Identifying genomic regions associated with C 4 photosynthetic activity and leaf anatomy in Alloteropsis semialata.

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.