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Characterization of homoeallelic base-identity in allopolyploids is difficult since homeologous subgenomes are closely related and becomes further challenging if diploid-progenitor data is missing. We present HANDS2, a next-generation sequencing-based tool that enables highly accurate (>90%) genome-wide discovery of homeolog-specific base-identity in allopolyploids even in the absence of a diploid-progenitor. We applied HANDS2 to the transcriptomes of various cruciferous plants belonging to genus  Brassica . Our results suggest that the three C genomes in  Brassica  are more similar to each other than the three A genomes, and provide important insights into the relationships between various  Brassica  tetraploids and their diploid-progenitors at a single-base resolution.

HANDS2: accurate assignment of homoeallelic base-identity in allopolyploids despite missing data