Cloning And Molecular Evolution Of GAD1 Regulating Grain Number,Grain Length And Awn Development In Rice

Asian cultivated rice (Oryza sativa L.) is domesticated from common wild rice (Oryza rufipogon Griff.). Both morphological traits and physiological characteristics changed remarkably during rice domestication. Wild rice typically produces a few, long grains per panicle, with long awns that are crucial for seed dispersal and deterring granivore predation. However, most current rice cultivars generate many grains per panicle and shorter grains and show no or only short awns. These characteristics facilitate rice storage and processing. Thus, the transition of these characteristics represents a critical event in rice domestication.In order to elucidate the molecular mechanisms responsible for these important changes, we obtained one introgression line (OIL31) from a set of introgression lines derived from a cross between indica variety NA93-11 and an accession of common wild rice (W2014, O. rufipogon Griff.). OIL31 produced fewer grains per panicle and longer grains as well as a high percentage of awned seeds, with long awns. Using a segregating population derived from a cross between OIL31 and NA93-11, we mapped the gene regulating grain number, grain length and awn development(GRAIN NUMBER, GRAIN LENGTH AND AWN DEVELOPMENT 1, GAD1) to a～6-kb region on the long arm of chromosome 8, in which there was only one predicted gene (Os08g0485500). Complementation test confirmed that the gene regulating grain number, grain length and awn development is GAD1. RNA in situ hybridization of OsHistone H4 indicated that GAD1 can regulate awn development by activating cell division. Cytokinin contents and OsCKX2, DST expression suggested a hypothesis that GAD1 activates DST and OsCKX2 to reduce the cytokinin level and cause less grain number per panicle in wild rice. GAD1 encodes a predicted small secreted cysteine-rich peptide, GAD1 is a homolog of EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) in Arabidopsis. Through sequence comparison between OIL31 and NA93-11, a frame-shift insertion in gad1 destroyed the conserved cysteine residues of the peptide, resulting in a loss of function, and causing the increased number of grains per panicle, shorter grains, and awnless phenotype characteristic of cultivated rice. Sequence analysis showed that the changing of the cysteine residues number caused by the frame-shift was highly associated with the presence or absence of awn in cultivated rice. GAD1 was a major target of artificial selection in rice domestication. We identified an interval of ～900-kb surrounding the GAD1 locus that exhibited a significantly decreased level of nucleotide diversity in cultivated rice relative to wild rice. Our study revealed that except several genes controlling rice domestication-related traits which encoded transcription factors or enzymes, small secretory peptide encoded by GAD1 also play roles in rice domestication. Identification of GAD1 not only provides a useful paradigm for revealing functions of peptide signal molecules in plant development, but also offers intriguing insights on the role of peptide signal molecules in rice domestication.