Without the ability to perform photosynthesis, G. elata depends completely on its symbiotic fungus for nutrition. It is thus obvious that the signaling pathways related to the establishment of this symbiotic relationship are crucial for G. elata. Some of the mechanisms underlying the symbiotic interaction between G. elata and A. mellea are similar to those for interactions between other plants and arbuscular mycorrhizal (AM) fungi22. The G. elata genome contains many of the genes known to participate in AM associations (Fig. 3a, Supplementary Table 38). Key genes for biosynthesis and secretion of strigolactone were expanded in G. elata (e.g., carotenoid cleavage dioxygenases, CCDs, for biosynthesis23 and ABC transporters, PDRs, for secretion24) (Supplementary Table 38). It is known that strigolactone can stimulate hyphal branching and development of arbuscular mycorrhizal fungi, which increases the chances of an encounter with a host plant24. We conducted growth assays and confirmed that strigolactone had similar branch-inducing effects in A. mellea (Fig. 3b, Supplemental Fig. 13). The expanded number of genes encoding CCDs and PDRs suggests that G. elata has enhanced its ability to interact with A. mellea to increase the efficiency of the establishment of the symbiotic relationship essential for its nutrition and metabolism. Calmodulin-dependent protein kinase genes of the does-not-make-infections 3 subfamily (DMI3) were also doubled or tripled in G. elata (10 genes) compared to P. equestris (3 genes), D. officinale (5 genes), and A. comosus (4 genes); these genes participate in the Ca2+ spiking process that has been shown to regulate the colonization of plants by fungi25.Fig. 3Strigolactone as a putative signal compound in G. elata in its mycoheterotrophic symbiotic relationship with A. mellea. a Overview of proposed signaling and nutrition transfer in G. elata. The red-labeled genes are expanded in the G. elata genome and P-value of Fisher’s exact test of gene number <0.05 (Supplementary Tables 38, 40, 41). ANT, ANT1-like aromatic and neutral amino acid transporters; ArgJ, glutamate N-acetyltransferase; CCD, carotenoid cleavage dioxygenases; COS, chitooligosaccharides; DMI3, does-not-make-infections 3 subfamily; LOS, lipochitooligosaccharides; LysM, LysM-receptor-like kinases; PDR, ABC transporter. P1, p-value of Fisher’s exact test of gene number in G. elata genome compared to P. equestris, D. officinale, A. comosus, and A. thaliana; P2, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. thaliana; P3, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. comosus; P5, p-value of Fisher’s exact test of gene number in G. elata, P. equestris, and D. officinale genome compared to A. comosus and A. thaliana. b Branching of A. mellea hyphae was significantly promoted after strigolactone treatment (Supplementary Fig. 13). Scale bar was 5 mm. c Cross sections and micrographs of immature G. elata tubers in association with A. mellea (A, epidermis; B, cortex; C, inner parenchyma cells, Supplementary Fig. 14). The black scale bar on the left was 100 μm and the white one on the right was 20 μm