two germplasms exhibited different responses to hormone treatment under LDs.PPIS femaleness was not responsive to the GA inhibitor,but it was inhibited by GA.PPS femaleness was recovered by the GA inhibitor.The single bioactive GA detected in PPIS seedlings,GA4,was accumulated in smaller aounts under both LDs and short days(SDs)than in PPS.GA regulated female flowering under LDs but not under SDs.Downregulation of DELLA targets,miR156 and miR159,in PPIS seedlings compared with PPS suggested that bioactive GA regulation of photoperiodic female flowering might be via an epigenetic pathway.This study provides a theoretical basis for further investigation of regulatory mechanism of photoperiod-insensitive female flower expression in pumpkin.