Location: Plant Science Research2019 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
This is a bridging project that was initiated March 2018, replacing 5062-21000-029-00D. Research is continuing utilizing the objectives of the previous project plan. Progress was made under Objective 1 to phenotype three soybean mutants. The gene mutation responsible for short trichomes (leaf hairs) was mapped to chromosome 6 using bulk segregate analysis followed by Array Comparative Genomic Hybridization. The candidate gene was validated using gene editing with CRISPR/Cas9. The mutation results in trichomes that are 400 um in length compared to 1100 um wild type trichomes with a smaller trichome base diameter. Mutant trichomes have reduced nuclear size and have not undergone the endoreduplication found in wildtype trichome nuclei. Mutant plants are reduced in plant height and have reduced seed yield compared to wildtype. Mutation of the orthologous gene in Arabidopsis results in the same phenotype as well as resistance to bacterial infection. A manuscript reporting this research was submitted. Mutations in LPS1 leading to shorter petioles was also investigated and found to be due to collapse of epidermal cells and smaller cortical cells. The mutation allows a higher field planting density but did not increase seed yield compared to the wildtype. A third mutation resulting in gnarled trichomes was mapped to a protein involved in cytoskeleton organization. The Arabidopsis mutant was complemented with the soybean gene validating the candidate gene identification. Investigations into the involvement of the gene in root hair curling during infection by symbiotic rhizobia is ongoing. Progress was also made in Objective 2 to support assembly and annotation of a reference alfalfa genome sequence. Material was isolated from alfalfa plants for DNA sequencing and for full length RNA transcripts. Progress under Objective 3 was made to develop alfalfa plants to hyperaccumulate phosphate (P) through mutation of PHO2 involved in P signaling and P homeostasis in alfalfa. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations were shown to be inherited when plants were self-pollinated and outcrossed. This is the first demonstration of inheritance of mutations from CRISPR gene editing in an autotetraploid species. A manuscript reporting these results is in preparation. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components, which yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Analysis of inheritance, gene expression, and P uptake is ongoing.