CULTIVAR DEVELOPMENT: ACCELERATED INTROGRESSION OF SYNTHETIC HEXAPLOID DERIVED DIVERSITY INTO AN APPLIED HARD WINTER WHEAT BREEDING PROGRAM

The major goals of this proposed research are to clone the Green (Gr) gene responsible to the green curd trait in cauliflower and to decipher its genetic and molecular basis in regulating chloroplast development for developing more nutritious crops.Objectives:Objective 1: Functional confirmation of the Gr gene via phenotypic complementation Our previous studies identified a few best candidates. In this Objective, we aim to confirm the identify of Gr via phenotypic complementation of curd color in cauliflower and in Arabidopsis ap1-1 cal-1 mutant that gives "curd-like" structure resembling cauliflower curd. Overexpression of the best candidates and CRISPR-Cas9 knockout of the endogenous candidate genes will be carried out to identify the gene that complements the phenotype in cauliflower curd and in Arabidopsis "curd-like" structure of ap1-1 cal-1 mutant.Objective 2. Molecular and microscopic characterization of the Gr geneIn this Objective, we will characterize the Gr gene to gain insights into better understanding of its role in conferring the green curd trait and in mediating chloroplast development. We will investigate the spatial expression pattern of Gr in various tissues of cauliflower, associate its expression with curd color in a germplasm collection of cauliflower, examine chloroplast numbers in various tissues of representative accessions, and discover the potential cause by analyzing the difference between the white and green alleles of Gr.Objective 3. Dissection of the regulatory mechanism of Gr in mediating chloroplast development Since none of the candidates is known to regulate chloroplast development, Gr represents a novel regulator in initiating chloroplast development. Gr is important as its variant promotes or abolishes chloroplast formation in cauliflower curd tissue. In this Objective, we will identify Gr-interacting proteins via several alternative and complementary strategies, which include yeast two-hybrid cDNA library screening, co-immunoprecipitation coupled with mass spectrometry analysis, and Gr protein association network analysis. We will also investigate how such interaction or the Gr-interacting protein(s) regulates chloroplast development to give the green curd phenotype in cauliflower.