Estimating the ploidy of Gracilariopsis lemaneiformis at both the cellular and genomic level.

The determination of the ploidy level of an organism is a prerequisite for studies of evolution, cellular function, and genomic construction. Identification of the ploidy of the economically important red alga Gracilariopsis lemaneiformis has been hindered by its small genome and large number of chromosomes. Therefore, in the current study, PloidyNGS, a tool that calculates the number of reads supporting different alleles at each position along the genome sequence, and fluorescence in situ hybridization coupled with tyramide signal amplification (TSA-FISH) were used to clarify the ploidy of G. lemaneiformis. In addition, flow cytometry (FCM) was used to estimate the ploidy of different somatic cells. The PloidyNGS results showed that most of the alleles in the gametophyte were monomorphic, whereas the TSA-FISH results showed that one hybridization signal was observed in gametophytic nuclei and two in tetrasporophytic nuclei when the nuclei were hybridized by single copy gene probes. These results confirmed that G. lemaneiformis is a haploid in the gametophytic generation and diploid in the sporophytic generation. Moreover, the FCM result suggested that G. lemaneiformis was not an endopolyploid. Based on previous studies, we hypothesize that the nuclear number is important for the cellular differentiation and function of this species. We also suggest that G. lemaneiformis evolved from a paleopolyploid, the genome of which has been diploidized, and that traces of genomic doubling are no longer apparent. Thus, this study provides important evidence for further studies on the evolution and genomes of red algae.