Identifying genomic regions associated with C₄ photosynthetic activity and leaf anatomy in Alloteropsis semialata

C₄ photosynthesis is a complex trait requiring multiple developmental and metabolic alterations. Despite this complexity, it has independently evolved over 60 times. However, our understanding of the transition to C₄ is complicated by the fact that variation in photosynthetic type is usually segregated between species that diverged a long time ago. Here, we perform a genome-wide association study (GWAS) using the grass Alloteropsis semialata, the only known species to have C₃, intermediate, and C₄ accessions that recently diverged. We aimed to identify genomic regions associated with the strength of the C₄ cycle (measured using δ¹³C), and the development of C₄ leaf anatomy. Genomic regions correlated with δ¹³C include regulators of C₄ decarboxylation enzymes (RIPK), nonphotochemical quenching (SOQ1), and the development of Kranz anatomy (SCARECROW-LIKE). Regions associated with the development of C₄ leaf anatomy in the intermediate individuals contain additional leaf anatomy regulators, including those responsible for vein patterning (GSL8) and meristem determinacy (GIF1). The parallel recruitment of paralogous leaf anatomy regulators between A. semialata and other C₄ lineages implies the co-option of these genes is context-dependent, which likely has implications for the engineering of the C₄ trait into C₃ species.