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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Hard Winter Wheat Genetics Research » Research » Research Project #431801

Research Project: Wheat Root Transcriptomics to Understand Nitrogen and Cadmium Response

Location: Hard Winter Wheat Genetics Research

Project Number: 3020-21000-010-35-I
Project Type: Interagency Reimbursable Agreement

Start Date: Jan 15, 2016
End Date: Jan 14, 2018

Objective:
Cereal crops with improved nitrogen use efficiency (NUE) are urgently needed for sustainable grain production; and pollution-safe cereal crops that can be grown in soils with high bioavailability of toxic minerals are needed to maintain agricultural production while safeguarding human health. Plant roots are the gateway through which nitrogen and minerals, both beneficial and toxic, are acquired and assimilated. The proposed research integrates molecular, biochemical, and whole-plant approaches to characterize the genetic networks regulating wheat root response to N and Cd. This research uses high and low NUE and high and low grain Cd winter wheat genotypes to develop the basic knowledge needed to breed wheat cultivars that better assimilate nitrogen and simultaneously exclude toxic levels of cadmium from grain. The supporting objectives are: 1) to characterize the winter wheat root’s transcriptome response to N limitation; 2) to identify root genes involved in differential accumulation of cadmium in winter wheat grain. The core tool is RNA sequencing, applied specifically to winter wheat roots, following N and Cd stress. The project is a conversion of a funded postdoctoral fellowship for research to address Foundational Program Area Priority Codes A1151 (Plant Photosynthetic Efficiency and Nutrient Utilization) and A1101 (Plant Growth and Development, Composition, and Stress Tolerance). The goal of the AFRI NIFA Fellowships Grant Program is addressed through: 1) developing the fellow’s expertise in plant physiology and bioinformatics; 2) developing grant writing skills; 3) developing teaching competencies through classroom instruction, workshops, and experiences teaching combined on-site/distance classes and mentoring undergraduate and graduate students. The grant is a transfer of funding of a previous AFRI-NIFA Postdoctoral Fellowship. The research objectives related to N deficiency are the primary focus of the funded period of this project.The teaching objectives were previously completed at the University of Nebraska. The research objectives related to Cadmium were largely completed at the University of Nebraska and will be prepared for publication under this project.

Approach:
The proposed research integrates molecular, biochemical, and whole-plant approaches to better characterize the network of wheat genes actively involved in wheat root response to N and Cd. Based on ongoing field studies, two pairs of winter wheat genotypes will be selected: 1) one pair that differs for NUE; 2) one pair that differs for grain Cd accumulation. The differential gene expression in roots of these pairs of genotypes in response to N limitation or Cd supplementation, respectively, in hydroponic conditions will be measured. IV.c.1. Project details/sequence Phase I: Determine appropriate level of N limitation and Cd supplementation in hydroponically grown plants. A range of N concentrations will be applied to the NUE+ and NUE- genotypes with the objective of identifying a minimum stress condition. Similarly, to determine a sub-toxic level of Cd, a range of Cd concentrations will be applied to the Cd+ and Cd- genotypes. A follow-up experiment will integrate the four winter wheat genotypes and the two pairs of stress treatments in a factorial treatment design to evaluate the effect of plant N status on Cd uptake across genotypes that vary for NUE and Cd accumulation in grain. Phase II: RNA-seq. RNA isolated from roots of the pairs of genotypes after N treatments (control, limitation) or Cd treatment (control, supplementation) will be sequenced using NGS technology. RNA sequencing will be conducted by the DNA Microarray and Sequencing Core Facility at the University of Nebraska Medical Center. Phase III: Data analysis and validation. A reference root transcriptome will be assembled using all sequence data from all four wheat genotypes and publicly available transcript sequences. Reads from each genotype x treatment combination will be mapped to the reference transcriptome and counted to determine differential expression. Differentially expressed reads will be annotated to Gene Ontology (GO) biological process, molecular function, and cellular component categories. Selected transcripts will be evaluated using RT-qPCR. SNPs among the four sequenced genotypes will be identified as potential targets for genetic markers. Phase IV: Data submission and publication. Sequence data will be submitted to the NCBI SRA. The non-redundant assembly will be made available to appropriate public wheat-related databases (e.g. GrainGenes). Potential SNP tags will be distributed to public-sector researchers to support ongoing marker development efforts for wheat. Results will be submitted for publication to refereed journals and disseminated through presentations at meetings of professional societies.