Location: Plant Gene Expression Center2013 Annual Report
1a. Objectives (from AD-416):
The long-term goal of this research is to identify and characterize genes that regulate growth and development in maize: Objective 1: Identify genes that regulate plant architecture in maize by using mutant analysis and positional cloning. Objective 2: Determine function of cloned genes with molecular, genomic and genetic techniques. Objective 3: Map and clone modifiers of plant architecture genes to understand their function. To truly understand the function of a gene, one needs a mutation. The mutation reveals how that gene functions. Mutations that result in a morphological phenotype identify genes that are critical for growth and development. Maize has a rich history of genetic analysis based on the ease of obtaining and analyzing mutants. Although many classically identified mutations have now been cloned, new ones are continually being discovered. Some of these new mutants are dominants, which are rare compared to recessives, and thereby have not been previously identified. Other mutants are background dependent and may have been overlooked because they appear different in each inbred and thus are difficult to track. We have several mutants and modifiers at different stages of analysis. In each case, we will identify the gene by recombination mapping. We will identify the lesion that causes the mutation and in the case of modifiers, we will seek to determine how the inbred allele differences affect the mutant phenotype. We will determine how the genes function in their normal context and in the mutant context by RNA, phenotypic, and genetic analysis. We will identify paralogs and determine their function when possible. The end goal is a clear understanding of each gene’s function and how it impacts growth and development.
1b. Approach (from AD-416):
Objective 1: Identify genes that regulate plant architecture in maize by using mutant analysis and positional cloning. Hypothesis: Genes defined by a morphological phenotype are important for growth and development. We have identified 4 new mutants by screening in different inbred backgrounds. All four mutants affect both the leaf and inflorescence. We will clone these genes by identifying recombinants. The goal is to reduce the interval to 1-2 genes and then sequence. The mutants have been made in defined inbred backgrounds making identification of the EMS induced lesion straight-forward. Objective 2: Determine function of cloned genes with molecular, genomic and genetic techniques. Hypothesis: Knowing how a gene functions provides important information about developmental processes. For each mutant we analyze and gene we clone, a series of steps are carried out. We will determine where the gene is expressed. We will determine the putative function of the gene. We will analyze closely related paralogs and for some proteins, we will identify interacting partners. Objective 3: Map and clone modifiers of plant architecture genes to understand their function. Hypothesis: Modifiers of mutants provide information about the genetic basis of natural diversity. For Lgn, we will identify recombinants to identify the modifer. We will also use RNAseq experiments and genetic screens. For nod, we will map the modifier after developing well introgressed material. It is possible that there is not a single modifier, but multiple genes. If that is the case, the RNAseq analysis should prove helpful.
3. Progress Report:
This report documents progress for Project Number 5335-21000-035-00D, which started in July 2013 and continues research from Project Number 5335-21000-034-00D, entitled “POSITIONAL CLONING IN MAIZE OF GENES THAT REGULATE PLANT ARCHITECTURE”. The three project Objectives fall under National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement. Progress on this project focuses on addressing Problem 3A – the need for fundamental knowledge of plant biological and molecular processes. Due to the previous work that leads into this project, we were able to make some progress in this short period of reporting. Objective 1. We have two alleles of narrow odd dwarf (nod), one from A619 and one from B73. We mapped nod to an interval of 18 genes on the top of chromosome 1. Upon sequencing one of the genes, we identified G to A mutations in both alleles. One allele changes a Proline to a Lysine in a conserved domain, the other allele is a stop codon. The gene encodes a conserved protein that has homology with the gene responsible for the fruit weight QTL in tomato, fw2.2. An ortholog was also identified in Arabidopsis as a gene that compliments a yeast Calcium transport defect. Using transient expression in tobacco, we determined that NOD localizes to the plasma membrane. Objective 2. We crossed the revertant of Wavy auricle in blade (Wab), which has upright tassel branches, to a number of mutants and selfed the F1 in the winter nursery. We are presently growing out the double mutants in our nursery and will evaluation at the end of the summer. Double mutants we will observe include wab-rev with liguleless1, wab-rev with ramosa2. We crossed nod into the PIN1a auxin transport reporter. This will be back-crossed this summer. Objective 3. Lgn mutants are very background and environmentally sensitive. We have now mapped the modifier, names Sol for sympathy for the ligule, to a 3 Mb region. We completed 500 DNA preps this summer and identified 5 crucial recombinants in the 3 Mb interval containing 9 genes. The recombinants will be phenotyped at the end of the summer and should help delimit the mapping interval of Sol even more. We also carried out RNAseq on 4 genotypes that segregate Lgn and the modifier. The RNAseq data revealed all the Mo17 B73 SNPs in the mapping interval. It also provided us with RNA levels of the candidate genes. The narrow odd dwarf (nod) mutant has been introgressed into A619, B73, W22 and Mo17. It is very severe in B73 and nearly normal in Mo17. This difference will allow us to test whether Sol also modifies nod and, if not, use the Intermated B73 x Mo17 (IBM) recombinant lines to map the modifier.
1. The role of calcium signaling in Maize development. Calcium is known to be an important signaling molecule for many plant processes; however, a clear role in development has not been demonstrated. ARS scientists identified the gene for narrow odd dwarf (nod) and found it encodes a protein that functions in calcium transport. The maize nod mutants are dwarfs with narrow leaves. In the B73 inbred, the mutants lose apical dominance and do not initiate a tassel. Leaves are narrow and often liguleless. Identification of the NOD protein provides insight into how calcium signaling functions in plant growth and development.