Harnessing the Wild Relatives of Rice for Novel Adaptive Phenotypes: Genetics and breeding for agricultural sustainability beyond the Green Revolution

Final report for GS21-241

Project Type: Graduate Student
Funds awarded in 2021: $16,500.00
Projected End Date: 08/31/2023
Grant Recipient: Texas Tech University
Region: Southern
State: Texas
Graduate Student:
Major Professor:
Dr. Benildo Reyes
Texas Tech University
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Project Information


Salinity is a major yield-limiting factor in rice production across the southern USA, especially in the Gulf Coast region, due to poor-quality irrigation water and intrusion of seawater. This study supports a breeding paradigm based on a holistic view of genomic interaction that explores the cryptic genetic contributions hidden in the wild progenitors of cultivated rice. The project maximized the full combining potential of wild genetic sources to create novel adaptive traits for sustainable production of rice under salinity-affected environments. We utilized the Chromosome Segment Substitution Lines (CSSL) of Oryza sativa (cv. Curinga) harboring introgression for almost the entire complement of genomic segments from Oryza rufipogon in the context of their effects in the creation of non-parental physiological attributes for salinity tolerance. This study led to the identification of novel salinity-tolerant inter-species introgression lines that can be utilized as donors for subsequent breeding of cultivars suitable to the limiting agroecology of the Southern USA’s rice industry. By exploring the cryptic genetic variation in O. rufipogon using a library of CSSLs, we identified novel salinity-tolerant phenotypes and their underlying genetic mechanisms by integrating genomics and transcriptomics datasets. Additionally, we mined the O. rufipogon genome for novel genes and alleles (nine novel genes and alleles for the BTB/POZ protein family) with potential application in wide introgression breeding. In conclusion, this study revealed the untapped cryptic genetic functions and novelties from the wild relatives of cultivated rice. The outcome of this project establishes the groundwork for widening the genetic base of the pre-breeding germplasm of rice that can be effectively used to supplement the inherent capacity of current cultivars for traits related to climate resilience and adaptation to marginal environments.


Project Objectives:

1) To perform a comprehensive physio-morphometric evaluation of two populations of chromosome segment substitution lines (CSSL) of Oryza sativa cv. Curinga harboring introgression for the almost entire complement of genomic segments from Oryza rufipogon and Oryza meridionalis in the context of tolerance to salinity. This study is designed to uncover novel stress tolerance attributes configured by wild chromosome segment introgression in certain CSSLs.

2) To perform comparative transcriptomics to shed light on the mechanism responsible for the novel salt stress tolerance from wild chromosome segment introgression in certain CSSLs.  This study will uncover global patterns of genetic reconfiguration, physiological, and/or biochemical enriched pathways in superior transgressive individuals. Outcomes are expected to facilitate the understanding of the gain or loss of stress tolerance attributes at the whole-plant level. This analysis will help identify genetic markers associated with the enriched pathways contributing to the tolerant phenotype. The identified makers will be used to develop a cost-effective PCR-based genotyping platform employing KASPTM (Kompetitive Allele-Specific PCR) technology.

3) To study the inheritance pattern of the novel genomic attributes and validation of KASPTM markers in recurrent crossed generations. An F2 mapping population, derived from the cross between specific CSSL(s) and recurrent parent (Oryza sativa cv. Curinga), will be used to validate the KASPTM markers. We will also perform qPCR (quantitative real-time PCR) analysis of the specific F2 lines to check the differential gene expression of specific genes.


Materials and methods:

Plant material:

Introgression line (IL) libraries harboring 97.6 % of the O. rufipogon (RUF) genome in the background of a tropical japonica cultivar, Curinga (CUR) (CSSLs) developed by Prof. Susan McCouch (Department of Plant Breeding and Genetics, Cornell University) have been screened for salinity tolerance in Lubbock, Texas. The chromosome segment substitution lines (CSSL) were developed by three rounds of marker-assisted backcrossing, and selected lines carrying specific introgression segments were made homozygous by doubled haploidization by anther culture (Arbelaez et al., 2015).

 Experimental setup and growing conditions

Our strategy for salinity stress experiments was based on the hydroponic screening pipeline established at IRRI (International Rice Research Institute). Seedlings were pre-grown in a greenhouse in standard Yoshida hydroponic medium. Salt stress was administered on 60-day-old plants with added sodium chloride (NaCl) at EC12 dSm-1.

 Morphophysiological parameters:

The entire CSSL population was examined for visual injury under salinity stress, electrolyte leakage index, chlorophyll content, total antioxidant capacity, lipid peroxidase activity, Na+ and K+ content in plant tissue, shoot and root length and biomass weight and gas exchange measurements.

 Analysis of genomic introgression and QTL mapping:

Genomic introgression of O. rufipogon into a select subset of CSSLs was mapped using SNP markers from the 6K Infinium and GBS platforms. The EnsemblPlants biomart tool was utilized to identify gene content within the introgressed segments. QTLs were identified through the QTL IciMapping v. 4.1, as described by Meng et al., 2015 and Arbelaez et al., 2015. Mapping  was conducted on the CSSL population to delineate the critical genomic segments with positive contributions to salinity tolerance traits using stepwise regression and likelihood ratio tests, with significance determined through a permutation test.

Global transcriptome analysis:

RNA was extracted from bulked shoot samples of various genotypes using the Spectrum Plant Total RNA Kit and assessed for purity before constructing RNA-Seq libraries. These libraries were sequenced using the NovaSeq 6000 System to generate paired-end sequences. Subsequently, the RNA-Seq data was processed and aligned to the reference genomes of Nipponbare and O. rufipogon using HISAT2. EdgeR was then used to perform differential expression analyses, and ShinyGO was utilized to analyze Gene Ontology (GO) terms for the genes that were found to be differentially expressed.

Transgenerational validation of the salt tolerance phenotypes :

To validate the novel phenotypes conferred by O. rufipogon genomic introgression, two novel salt-tolerant CSSLs were crossed with the recurrent parent (Oryza sativa cv. Curinga). F1 plants were grown to maturity to obtain F2 seeds. Subsequently, F2 plants were subjected to salinity stress screening (as described earlier) to identify tolerant lines.

Research results and discussion:

The CSSL library exhibited extensive phenotypic variation across the population for all examined traits, specifically SES, ELI, Na+ content, K+ content, the Na+/K+ ratio, chlorophyll content, as well as reductions in root and shoot biomass and stomatal conductance. Two transgressive CSSLs that are exceptionally tolerant, CR1 and CR17, were identified for in-depth characterization because they show less growth penalty under salinity stress when compared to both the more superior and inferior parents, and also relative to known salinity tolerance standards (check). Results of the analysis of gas exchange parameters implied that the parent Oryza rufipogon and its tolerant derivatives (CR1 and CR17) had better photosynthetic capacity under extreme salinity stress.

Using single nucleotide polymorphism genotypic data obtained through the 6K Infinium genotyping platform, we conducted a stepwise regression analysis and found five genomic regions across chromosomes 1, 2, 4, and 11 (LOD > 2.75), which with statistically significant association with four introgressed traits: stomatal conductance, shoot weight, K+ ion concentration, and Na/K ratio. Additional investigation utilizing global transcriptome signatures revealed the expression quantitative trait loci (eQTLs) associated with the transgressive salinity stress response of the critical CSSLs. Noteworthy, the eQTLs included genes encoding cytokinin dehydrogenases and MYB transcription factors that have been previously implicated with shoot weight and functions of transmembrane proteins for stomatal conductance.

Furthermore,  we crossed the transgressive CR1 and CR17 with their recurrent parent O. sativa ssp. Japonica cv. Curginga to investigate the inheritance of the novel salinity tolerance phenotypes. The hybridity of the F1 plants was confirmed using SSR markers. The true F1 plants were selfed, and the resulting F2 populations were screened for salinity response using the same method we screened our CSSL library. Both populations showed segregation for SES score under salinity stress, resulting in a normal distribution, indicating that the novel (transgressive) salinity tolerance phenotype conferred by alien introgression from O. rufipogon into O. sativa ssp. Japonica cv. Curinga was due to the coupling and uncoupling of many core and peripheral genes.

Overall, the results of this study revealed the evidence that cryptic gene functions reside in the genome of O. rufipogon and these cryptic gene functions could be expressed in a recombinant genetic background with O. sativa, indicating a positive complementation between the two distant genomes. The outcome of this complementation is novel adaptive phenotypes that transgress the parental phenotypic boundaries. Such novel adaptive traits exhibited by the transgressive progenies of O. rufipogon and O. sativa (CR1, CR17) will serve as donor germplasm that can be to more deeply examine the combining abilities of the critical loci in other genetic backgrounds that are commonly used in rice breeding programs.


Participation Summary

Educational & Outreach Activities

2 Webinars / talks / presentations

Participation Summary:

Education/outreach description:

Published Abstracts: 

Mandal, S. N., Kitazumi, A., Van-Beek, C. R., Bello, O. C. M., Pabuayon, I. C. M., & de los Reyes, B. G. (2023) Unlocking of Cryptic Gene Functions in Wild Species O. rufipogon By Alien Introgression into the Genome of Domesticated O. sativa Ssp. japonica to Develop Novel Salinity-Tolerant Phenotypes. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO. https://scisoc.confex.com/scisoc/2023am/meetingapp.cgi/Paper/154259

Mandal, S. N., Sanchez, J., Bhowmick, R., Bello, O. R., Van-Beek, C. R., & de los Reyes, B. G. (2023) BTB/POZ Proteins and Their Gene Family in Oryza rufipogon: Identification of Novel Genes and Alleles from the Progenitor of Domesticated Japonica Rice (O. sativa L.). Plant and Animal Genome XXX Conference, San Diego, CA. Poster P49839.

Mandal, S. N., Kitazumi, A., Pabuayon, I. C. M., & de los Reyes, B. G. (2023) Investigating Variation in Transcriptomic Fluxes Across Rice Genotypes Representing a Spectrum for Salinity Tolerance and Susceptibility. Plant and Animal Genome XXX Conference, San Diego, CA. Poster P49833.

Mandal, S. N., Bello, O. C. M., Sanchez, J., Kitazumi Ai., Van-Beek, C. R., Pabuayon, I. C. M., & de los Reyes, B. G. (2022)Genomic interactions governing novel salinity tolerance mechanisms in chromosome segment substitution lines of Oryza sativa x Oryza rufipogon. Plant and Soil Science Student Research Symposium, Texas Tech University,  April 18, 2022, Lubbock, TX.

Mandal, S. N., Bello, O. C. M., Sanchez, J., Cushman, K., Van-Beek, C. R., Pabuayon, I. C. M., & de los Reyes, B. G. (2021) Transgressive Salinity Tolerance Phenotypes Configured by Chromosome Segment Substitution of Oryza rufipogon in Oryza sativa ssp. Japonica. ASA, CSSA, SSSA International Annual Meeting, Salt Lake City, UT, Nov 7-10, 2021. https://scisoc.confex.com/scisoc/2021am/meetingapp.cgi/Paper/138614.


Mandal, S. N., Sanchez, J., Bhowmick, R., Bello, O. R., Van-Beek, C. R., & de Los Reyes, B. G. (2023). Novel genes and alleles of the BTB/POZ protein family in Oryza rufipogon. Scientific Reports, 13(1), 15466.

Mentoring of undergraduate students: This project provides training support on research plan development, literature survey, experimental setup, analytical techniques, data analysis, and research presentation to undergraduate students at Reyes Genetics Lab, Texas Tech University.

Project Outcomes

1 Grant received that built upon this project
Project outcomes:

The project made notable progress in the development of inter-species genetic donor germplasm for tolerance to high saline environments. We investigated the possibility of introducing genes from the wild species Oryza rufipogon into the cultivated O. sativa ssp. Japonica cv. Curinga to enhance salinity tolerance in order to address the challenge of developing climate-resilient and marginal environment-adapted modern cultivars of rice.  We evaluated a population of 48 CSSLs (containing 96.6% of the donor genome) and compared their responses to salinity stress (EC =12) at the mid-vegetative growth stage relative to the donor and recipient parents. We revealed that the introgression of foreign genomic segments from O. rufipogon confers novel adaptive traits under extreme salinity stress as manifested by reconfigured physiological properties of the critical introgression lines. Two critical introgression lines (CR1, CR17) demonstrated transgressive traits by virtue of their unique abilities to withstand extreme salinity far better than the donor parent O. rufipogon and recipient parent O. sativa ssp. Japonica cv. Curinga. These transgressive CSSLs exhibited higher photosynthetic capacities and lower growth penalty compared to the other known tolerant derivatives of a well-known donor of salt tolerance mechanisms, O. sativa ssp. Aus cv. Pokkali (i.e., elite introgression lines FL478 and FL510). Additionally, the putative expression quantitative trait loci (eQTLs) linked to the novel phenotypes of the critical CSSLs were also discovered. These eQTLs encoded  a MYB transcription factor, transmembrane transporters, and proteins involved in the degradation of cytokinin. We hypothesized that these genes are involved in mechanisms that preserve shoot biomass, through the regulation of stomatal conductance. Furthermore, we revealed that the transgressive CSSLs exhibited altered expression of several cryptic genes when compared to either the donor or the recurrent parents. Novel expression patterns appeared to be the consequence of modified regulation brought about by the blending of O. rufipogon genomic segments with the genomic background of the recipient O. sativa cv. Curinga.

To determine the inheritance of the transgressive salinity tolerance phenotypes, CR1 and CR17 were crossed with the recurrent parent Curinga. Using SSR markers, F1 plants were confirmed to be true hybrids. Selfing of the true F1 plants produced F2 populations that were salinity-screened using the same method used for screening the CSSL library. Both populations showed segregation for SES score under salinity stress, resulting in a normal distribution for polygenic traits indicating that alien introgression from O. rufipogon into O. sativa ssp. japonica caused the coupling and uncoupling effects of numerous core and peripheral genes and alleles from the donor and recurrent genomes, resulting in transgressive salinity tolerance phenotypes. It appears that the impact of O. rufipogon genome is not a mere addition of new functions but rather more on the global scale through the reconfiguration of global gene regulation either genetically or epigenetically.

Furthermore, our research offered a thorough examination of the BTB/POZ protein family in O. rufipogon, identifying 110 encoding gene loci and nine new genes that are absent from its domesticated counterpart, O. sativa ssp. japonica. When introduced into O. sativa, these novel genes may serve as potential drivers of novel functions in the genomic backgrounds of cultivars. In addition, these results provide insight into the evolution and functional divergence of important regulatory genes, establishing a baseline for functional genomics research and allele mining aimed at broadening the genetic base of rice cultivars.

Knowledge Gained:

Our study highlights the substantial contribution of wild relatives in enriching genetic diversity and the potential of incorporating alien/wild genetic segments to generate novel phenotypes. We have identified putative candidate expression quantitative trait loci (eQTLs) that regulate the novel salinity-tolerant phenotypes by integrating comprehensive phenotyping, genomics, and transcriptomics. These salinity-tolerant CSSLs (CR1, CR 17) are valuable for enhancing rice's adaptive capacity and sustaining rice production in salinity-affected agroecosystems. Based on the outcomes of this study, it is possible to create novel adaptive traits through transgressive segregation, facilitated by the positive genomic complementation of distant species of rice such as O. rufipogon and O. sativa ssp. Japonica.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.