Gene expression profiles in early leaf of rice(Oryza sativa)and foxtail millet(Setaria italica)

Plant anatomy is patterned early during leaf development which suggests studying the spatial–temporal transcriptomes of primordia will help identify critical regulative and functional genes.We successfully isolated the leaf primordia tissues from the C3grass rice and the C4grass foxtail millet by laser capture microdissection(LCM)and studied the gene expression throughout leaf developmental stages.Our data analysis uncovered the conserved expression patterns of certain gene clusters both in rice and foxtail millet during leaf development.We revealed genes and transcription factors involved in vein formation,stomatal development,and suberin accumulation.We identified 79 candidate genes associated with functional regulation of C4anatomy formation.Screening phenotype of the candidate genes revealed that knock-out of a putative polar auxin transport related gene NAL1 resulted significantly reduced veinal space in rice leaf.Our present work provides a foundation for future analyses of genes with novel functions in grasses and their role in leaf development,in particular the role in leaves with a contrasting C3vs.C4biosynthetic pathway.

A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.