Chromosome-level assemblies of Amaranthus palmeri, Amaranthus retroflexus, and Amaranthus hybridus allow for genomic comparisons and identification of a sex-determining region.

Amaranthus palmeri (Palmer amaranth), Amaranthus retroflexus (redroot pigweed), and Amaranthus hybridus (smooth pigweed) are troublesome weeds that are economically damaging to several cropping systems. Collectively referred to as "pigweeds," these species are incredibly adaptive and have become successful competitors in diverse agricultural settings. The development of genomic resources for these species promises to facilitate the elucidation of the genetic basis of traits such as biotic and abiotic stress tolerance (e.g., herbicide resistance) and sex determination. Here, we sequenced and assembled chromosome-level genomes of these three pigweeds. By combining the haplotype-resolved assembly of A. palmeri with existing restriction site-associated DNA sequencing data, we identified an approximately 2.84 Mb region on chromosome 3 of Hap1 that is male-specific and contains 37 genes. Transcriptomic analysis revealed that two genes, RESTORER OF FERTILITY 1 (RF1) and TLC DOMAIN-CONTAINING PROTEIN (TLC), within the male-specific region were upregulated in male individuals across the shoot apical meristem, the floral meristem, and mature flowers, indicating their potential involvement in sex determination in A. palmeri. In addition, we rigorously classified cytochrome P450 genes in all three pigweeds due to their involvement in non-target-site herbicide resistance. Finally, we identified contiguous extrachromosomal circular DNA (eccDNA) in A. palmeri, a critical component of glyphosate resistance in this species. The findings of this study advance our understanding of sex determination in A. palmeri and provide genomic resources for elucidating the genetic basis and evolutionary origins of adaptive traits within the genus.