Large-Scale Screening Of Setaria Viridis Mutants With Defects In The C4Photosynthetic Process And Whole Transcriptome Analysis Of S.viridis To Support C4Research Analysis

Ever since the discovery of C4photosynthesis, attempts of engineering the current majorstaple crops to perform C4photosynthesis have never stopped. Unfortunately, relatively littlesuccess has been achieved. So far, which has created tremendous doubt in research communityand policy makers alike regarding whether it is in the end possible to engineer C3crops toperform C4photosynthesis. Paramount of evidences with the rapid advances in the nextgeneration sequencing technologies and new approaches in genetic engineering suggest the C4engineering is a tangible goal. However, reaching this goal depends on understanding of themolecular mechanisms controlling different features of C4photosynthesis. In this study,we usea new C4model species Setaria viridis, which has the following advantages: small genome of⁵10Mb; small plant （10-15cm）; simple growth conditions; a short life cycle （6-9weeks） andprolific seed production. We obtained8000individual EMS or γ-ray mutagenized M1plants andperformed M2generation screening in the field, By observing the plant type, leaf color, leafveins, leaf and ear traits to find screening C4defect mutants. we characterized a total of206mutants with defects in the plant type, leaf color, leaf veins, leaf and ear traits including158EMS mutagenesis,48γ mutagenesis including leaf curl, leaf notch, short stature, the veinschange and ear abnormalities variety of mutant phenotypes. In addition, S.viridis has a typicalKranz anatomy and greater vein density than C3plants, therefore we use vein spacing as ascreening indicators to find the mutant associated with the development of Kranz anatomy. Weused hand-held microscope to count the number of veins and found75mutants which reduced inthe number of veins. These mutants are in further analysis.As a typical C4plant, S.viridis has a very low CO₂compensation point （around5ppm）. Ifthe mutation cause S.viridis lose its C4characteristics, the CO₂compensation point is supposedto increase. Therefore we performed mutant screening in low CO₂chamber for slow growth orloss of green individuals. The mutants identified were shifted to normal air with a CO₂concentration of380ppm for growth rescue. Until now we have screened3000EMSmutagenized M2plants and found some important mutants, further characterization of theseinteresting mutants are performing in our lab. S.viridis is an emerging model species for genetic studies of C4photosynthesis. Many basicmolecular resources need to be developed to support for this species. In this paper, we performeda comprehensive transcriptome analysis from multiple developmental stages and tissues ofS.viridis using next-generation sequencing technologies. Sequencing of the transcriptome frommultiple tissues across three developmental stages （seed germination, vegetative growth, andreproduction） yielded a total of71million single end100bp long reads. Reference-basedassembly using Setaria italica genome as a reference generated42,754transcripts. De novoassembly generated60,751transcripts. In addition,9,576and7,056potential simple sequencerepeats （SSRs） covering S. viridis genome were identified when using the reference basedassembled transcripts and the de novo assembled transcripts, respectively. This identifiedtranscripts and SSR provided by this study can be used for both reverse and forward geneticstudies based on S. viridis..