Progress report for GS21-241
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 will maximize the full combining potential of wild genetic sources to create novel adaptive traits for sustainable production of rice under salinity-affected environments. We will utilize Chromosome Segment Substitution Lines (CSSL) of Oryza sativa (cv. Curinga) harboring introgression for almost the entire complement of genomic segments from Oryza rufipogon and Oryza meridionalis in the context of their effects in the creation of non-parental physiological attributes for salinity tolerance. The direct outcome of this research will be the identification of novel salinity tolerant introgression lines that can be used for cultivation in the Southern USA and will also serve as donors for subsequent breeding of salinity tolerance in elite cultivars for sustainable production under marginal environments. Using genomic technologies, we seek to reveal the mechanism that underpins tolerance in CSSLs, identify genetic variations that may serve as markers for salinity tolerance, and identify critical gene pathways connected to salinity tolerance. We are moving to develop a cost-effective PCR (polymerase chain reaction) toolkit useful for marker-assisted breeding of salinity tolerance.
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.
Introgression line (IL) libraries harboring 97.6 % of the O. rufipogon (RUF) and 76.73 % of the O. meridionalis (MER) genome in the background of a tropical japonica cultivar, Curinga (CUR) developed by Prof. Susan McCouch (Department of Plant Breeding and Genetics, Cornell University) have been screened for salinity tolerance by our laboratory. The chromosome segment substitution lines (CSSL) were developed by three rounds of marker-assisted backcrossing and selected lines were made homozygous by doubled haploidization by anther culture (Arbelaez et al., 2015).
Experimental setup and growing conditions
Our strategy for salinity stress experiments is based on the hydroponic screening pipeline established at IRRI (International Rice Research Institute). Seedlings are pre-grown in a greenhouse in standard Yoshida hydroponic medium. Salt stress is administered on 60-day old plants with added sodium chloride (NaCl) at EC12 dSm-1.
We have initially screened the set of two introgression line libraries under salinity stress. Based on our screening, we have identified two introgression lines performing as highly tolerant under salinity stress. These lines were superior to both parents in response to salinity stress thereby making them products of transgressive segregation. Thus, these two CSSLs represent unique genetic combinations of positive and negative traits from both parents. Genomic dissection of these lines will help unravel the basis for the transgressive salinity tolerance and lead to the mapping of the critical genomic regions that are important for the expression of non-parental salinity tolerance attributes.
Global transcriptome analysis and identification of SNP:
Transcriptome analysis will help reveal differentially expressed genes across the introgressed regions and how these genes interact with others in the genetic background to drive the novel response to salinity. Elucidation of a genetic mechanism responsible for salinity tolerance as well as identification of sequence variation associated with salinity tolerance will be made possible by this type of analysis. These variations will then be used to develop genetic markers useful in marker-assisted selection for salinity tolerance. RNA sequencing will be performed from frozen plant tissue samples collected at different time points after stress induction. Total RNA will be isolated from frozen plant tissues of control and salt-stressed plants using the miRVana (Ambion, Thermo Fisher Scientific, USA) extraction kit. RNA extracted from each sample will be used in two replicates to construct RNA-Seq libraries using the Illumina TruSeq RNA Library Prep kit V2 according to manufacturer's protocols (Illumina Inc., USA). Sequencing will be performed at 150-bp paired-end reads using Illumina HiSeq3000. Based on our experience, ~ 100 million paired-end reads at 150 bp provide sufficient coverage for a comprehensive picture of rice transcriptome by both reference-guided and de novo assembly methods.
Output of the RNA-Seq will be processed by using Cutadapt (Martin 2011). The assembly of the RNA-Seq datasets will be performed using a proven transcriptome analysis pipeline established in the De los Reyes laboratory at Texas Tech University. According to the IRGSP-1.0 reference genome annotation, unique locus or transcript identifiers (Loc ID) will be assigned to assembled transcript contigs. SNP detection and visualization will be performed with Samtools, GATK, and IGV tools.
Development of KASP Assay Markers:
The selected SNP markers will be designed following the KASP-by-Design assay mix (LGC Genomics, Beverly, MA), and the common reverse primer designed using the Primer 3 software (Untergasser, 2012).
Transgenerational validation of the salt tolerance using KASP SNP markers:
To validate that the genetic markers are associated with salinity tolerance and, thus, useful for marker-assisted breeding, the two novel salt-tolerant CSSLs harboring introgression from wild rice will be crossed with the recurrent parent (Oryza sativa cv. Curinga). F1 plants will be grown to maturity to obtain F2 seeds. F2 plants will be subjected to salinity stress screening (as described earlier) to identify tolerant lines. These lines will then be genotyped (fingerprinted) using the developed KASP SNP markers. The genotyping protocol involves isolating genomic DNA from the selected salinity tolerant F2 plants using CTAB (cetyltrimethylammonium bromide) DNA extraction protocol. A BioRad CFX-96 RT-PCR thermal cycler will be used to conduct the PCR amplification for the SNP genotyping.
Educational & Outreach Activities
Poster presentation: 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.
Oral presentation: 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.
Manuscript: Mandal, S. N., Sanchez, J., Bhowmick R., Bello, O. C. M., Van-Beek, C. R., and de los Reyes, B. G. (2022) The BTB/POZ proteins and their gene family in Oryza rufipogon: Implications to allele mining from the progenitor of the domesticated japonica rice (O. sativa L.). (Submitted for review)
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. The graduate student supported by this grant also participated in teaching of an on-line course in genetics (PSS 3421 – Fundamental Principles of Genetics). Students this class are in the undergraduate programs in Crop Science with concentration in Genetics and Breeding.
The southern region of the United States is the major producer of rice. Of the top six rice-producing states in the USA, at least four (Arkansas, Louisiana, Mississippi, Texas) are facing the impacts of agricultural marginalization due to poor irrigation water quality or direct soil salinity. Thus, the direct impacts of salinity stress are to rice farms in the irrigated and coastal areas of the southern U.S. Irrigation with brackish water, harsh climatic conditions, and sporadic rainfall are major contributors to the encroachment of salinity into affected areas. Additionally, after hurricanes Katrina, Rita, Gustav, and Ike, soil salinity increased in the rice-producing areas of the Gulf region because of saltwater intrusion(Saichuk and Gauthier, 2011).
The development of salinity tolerant rice cultivars will aid in maintaining the economic viability of farms across salinity-affected areas, thus impacting the economic sustainability and quality of life of local rice-producing communities. Continued rice production in salinity-prone systems will prevent the abandonment of such systems which would inevitably impact economic and quality of life, but also have negative ramifications for environmental sustainability and biodiversity. Rice is a small grain crop with vigorous root growth and high production of biomass that positively affects soil carbon sequestration through increased organic matter. Rice cultivation has been shown to increase soil organic matter accumulation which helps in improving soil properties such as soil aggregation and soil microbial activity and diversity (Wang et al., 2015). Flooded rice fields, which mostly have a light layer of water above ground, also help in preventing soil erosion and provide natural suppression of weeds. Rice fields also serve as a home for wildlife providing natural habitats and food sources for migratory and local waterfowl. In the United States, the biodiversity created by overwintering flooding rice fields is worth $3.4 billion (Anonymous, 2018).
To improve and sustain rice production in salinity-affected agroecosystems, modern plant breeding will need to create germplasm capable of limiting yield and growth reductions associated with salinity stress. Currently, breeding is plagued by the reductionist paradigm of single gene manipulation for large genetic effects on complex traits. Because growth and yield are complex traits that cannot be explained by single-gene changes, modern breeding must employ the paradigm of transgressive segregation where novel genetic combinations are formed across the entire genome of germlines from bi-parental crosses. Further, gene pools from wild rice are excellent sources for improving tolerance to salinity stress in modern, elite rice cultivars.
Our proposed research will help increase the adaptive capacity of rice through the creation of improved salinity tolerant phenotypes required to address salinity stress tolerance in the southern USA. Our primary output from this project would be introgression lines harboring salinity tolerant attributes that have not previously been utilized in rice breeding. The rice stocks to be characterized will be made available to breeders for use as genetic donors for creating locally-adapted rice cultivars. Further impacts of the research include generating scientific knowledge and information that are beneficial and transformational to the scientific and farming communities. The genomics datasets generated will be made available to the broader scientific community through public data repositories. The novel SNP markers developed in this study will be useful for marker-assisted selection of salinity tolerance. Capitalizing on the transformative knowledge resulting from the investigation, similar research will be possible in other cereal crops of economic importance due to the orthology (i.e. similarities) of genes in cereals.