Integrated GWAS and transcriptome analyses reveal candidate genes associated with betalain-related pigmentation under heat stress in Amaranthus tricolor

Amaranthus tricolor is a climate-resilient orphan crop whose betalain pigments contribute to nutritional quality and stress tolerance. However, breeding efforts have traditionally relied on field yield trials due to the limited availability of genomic information and genetic resources. To address this gap, we first used the Illumina and PacBio sequencing platforms to assemble a novel reference genome of A. tricolor, using the Japanese cultivar Bayam. By combining this reference genome with a core collection developed in our previous study, we aimed to identify genomic regions associated with pigmentation-related traits across winter and summer seasonal environments. Betalains are antioxidant phytonutrients that contribute to abiotic stress tolerance, making the regulation of their biosynthesis under heat stress particularly relevant for developing climate-resilient crops such as A. tricolor. As a result, ATR1.0CH03_5131103, which is associated with ATR1.0ch03g000565, was identified under the winter seasonal environment. Meanwhile, transcriptomic profiles in this study revealed that ATR1.0ch03g000565 was differentially expressed under both control and heat stress conditions, suggesting that it may be associated with the regulation of CYP76AD1 expression under optimal conditions. Under the elevated-temperature treatment, transcript levels of CYP76AD1 and 5GT decreased in Am087, coinciding with lower betacyanin-dominated absorbance indices. cDOPA-like homologs were upregulated; however, pigmentation-related absorbance remained reduced. Further molecular experiments, such as CRISPR-Cas9 knockouts or overexpression studies, are necessary to validate the regulatory mechanisms of CYP76AD1 and 5GT under heat stress.