Performing Department
(N/A)
Non Technical Summary
Expansion of soybean cultivation to the northern regions of United States and attempt to plant soybean earlier to avoid drought and heat stresses at the flowering stage require research effort to address the adverse effects of weather conditions (wet/waterlogging and cold) in late spring-early summer. Climate change has caused more temperature fluctuations in the soybean planting season, accompanied by the increasing frequency of heavy precipitations in late spring-early summer. An unexpected heavy cold rain after soybean planting can lead to severe problems in seedling establishment posing significant economic loss to farmers. Thus, it is imperative to develop waterlogging-tolerant and/or cold-tolerant soybean varieties through soybean breeding and genetic engineering strategies. Our groups have developed soybean pan-genome genomic resource for gene discovery and discovered the genetic diversity of soybean germplasm in terms of their responses to waterlogging and/or cold stresses. In this proposal, we aim to characterize genetic diversity in soybean germplasm in responses to waterlogging, cold and their combination, identify stress tolerance-related genes, reveal molecular mechanisms underlying the observed stress tolerance-associated traits through transcriptomics and metabolomics analyses, and develop strategies to develop new soybean varieties with improved tolerance to waterlogging and/or cold stresses at early growth stage. Outcomes will include a comprehensive understanding of genetic diversity and architecture of soybean tolerance to the early season waterlogging and/or cold stresses and knowledge on the physiological and molecular regulation of the tolerance-related traits to design strategies for developing future climate-smart soybeans. New stress-resilient soybean lines are expected by completing this project.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Expansion of soybean cultivation to the northern regions of United States and attempt to plant soybean earlier to avoid drought and heat stresses at the flowering stage require research effort to address the adverse effects of weather conditions (wet/waterlogging and cold) in late spring-early summer. Climate change has caused more temperature fluctuations in the soybean planting season, accompanied by the increasing frequency of heavy precipitations in late spring-early summer. An unexpected heavy cold rain after soybean planting can lead to severe problems in seedling establishment posing significant economic loss to farmers. Thus, it is imperative to develop waterlogging-tolerant and/or cold-tolerant soybean varieties through soybean breeding and genetic engineering strategies. Our groups have developed soybean pan-genome genomic resource for gene discovery and discovered the genetic diversity of soybean germplasm in terms of their responses to waterlogging and/or cold stresses. In this proposal, we aim to characterize genetic diversity in soybean germplasm in responses to waterlogging, cold and their combination, identify stress tolerance-related genes, reveal molecular mechanisms underlying the observed stress tolerance-associated traits through transcriptomics and metabolomics analyses, and develop strategies to develop new soybean varieties with improved tolerance to waterlogging and/or cold stresses at early growth stage. Outcomes will include a comprehensive understanding of genetic diversity and architecture of soybean tolerance to the early season waterlogging and/or cold stresses and knowledge on the physiological and molecular regulation of the tolerance-related traits to design strategies for developing future climate-smart soybeans. New stress-resilient soybean lines are expected by completing this project.
Project Methods
Aim 1: Characterize the genetic diversity and architecture of soybean germplasm lines in responses to early season waterlogging, cold and their combinationAim 1.1.Confirm waterlogging tolerance in a diverse set of soybean lines in the greenhouseWe will confirm the tolerance performance of these 30 lines (15 most tolerantvs15 most sensitive lines) in the flooded soil bed built in the greenhouse facility at the University of Missouri. The same experiment will be conducted without waterlogging treatment again to estimate the final seedling establishment rates under control conditions. Aim 1.2. Characterize a set of diverse soybean lines for cold tolerance during seedling establishment stageWe will confirm the cold tolerance of 309 soybean lines to identify the top 15 tolerant and top 15 sensitive genotypes using the growth chamber (Conviron CMP6060, Winnipeg, Canada) facility at Texas Tech University. Aim 1.3. Evaluate the selected set of soybean lines for their tolerance performance under the combined early season waterlogging and cold stress?The same set of 309 diverse soybean lines will be used to test their performance under the combined early season waterlogging and cold stress in the control environment using walking chamber at Texas Tech University. The lines with contrasting performance (goodvs.poor seedling establishments) under the combined waterlogging and cold stress will be selected for breeding, genetic studies, and omics analysis in the subsequent objectives. Aim 1.4. Identify the genetic architecture and candidate genes associated with tolerance to early season waterlogging and/or cold stresses.GWAS will be conducted with the phenotypic data collected in the greenhouse and growth chamber experiments.Linkage disequilibrium(LD) blocks will be defined using PLINK across the chromosomes to find the SNPs strongly linked to each other by calculating ther2value of each SNP, and such LD blocks will be further used for haplotype analysis for detailed analysis of markers identified from GWAS analysis. Haplotype analysis will be performed by using tools, such as HaplotypeMiner in R, LDBlockShow will be used for the LD block visualization. With the assistance of the whole-genome sequencing data and high-density SNP matrix, we expect to narrow down the QTL confident region at a scale of 20 to 50 Kb regions with a high possibility to pinpoint the candidate genes and identify DNA sequence variations leading to gene functional changes, as shown in our previous study (Marsh et al., 2022). Aim 2: Reveal the molecular mechanisms, regulatory networks underlying their responses to the early season waterlogging, cold and their combination through transcriptomics and metabolomics analyses.Comparative bulk transcriptomics of roots from soybean plants under individual and combined cold andwaterloggingstresses. Comparative bulk transcriptomics of root samples from top 5 tolerant and top 5 sensitive lines will be performed using bulk RNA-seq analysis.Identification of key DEGs from bulk 'tolerantvs. sensitive soybean lines' comparison. We aim to identify DEGs and their expression patterns, which will allow us to identify and compare genes mediating the responses of soybean seedlings to individual and combined cold and waterlogging stresses.GO and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analyses. To obtain an overview of the physiological processes in which the individual and combined cold and waterlogging stress-responsive genes are involved, GO enrichment and KEGG pathway analyses will be performed using 'ShinyGO' (bioinformatics.sdstate.edu/go) and 'clsuterProfiler' package in R (Wu et al., 2021).Co-expression network analysis.To identify gene modules underlying the responses of soybean seedlings to individual and combined cold and waterlogging, gene regulatory networks will be built from identified DEGs using the 'WGCNA' package (Langfelder & Horvath, 2008).qRT-PCR analysis.At least 20 DEGs from 'combined cold and waterlogging-tolerantvs. cold and waterlogging-sensitive' comparison will be selected to validate RNA-seq data by qRT-PCR analysis.Comparative bulk metabolomics of roots from soybean plants under individual and combined cold andwaterloggingstresses. Bulk metabolomics of root samples of top 5 tolerant and top 5 sensitive soybean lines will be conducted using untargeted metabolite profiling. Root samples will be collected from single and combined cold and waterlogging treated and non-treated lines. Differentially produced metabolites (DPMs) will be analyzed to identify the metabolic enrichment pathways.Identification of hormonal changes. Hormonal profiles (top 5 tolerant and top5 sensitive soybean lines,) will be conducted using the root samples collected in parallel as those for transcriptomics (Aim 2.1).KEGG enrichment pathway analysiswill be performed using 'clusterProfiler' package in R .Gene-metabolite correlation analysisof the data obtained from different treatment combinations will be performed.Aim 3: Develop breeding and genetic engineering strategies for improving the early season waterlogging and/or cold tolerance in soybean varieties.Analyze DNA sequences and develop DNA markers for Kompetitive Allele Specific PCR (KASP) assay:Based on the GWAS results, the soybean genome sequence and genetic-physical map resources will be used to identify SNPs linked to the discovered tolerance QTL/genes. The genomic sequences will be aligned between the two parents and the SNPs in the QTL regions and will be used to design KASP assay-based DNA markers.Backcrossing with the aid of Marker-assisted selection (MAS):Backcrossing with MAS will also be used as an alternative breeding strategy to introgress the QTLs/genes into the best yielding lines at the two breeding groups. Three elite lines of MG II and III will be selected to cross with the three most tolerant lines to the combined waterlogging/cold stress.Forward breeding by advancing the F1to F5generation within 2 years:The F1plants derived above will be advanced to F5generation. Populations from F4:5and subsequent generations will be evaluated for stress tolerance as described in Aim 1 and the most tolerant lines will be selected for further field evaluation after completion of the proposed project. These genetic materials will be used for genetic study and development of breeding materials for future research and breeding with the project continuation.Gene function characterization: throughAims 1 and 2, we will identify the top 10 crucial genes associated with soybean tolerance to combined cold and waterlogging. Target genes will be individually cloned and transformed intoA. thaliana[Columbia (Col-0) ecotype] through the floral-dip method. Transgenic lines will be assessed under individual and combined cold and waterlogging stresses as previously described inAim 1. In addition toA. thalianatransgenic system, we will use soybean composite transgenic plant system (with transgenic hairy roots) to characterize genes' function if the candidate genes are involved in root growth and development pathways. Transgenic constructs with overexpressing, RNAi silencing, and gene-editing for the selected candidate genes will be constructed accordingly. Transgenic roots will be retrieved from soil pots and phentoyped by our lab protocols for total root length, root tip numbers, root surface area and volume, root diameter and thickness, and root anatomy (Prince et al., 2017; Ye et al., 2018). In year 3 of the proposed project, the genes with identified functions in stress tolerance and root traits will be selected to be further validated in the stable soybean transgenic lines.?