Exploratory Stage-Resolved Transcriptomic Profiling of Seed Development in Amaranthus hypochondriacus var. Revancha Prioritizes Candidate Genes for Seed Maturation

Seed development influences reserve accumulation, grain quality, seed maturation, and subsequent crop performance, yet stage-associated transcriptional changes remain poorly characterized in grain amaranth. Here, we performed exploratory transcriptomic profiling of three reproductive stages of Amaranthus hypochondriacus var. Revancha: ovule (OV), immature seed (IS), and mature seed (MS). One composite RNA-seq library was generated for each stage from pooled tissues collected during three independent sampling events. The composite-sampling strategy did not provide independent biological replication or allow estimation of biological dispersion. Transcript-abundance patterns were evaluated using exploratory expression contrasts, functional annotation, Gene Ontology and KEGG analyses, and targeted expression and chromatin assays. OV was characterized by transcripts associated with structural organization and early developmental processes, whereas IS showed increased representation of metabolic and biosynthetic functions consistent with active reserve accumulation. MS displayed transcript-abundance patterns associated with stress responses, redox homeostasis, abscisic acid-associated processes, and desiccation-related protection. Selected genes related to gibberellin metabolism, reserve regulation, developmental control, and cellular protection showed stage-associated expression patterns. Across developmental-stage comparisons, 1083 genes showed higher and 2801 lower transcript representation in OV relative to IS, whereas 5692 and 4424 genes, respectively, differed between OV and MS, and 6399 and 4128 between IS and MS. Semi-quantitative RT-PCR and targeted H3K4me3 ChIP-PCR provided complementary evidence for selected loci. Because each developmental stage was represented by a single composite RNA-seq library, the transcriptomic contrasts are interpreted as exploratory and hypothesis-generating rather than as statistically replicated developmental effects. Therefore, this study provides a stage-resolved transcriptomic resource for grain amaranth and prioritizes candidate genes and biological processes for future functional, physiological, and breeding-oriented studies of seed maturation and composition.