Insights into the architecture of earthworm metallothionein genes, powered by long-read genomics and transcriptomics.

The metal-handling metallothionein gene family is subjected to a strong selection pressure in earthworms inhabiting metalliferous soils. The resultant single nucleotide polymorphisms and structural variants within metallothionein's characteristically short exons challenge standard gene prediction and genome assembly tools and complicate phylogenetic analysis of earthworm metallothioneins (wMTs). Here, the genomic origin of wMTs was defined by sequencing four wMT-containing Bacterial Artificial Chromosomes (BACs). This yielded sequence contigs of lengths from 97 to 123 kb and enabled further genetic analyses, supporting the creation of a BLAST-based software for visualizing long-read RNA-seq alignments-lrRNAseq TAST (Total Alignment Search Tool). Phylogenetic analysis of wMT proteins from long-read RNA-seq data grouped wMTs into three distinct clades. Tissue-specific lrRNAseq and metallomic mapping, conducted in context of lead toxicity, revealed enrichment of wMT-1 and wMT-2 in tissues of heavy metal detoxification, and colocalization of wMT with lead in the chloragog. The generation of rare wMT isoforms was attributed to errors in expression, but the erroneous transcripts may possess a degree of order and purpose. A conserved region in the 3' UTR of wMT transcripts was found completely distinct between wMT homologues, allowing classification without inferring phylogenetic trees.