Identifying leaf anatomy and metabolic regulators that underpin C4 photosynthesis 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. 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. 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 ), non-photochemical quenching ( SOQ1 ), and the development of Kranz anatomy ( SCARECROW-LIKE ). Regions associated with the development of C4 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 C4 photosynthesis in A. semialata required a few loci of large effect. The candidate genes could prove to be relevant for engineering C4 leaf anatomy in C3 species. ### Competing Interest Statement The authors have declared no competing interest.