Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress.

Natural selection drives local adaptations of species to biotic or abiotic environmental stresses. As a result, adaptive phenotypic divergence can evolve among related species living in different habitats. However, the genetic foundation of this divergence process remains largely unknown. Two closely related alpine grass species, Stipa capillacea and Stipa purpurea, are distributed in different rainfall regions of northern Tibet. Here, we analyzed the drought tolerance of these two closely related Stipa species, and found that S. purpurea was more resistance to drought stress than S. capillacea. To further understand the genetic diversity behind their adaptation to drought environments, a comprehensive gene repertoire was generated using PacBio isoform and Illumina RNA sequencing technologies. Bioinformatics analyses revealed that differential transcripts were mainly enriched in the wax synthetic pathway, and a threonine residue at position 239 of WSD1 was identified as having undergone positive selection in S. purpurea. Using heterologous expression in the Saccharomyces cerevisiae mutant H1246, site-directed mutagenesis studies demonstrated that a positive selection site results in changes to the wax esters profile. This difference may play an important role in S. purpurea in response to drought conditions, indicating that S. purpurea has evolved specific strategies involving its wax biosynthetic pathway as part of its long-term adaptation to the Qinghai-Tibet Plateau.