Identifying leaf anatomy and metabolic regulators that underpin C₄photosynthesis inAlloteropsis 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. 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. 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 ), non-photochemical quenching ( SOQ1 ), and the development of Kranz anatomy ( SCARECROW-LIKE ). Regions associated with the development of C 4 leaf 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 C 4 photosynthesis in A. semialata required a few loci of large effect. The candidate genes could prove to be relevant for engineering C 4 leaf anatomy in C 3 species.