Location: Plant Science Research
2020 Annual Report
Objectives
The overall goal of this project is to reduce nutrient inputs, particularly nitrogen (N) and phosphorus (P), in legume crops through the identification of germplasm having root architectural diversity and the discovery of genes that may contribute to that diversity. Desired outcomes from the research proposed herein include identification of unique germplasm with altered root morphology that may reduce costly fertilizer inputs, novel genes that regulate root development and function, and fundamental insight into the biochemical processes that affect nutrient acquisition. To achieve these goals and outcomes, three integrated objectives will be pursued.
Objective 1: Phenotype and evaluate root architecture changes in soybean, common bean and Medicago mutants, determine relationships between root architecture and improved nutrient acquisition, and define genome lesions.
Objective 2: Evaluate whole genome transcript analysis of common bean and alfalfa through RNA-seq analysis of roots, root nodules, leaves and seeds to compare wild-type and mutants.
Objective 3: Identify genes contributing to root architecture and nutrient acquisition in legumes and determine their function.
Approach
Identify mutant plants derived from fast neutron and Tnt1 mutagenized populations which affect root architecture and development, and define genetic lesions through next generation sequencing. Conduct RNA-seq transcript expression studies for the organs of wild type and mutant legume species such as alfalfa, common bean, and soybean to identify genes involved in unique adaptations displayed by these species. Utilize RNAi, zinc finger nuclease modification and/or antisense constructs to silence expression of selected root-specific/enhanced genes affecting root architecture and/or nutrient acquisition.
Progress Report
This is a bridging project that was initiated March 2018. Significant progress has been made in two objectives (1 & 3). In support of Objective 1, targeted mutagenesis was carried out on alfalfa plants using gene editing reagents targeting alleles of phosphate uptake related genes. Approximately 90-100 plants were recovered from tissue culture and screened for the presence of the reagent. Reagent positive plants were further analyzed for mutations at individual alleles. However, the high level of sequence homology combined with insufficient genomic resources has made the identification of mutations challenging. Through collaboration with the Noble Research Institute and the National Center for Genome Resources (NCGR) the lab gained access to the unpublished alfalfa genome assembly to assist in the identification of all the alleles. Using this sequence information, a Pacbio amplicon sequencing assay will be carried out on T0 and T1 plants transformed with the gene editing reagent. This experiment will provide an unprecedented coverage of the mutation landscape of the alleles from each gene and will enable the selection of germplasm for phosphate uptake experiments. A manuscript reporting these results is in preparation. In support of Objective 3, a candidate list of root-architecture associated gene targets is currently under-construction and continues to be refined. Targets include legume orthologs of genes associated with a deep rooting and other root elongation traits, genes that contribute to variation in root architecture identified from a published wild soy quantitative trait locus (QTL) analysis and several other root meristem-associated genes involved in the lateral root initiation pathway recently identified in Arabidopsis. In addition, the lab has been planning and developing in-house soybean and alfalfa transformation protocols to enable the delivery gene editing reagents to a wide range soybean and alfalfa cultivars. These assays will enable the lab to generate mutants for the phenotypic validation of these genes.
Accomplishments
