Unravelling genome organization of neopolyploid flatworm Macrostomum lignano

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of genome doubling through allo- or autopolyploidization could lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are undoubtedly important for understanding one of the crucial stages of genome evolution. However, assembling neopolyploid appears to be a challenging task because its genome consists of two homologous (or homeologous) chromosome sets and therefore contains the extended paralogous regions with a high homology level. Post-WGD evolution of polyploids includes rediploidization, first part of which is cytogenetic diploidization led to the formation of species, whose polyploid origin might be hidden by disomic inheritance and diploid-like meiosis. Earlier we uncovered the hidden polyploid origin of free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Despite the different mechanisms for their genome doubling, cytogenetic diploidization in these species accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we reported unusual subgenomic organization of M. lignano through generation and sequencing of two new laboratory sublines of DV1 that differ only by a copy number of the large chromosome MLI1. Using non-trivial assembly-free comparative analysis of their genomes, including adapted multivariate k-mer analysis, and self-homology within the published genome assembly of M. lignano, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists in (1) presence of three subgenomes, emerged via formation of large fused chromosome and its variants, and (2) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.