Cross-species dissection of saline-related genes by genetically deciphering a euryhaline microalga Chlorella sp. MEM25

Deciphering habitat shifts across salinity boundary, necessitates discovery of the "lost" and "acquired" saline genes. By assembling a telomere-to-telomere genome, we propose that the euryhaline Chlorophyta Chlorella sp. MEM25 represents an early-diverging saltwater species that has evolved numerous genes essential for saltwater-freshwater transitions. Genome comparison to those of Viridiplantae identified green-plant-heritage genes and evolutionary novelties related to salinity adaptation. Saline-related features may arise through genes expansion, horizontal gene transfer, and multi-levels choreography. Loss-of-function mutants of the proposed salt-sensitive genes in algae and plants exhibited increased salt resistance, highlighting the potential of the MEM25 genome as breeding resources. This resource will enhance a wide range of studies, from exploring the evolutionary history of saltwater-freshwater shifts to uncovering the mechanisms of osmoregulation, which can be utilized for crop breeding.To validate the salinity-related genes uncovered through genome expansion, genome-wide association studies (GWAS) was conducted. An ethyl methane sulfonate (EMS) mutant population was generated using MEM25 as the parent strain. Out of 50,000 independent mutant lines, 536 mutants with altered OD750 under either low or high salinity were selected for further phenotyping under the high-salinity conditions. Subsequently, 365 mutants with stable phenotypes after ten generations were chosen to assess variations in cell number, cell size, and dry weight (DW) under the high-salinity conditions. Lastly, the genomes of 195 saline-related mutants and three WT samples were resequenced with 30x coverage (n=198).