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Despite the importance of secondary dormancy for plant life cycle timing and survival, there is insufficient knowledge about the (epigenetic) regulation of this trait at the molecular level. Our aim was to determine the role of (epi)genetic processes in the regulation of secondary seed dormancy using natural genotypes of the widely distributed Capsella bursa-pastoris. Seeds of nine ecotypes were exposed to control conditions or histone deacetylase inhibitors [trichostatin A (TSA), valproic acid] during imbibition to study the effects of hyper-acetylation on secondary seed dormancy induction and germination. Valproic acid increased secondary dormancy and both compounds caused a delay of t50 for germination (radicle emergence) but not of t50 for testa rupture, demonstrating that they reduced speed of germination. Transcriptome analysis of one accession exposed to valproic acid versus water showed mixed regulation of ABA, negative regulation of GAs, BRs and auxins, as well as up-regulation of SNL genes, which might explain the observed delay in germination and increase in secondary dormancy. In addition, two accessions differing in secondary dormancy depth (deep vs non-deep) were studied using RNA-seq to reveal the potential regulatory processes underlying this trait. Phytohormone synthesis or signalling was generally up-regulated for ABA (e.g. NCED6 , NCED2 , ABCG40 , ABI3) and down-regulated for GAs (GA20ox1 , GA20ox2 , bHLH93), ethylene (ACO1 , ERF4-LIKE, ERF105 , ERF109-LIKE), BRs (BIA1 , CYP708A2-LIKE , probable WRKY46 , BAK1 , BEN1 , BES1 , BRI1) and auxin (GH3.3 , GH3.6 , ABCB19 , TGG4 , AUX1 , PIN6 , WAT1). Epigenetic candidates for variation in secondary dormancy depth include SNL genes, histone deacetylases and associated genes (HDA14 , HDA6-LIKE , HDA-LIKE , ING2 , JMJ30), as well as sequences linked to histone acetyltransferases (bZIP11 , ARID1A-LIKE), or to gene silencing through histone methylation (SUVH7 , SUVH9 , CLF). Together, these results show that phytohormones and epigenetic regulation play an important role in controlling differences in secondary dormancy depth between accessions. [ABSTRACT FROM AUTHOR]