Identification of genes associated with mycelial growth in Flammulina filiformis using high-throughput screening and a T-DNA mutant library

In Flammulina filiformis , the mycelial growth rate is an important vegetative trait that influences the cultivation cycle. However, the genetic networks underlying mycelial growth remain poorly understood. To address this gap, this study aimed to generate a high-quality reference genome of F. filiformis , construct a T-DNA insertion mutant library, and establish a candidate co-expression network governing mycelial growth through integrative analysis of mutant phenotypes and transcriptomic data. A chromosome-level genome assembly of F. filiformis monokaryotic strain 6–3 was generated using third- and next-generation sequencing, Hi-C chromosome conformation capture, and a genetic linkage map constructed from 142 monosporic strains. A library of 210 T-DNA insertion mutants was constructed via Agrobacterium tumefaciens -mediated transformation, with integration sites precisely mapped by whole-genome resequencing. Phenotypic screening was performed by measuring mycelial growth rates on both potato dextrose agar and sawdust-based cultivation media, and transcriptome sequencing was conducted on selected growth-defective mutants, followed by differential expression and functional enrichment analyses. The chromosome 08 exhibited high variation, repetitive sequence clustering, cold spots for exogenous insertions, and enrichment regions for mating-type factors. Six distinct integration patterns were characterized, revealing non-random chromosomal hotspots. Phenotypic screening results showed that 37.1% of mutants exhibited significant mycelial growth inhibition. Transcriptomic analysis of selected growth-defective mutants highlighted the importance of ABC transporters and pentose and glucuronate interconversions in regulating mycelial growth. By integrating mutant phenotypes with transcriptome data, we constructed a candidate co-expression network of 25 genes associated with mycelial growth, including nine novel genes and two genes not previously associated with this trait. This study provides a chromosome-level genome assembly, characterized T-DNA insertion mutant library, and integrated framework for identifying candidate genes associated with mycelial growth in F. filiformis . These resources may facilitate functional genomic studies and support the molecular breeding of edible fungi.