Uncovering the Genetic Basis of Heterodichogamy in Pterocarya and Cyclocarya Using a Low-Input Pan-Genomic Approach

The heterodichogamous mating system, characterized by two distinct mating types (protogyny and protandry), is rare among flowering plants, but it is present in nearly all species in Juglandaceae (the walnut family). Recent studies have identified distinct structural variations underlying heterodichogamy in Juglans and Carya . To verify the independent origins of this trait in Juglandaceae and investigate whether structural variations also drive heterodichogamy in Juglans closely related genera, we explored its genetic basis in Pterocarya and Cyclocarya . Using a pan-genome graph approach, we identified a structural variation region associated with mating types across the Pterocarya genus. This region includes 30 kb tandem repeats in the dominant allele and an insertion in the recessive allele, with shared polymorphisms spanning 78 kb from the 3′UTR of S12e , covering a FAF-like gene, to a Gypsy transposable element. Downstream analyses suggest that the specific expression of FAF-like gene and small RNAs uniquely expressed from the tandem repeats of dominant allele regulate heterodichogamy. Further investigation in Cyclocarya identified nine candidate loci associated with heterodichogamy, which are non-homologous regions with those found in Pterocarya , Juglans , and Carya . These findings provide novel evidence for the multiple independent originations of convergent genetic basis in regulating heterodichogamy in Juglandaceae and highlight the utility of pan-genome approaches in deciphering structural variation-associated traits.