Identifying leaf anatomy and metabolic regulators that underpin C₄photosynthesis inAlloteropsis semialata

C4photosynthesis 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 C4is complicated by the fact that variation in photosynthetic type is usually segregated between species. Here, we perform a genome wide association study (GWAS) using the grassAlloteropsis semialata, the only known species to have C3, intermediate, and C4accessions. We aimed to identify genomic regions associated with the strength of the C4cycle (measured using δ13C), and the development of C4leaf anatomy. Genomic regions correlated with δ13C include regulators of C4decarboxylation enzymes (RIPK), non-photochemical quenching (SOQ1), and the development of Kranz anatomy (SCARECROW-LIKE). Regions associated with the development of C4leaf anatomy in the intermediate accessions contain additional leaf anatomy regulators, including those responsible for vein patterning (GSL8) and meristem determinacy (GRF1). The detection of highly correlated genomic regions with a modest sample size indicates that the emergence of C4photosynthesis inA. semialatarequired a few loci of large effect. The candidate genes could prove to be relevant for engineering C4leaf anatomy in C3species.