Location: Plant Science Research
2021 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 and 3). In support of Objective 1, targeted mutagenesis was carried out on alfalfa plants using gene editing reagents targeting haplo-alleles of phosphate uptake related genes. Transgenic plants recovered from tissue culture were screened for gene edits using next generation sequencing technologies. This analysis successfully identified a suite of single, double, triple and quadruple mutant plants. A phosphate analytical assay was developed to quantify Pi concentration in the mutant and wild-type plants. This analysis confirmed a five- to ten-fold increase in the concentration of Pi in the mutant plants compared to wild-type confirming the hyper-accumulation trait. An invention disclosure was submitted and a CRADA agreement with a commercial partner is in preparation. A manuscript reporting these results will be submitted this FY. In support of Objective 3, a candidate list of root-architecture associated gene targets is currently being identified. Laser ablation tomography was used to phenotype 600 root samples from 260 accessions of Medicago truncatula. The image data was processed using image analysis software and the data from multiple root traits called ‘phenes’ was collected, including root cross-section measurements, the number of xylem vessels, and root conductance calculations. The data will be used in a genome wide association study to generate further root architecture related gene targets. Targeted mutagenesis of a select number of these candidate genes will be carried out along with other legume orthologs of genes associated with a deep rooting and root elongation traits identified from published genomic analyses. One of the challenges in identifying new genes related root architecture and nutrient uptake is not their discovery, but their validation. There are many excellent published genomics studies in recent years that have identified potential candidate genes. However, these genes are typically in exotic germplasm that are often recalcitrant to genetic transformation. Many laboratories lack the required specialized transformation skills and rightly balk at the idea of spending two to three years trying to generate candidate mutants for validation. Over the last year, ARS has obtained improved gene editing reagents including novel mutant Agrobacterium strains and developmental regulator constructs for increased transformability. In addition, specialized equipment necessary for improved transformation has been acquired along with the recruitment of industry trained and experienced tissue culture personnel. These pieces will help the development of in-house legume transformation assays that will enable the delivery gene editing reagents to a wide range of soybean, cowpea, common bean and alfalfa cultivars.
Accomplishments
1. Tools for genome editing in alfalfa. Alfalfa is the third most widely grown crop in the U.S., but breeding for increasing herbage yield, quality, and stand persistence has been slow due to the outcrossing breeding system of alfalfa. Modifications to the alfalfa genome through genome editing can accelerate development of more productive and environmentally resilient cultivars; however, genome editing is complicated by the multiple copies of each gene in alfalfa. ARS scientists in Saint Paul, Minnesota, developed novel genome editing reagents that more efficiently edited a phosphate uptake regulatory gene. Mutations were identified for the first time through targeted sequencing of the mutated areas, significantly accelerating identification of mutant plants and reducing costs of identifying mutant plants by tens of thousands of dollars. Alfalfa plants with mutations in one to four copies of the gene were identified and were shown to hyperaccumulate phosphate at varying levels according to the number of mutant haplo-alleles. DNA sequences were identified that will facilitate the tracking of mutations when cross pollinated to other alfalfa backgrounds. These tools will accelerate development of alfalfa cultivars for bioremediation, animal nutrition, and nutrient uptake.
