Fine mapping of QTL and identification of candidate genes responsible for tuberous stem formation in Kohlrabi (Brassica oleracea var. gongylodes L.)

Tuberous stem is a key determinant of yield and quality in kohlrabi, and it is a prime target of artificial selection for breeding and domestication. To dissect the genetic basis of stem tuberization, a previously identified quantitative trait locus, REnBo2 on chromosome C03, was fine mapped to an 87.5-kb interval using recombinant populations from three successive backcross generations derived from double haploid lines of kohlrabi and broccoli. The fine-mapped interval contained three genes: BolC03g081960.2 J (encoding CIPK6 ), BolC03g081970.2 J ( FAD6 ) and BolC03g081980.2 J ( WRKY32 ). Integrated analysis of gene annotations, allelic variations, expression profiles, and previously generated transcriptome and endogenous phytohormone datasets identified CIPK6 as the most promising candidate underlying REnBo2 , whereas WRKY32 remains a potential secondary candidate requiring further functional characterization. CIPK6 exhibited allelic variation and significantly lower expression in kohlrabi stems during tuber initiation and development. Functional validation in Arabidopsis further supported the role of CIPK6 , as the atcipk6 knockdown mutant displayed swollen hypocotyls along with impaired root development. Heterologous expression of the broccoli CIPK6 allele retrieve the non-swollen hypocotyl morphotype with a developed root system similar to the wild-type plants, whereas the kohlrabi CIPK6 allele failed to restore the wild-type phenotypes despite high transgene expression, indicating allele-specific functional divergence. Collectively, our results identify CIPK6 as a prominent candidate gene regulating stem tuberization in kohlrabi and provide a new insight into the auxin-associated molecular mechanisms controlling storage stem development in Brassica and related species.