Harnessing Bulk‐Segregant Mapping to Identify Trait‐Associated Genes in the Allopolyploid Model Plant <i>Nicotiana benthamiana</i>

Forward genetics has been instrumental in identifying genes underlying desirable traits, yet its application to polyploid plants, many of which are key agricultural crops, remains challenging due to their genomic complexity. Therefore, we developed BenthMap, a bulk segregant analysis platform for high-throughput trait mapping and gene discovery, in the allotetraploid model plant Nicotiana benthamiana. BenthMap leverages high-quality genome assemblies of two genetically and phenotypically distinct strains, LAB and QLD. To validate the pipeline, we investigated their contrasting anthocyanin responses. Transient overexpression of AcMYB110, an activation regulator of anthocyanin biosynthesis, induces robust anthocyanin production in QLD leaves but gives a detrimental, often necrotic, response in LAB. Using BenthMap and a population derived from selfing the F1 hybrid of a LAB × QLD cross (F1S1 population), with genome coverage as low as 10×, we mapped the necrotic LAB response to a 3.5 Mb homozygous region on chromosome 10. This region contains a leucoanthocyanidin dioxygenase gene. Transiently expressing the QLD version of this gene, along with AcMYB110, restored robust anthocyanin accumulation in LAB, validating the causal gene. These findings demonstrate BenthMap's utility for rapid trait-gene identification in N. benthamiana and have potential for application to other allopolyploid plants.