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

Summary C 4 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 C 4 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 C 3 , intermediate, and C 4 accessions that recently diverged. We aimed to identify genomic regions associated with the strength of the C 4 cycle (measured using δ 13 C), and the development of C 4 leaf anatomy. Genomic regions correlated with δ 13 C include regulators of C 4 decarboxylation enzymes ( RIPK ), nonphotochemical quenching ( SOQ1 ), and the development of Kranz anatomy ( SCARECROW‐LIKE ). Regions associated with the development of C 4 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 C 4 lineages implies the co‐option of these genes is context‐dependent, which likely has implications for the engineering of the C 4 trait into C 3 species.