Recipient Organization
TEXAS TECH UNIVERSITY
(N/A)
LUBBOCK,TX 79409
Performing Department
(N/A)
Non Technical Summary
Ideotype breeding during the Green Revolution of the mid-20th century created the first-generation high-yielding rice cultivars with high productivity underunpolluted environments, with ample supply of cleanwater for irrigation and nutrients from inorganic fertilizers.This approach is based on the paradigm of stackingindividual morphological and physiological traits that act synergistically to create a plant architecture that functions optimally under certain environments for maximal yield.In the 21st century, the challenge to food security is much grander thanduring the Green Revolution as more foods need to be produced under steadilymarginalizing environments and changing climate. New realities surrounding the changing global ecological dynamics and limiting resourcespainta new picture of the much grander challenges ahead thatrequire bolder yet pragmatic new approaches for crop breeding. New technologies are needed to re-optimize the growth, morphology, physiology and reproduction of crop plants to maintain yield underless ideal environments and limited natural resources.Using a modern andintegrarive approachto the dissection of the genetic and physiological bases of traits, this project takes a much deeper look on the classic genetic phenomenon of transgressive segregation in plants. This phenomenon will be usedas the core component of a holistic strategy to createnovel ideotypesfor the next-generation of rice cultivars with robustyield under environments affected by salinity and marginalized water resources. Transgressive segregation occurs when a small minority of progenies derived from the mating of two genetically widely contrastingparents exhibit traits that are either superior to the better of the two parents or inferior to the worse of the two parents.This project will elucidate the genetic and physiological underpinnings behind the non-parental super-tolerance (positive transgressive) or super-sensitivity (negative transgressive) to salinity across the advanced generation progenies ofIR29 (indica) x Pokkali (aus) rice, beyond the effects of the known major defense mechanism that wasdeployed into cultivars bybreeding during the last four decades. This project seeks to reveal the genetic and physiological causes of the adaptive novelties of transgressive segregants under salinity stress, which could create ideotypes that are far better than the superior parent (net genetic gain) or far worse than the inferior parent (net genetic penalty). The specific objectives of the proposed project will be accomplished by a comprehensiveand integrative approach to geneticsthat includes digital analysis of plant growth, development, and morphology, profiling of the changes in gene expression, and analysis of gene function bytransgenics and gene editing.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
Guided by the Omnigenic Theory, the overarching goal of this project is to explore a classic phenomenon in plant breeding, i.e.,transgressive segregation, as a non-reductionist approach for stacking optimal physiological properties that synergize towards novel mechanisms of salinity tolerance, beyond the full potentials of individual parents. We aim to integrate a classical concept in plant breeding with modern OMICS-guided biology towards a new paradigm for examining the physiological basis of integrated growth, developmental, and defense processes that define the mechanisms by which plants adapt to stress environments affecting agriculture today and in the future. With rice under salinity stress as experimental system, this project aims to build the foundation for a holistic approach for creating the new generation of climate- and marginal environment-resilient ideotypes, with broad applications to other crops of importance to the U.S. Translation of this foundational project to plant breeding is expected to complement the single-gene approaches of functional genomics to define the profiles of the 21st century crop ideotypesand their growth, developmental, and defense components. The specific objectives are as follows:1) Elucidate the growth processes contributing to the superiority of a positive transgressive segregant (propelling effects) and inferiority of a negative transgressive segregant (dragging effects) in relation tosalinity tolerance (Growth Sustenance Hypothesis). 2)Elucidate the mechanisms regulating plant morphology and architecture that lead to super-tolerancein positivetransgressive segregant and super-sensitivity in nagative transgressive segregant(Adaptive Plant Architecture Hypothesis).3)Reveal other mechanisms underlying the optimal and sub-optimal defense capacities of positive and negative transgressive segregants, respectively,beyond the effects ofsodiumexclusionmechanism (Defense-Beyond-SalTol Hypothesis).
Project Methods
Phenomics Experiments: With the LemnaTec digital phenotyping platform, we will compare in real-time the growth and plant architectural features across the comparative panel of recombinant inbred lines and parents, from early vegetativeto early reproductivestages of growth, acrosstemporal windows that capture each physiologically distinct stages of development under optimal and salinity stress conditions. Standard procedures for salinity treatment involve a two-step increment over a three-day period to a final EC= 9 dS m-1 (90 mM NaCl), with supplemental Ca2+ at 10:1 ratio with NaCl to minimize salt shock effects and mitigate Na+-induced Ca2+ deficiency. Control and salinity experiments will be performed in parallel with image-capture of five plants per genotype (n=5) on the conveyor belt. We will use separate plants in parallel for ion analysis and measurements of photosynthesis and respiration across five time-points using the LiCOR6800 system. Plants will be imaged at multiple side-views with RGB, fluorescence, and hyperspectral cameras on the Lemnatec 3D Scanalyzer. Image analysis and their associated statistical analyseswill be through a well-established and open-source pipeline PhenoImage developed at UNL. All whole-plant-level phisological studies will be conducted in the same experimental system used for digital phenotyping.Efforts:The phenomics experiments will create high-resolution profiles of plant physiological status (Growth Physiology Atlas) by revealing common and genotype-specific changes as each genotype go through the critical stages of vegetative and reproductive growth under salinity. Results of these experiments will illuminate the physiological significance of novel morpho-anatomical, transcriptomic, and metabolomic signatures of transgressive phenotypes. Results of these studies will be published in refereed journals.Transcriptomics Experiments:Transcriptomes will be examined comprehensively at each physiologically distinct phases of development and will be compared between control and salinity across genotypes using the third and fourth leaves of the primary tiller for early vegetative growth stages andall tillers and flag leaves at mid-vegetative and late vegetative growth stages, respectively. Transcriptome experiments will be performed by RNA-Seq (TruSeq libraries; NovaSeq6000 at 150-bp paired-ends with coverage of 20 to 30 million reads per library), through the standard in-house analytical pipeline in the PI's lab that makes use of a suite of open-source data analytics that include reference-guided and de novo assembly, annotation, normalization, and differential expression that includesCutadapt48, TopHat2, Cufflinks, Cuffdiff, Augustus, Trinity, EdgeR, DESeq, K-means, PCA, MBCluster, KEGG, KappaView) and other R-based custom scripts for network modeling.Efforts:Parallel to the non-destructive digital growth and physiological profiling, we will perform experiments with identical developmental timing and experimental designunder optimal and salinity stress conditions for comprehensive and integrative transcriptome profiling. These experiments will integrate whole-plant and cellular level physiological, biochemical, and transcriptional profiles to reveal both the large-scale and fine-scale differences in the physiology between the transgressive segregants andtheir parents and siblings.Results of these studies will be published in refereed journals.Metabolomics Experiments:Comprehensive metabolite profiling across the comprative panelwill follow the temporal design, sample-pooling, and replication schemes of transcriptomic experiments using the services of OmicsCraft LLC (Washington, DC) through their sample-to-solution packages for LC-MS/MS and GC-MS, metabolite annotation, differential analysis, and integration with other OMICS datasets. OmicsCraft pipeline is facilitated by cloud-based tools (MetaboQuest, MetCraft, SysMet) using statistical, deep-learning, network-based, and fuzzy logic methods. LC-MS/MS will be performed in Vanquish Flex UHPLC System coupled to Q-Exactive MS, operating in positive and negative polarity.GC-MS will be performed with LECO Pegasus HT GC-TOF-MS system coupled to Agilent 7890A GC with ChromaTOF and MetaboliteDetector for data preprocessing. Metabolites will be annotated by spectral matching of MS/MS data against spectral databases such as RefMet, Compound Discoverer, METLIN, and MetaboQuest.Efforts:Parallel to the non-destructive digital growth and physiological profiling (phenomics), and destructive transcriptome profiling, we will perform metabolomics profiling across the comparative panelwith identical developmental timing and experimental designas the transcriptomics experiments. Parallel and integrative analyses of the phenomics, transcriptomics, and metabolomics datasetswill illuminate thewhole-plant and cellular level physiological, biochemical, and transcriptional profiles to reveal both the large-scale and fine-scale differences in the physiology between the transgressive segregants andtheir parents and siblings.Results of these studies will be published in refereed journals.Reverse Genetics Experiments by Transgenics and Gene Editing:We will examine the functional significance of candidate genes selected for their potential involvement in network rewiringto create the morpho-anatomical novelties of transgressive segregants. Two approaches will be used to achieve this goal.First, we will create transgenic overexpression lines (ox-lines; pCambia1300-IRS154-OsMPSandpCambia1300-IRS154-OsHK5) through the constitutive promoter of maize ubiquitin gene (IRS154) in the genetic backgrounds of the vigorous but salt-sensitive parent IR29, and super-sensitive sibling FL499. Standard procedures forAgrobacterium-mediated transformation of immature rice embryos with pCAMBIA1300 vectors that are very well-established in the PI's lab will be followed.Second, we will utilize the established capabilities for CRISPR-Cas gene editing in Co-PI's lab to recreate the novel FL510 allele in the genetic backgrounds of IR29 and FL499 to mimic the effects of the novel superior allele and its network. Promoter edits and ox-lines will be examined for growth, morphological, physiological, and molecular signatures associated with the positive transgressiveallele.Efforts: The impacts of the altered candidate regulatory genesin mimicking the unique phenotypes of the superior transgressive segregant will be examined by virtue of physiological and growth signatures and expression of networks. We expect to identify additional regulatory genes from these studies.Results of these studies will be published in refereed journals.