High-quality assembly of the Chinese white truffle genome and recalibrated divergence time estimate provide insight into the evolutionary dynamics of Tuberaceae.

The genus (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 ECM fungi, including Tuberaceae. Repetitive sequences constitute a principal driver of genome evolution shaping its architecture and regulatory networks. In this context, Tuberaceae can become an important model system to study their genomic impact; however, the family lacks high-quality assemblies. Here, we investigate the interplay between TEs and Tuberaceae genome evolution by producing a highly contiguous assembly for the endangered Chinese white truffle , along with a recalibrated timeline for Tuberaceae diversification and comprehensive comparative genomic analyses. We find that, concurrently with a Paleogene diversification of the family, pre-existing Chromoviridae-related Gypsy clades independently expanded in different truffle lineages, leading to increased genome size and high gene family turnover rates, but without resulting in highly rearranged genomes. Additionally, we uncover a significant enrichment of ECM-induced gene families stemming from ancestral duplication events. Finally, we explore the repetitive structure of nuclear ribosomal DNA (rDNA) loci for the first time in the clade. 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 establishes a fundamental genomic resource for future research on truffle genomics and showcases a clear example of how establishment and self-perpetuating expansion of heterochromatin can drive massive genome size variation due to activity of selfish genetic elements.