Martelossi, J.; Vujovic, J.; Huang, Y.; Tatti, A.; Xu, K.; Puliga, F.; Chen, Y.; Rota Stabelli, O.; Ghiselli, F.; Zhang, X.; Zambonelli, A.

The genus Tuber (family: Tuberaceae) includes the most economically valuable ectomycorrhizal (ECM), truffle-forming fungi. Previous genomic analyses revealed that massive transposable element (TE) proliferation represents a convergent genomic feature of mycorrhizal fungi, including Tuberaceae. Repetitive sequences are one of the major drivers of genome evolution shaping its architecture and regulatory networks. In this context, Tuberaceae represent an important model system to study their genomic impact; however, the family lacks high-quality assemblies. Here, we tested the interplay between TEs and Tuberaceae genome evolution by producing a highly contiguous assembly for the endangered Chinese truffle Tuber panzhihuanense, along with a novel timeline for Tuberaceae diversification and comprehensive comparative genomic analyses. We found that concurrently with a Paleogene diversification of the family, pre-existing Chromoviridae-related Gypsy clades independently expand in different truffle lineages leading to increased genome size and high gene family turnover rates, but without resulting in highly scrambled genomes. Additionally, we found an enrichment of ECM-induced gene families among ancestral duplication events. Finally, we explored the repetitive structure of nuclear ribosomal DNA (rDNA) loci for the first time in the clade. We found that most of the 45S rDNA paralogues are undergoing concerted evolution, though an isolated divergent locus raises concerns about potential issues for metabarcoding and biodiversity assessments. Our study provides a fundamental genomic resource for future research on truffle genomics and showcases a clear example on how establishment and self-perpetuating expansion of heterochromatin can drive massive genome size variation due to activity of selfish genetic elements.