Comparative quantitative trait loci analysis framework reveals relationships between salt stress responsive phenotypes and pathways.

Soil salinity is a complex abiotic stress that involves several biological pathways. Hence, focusing on a specific or a few salt-tolerant phenotypes is unlikely to provide comprehensive insights into the intricate and interwinding mechanisms that regulate salt responsiveness. In this study, we develop a heuristic framework for systematically integrating and comprehensively evaluating quantitative trait loci (QTL) analyses from multiple stress-related traits obtained by different studies. Making use of a combined set of 46 salinity-related traits from three independent studies that were based on the same chromosome segment substitution line (CSSL) population of rice (Oryza sativa), we demonstrate how our approach can address technical biases and limitations from different QTL studies and calling methods. This allows us to compile a comprehensive list of trait-specific and multi-trait QTLs, as well as salinity-related candidate genes. In doing so, we discover several novel relationships between traits that demonstrate similar trends of phenotype scores across the CSSLs, as well as the similarities between genomic locations that the traits were mapped to. Finally, we experimentally validate our findings by expression analyses and functional validations of several selected candidate genes from multiple pathways in rice and Arabidopsis orthologous genes, including OsKS7 (ENT-KAURENE SYNTHASE 7), OsNUC1 (NUCLEOLIN 1) and OsFRO1 (FERRIC REDUCTASE OXIDASE 1) to name a few. This work not only introduces a novel approach for conducting comparative analyses of multiple QTLs, but also provides a list of candidate genes and testable hypotheses for salinity-related mechanisms across several biological pathways.

Functional Bph14 from Rathu Heenati promotes resistance to BPH at the early seedling stage of rice (Oryza sativa L.) as revealed by QTL-seq.

KEY MESSAGE: A QTL associated with BPH resistance at the early seedling stage was identified on chromosome 3. Functional Bph14 in Rathu Heenati was associated with BPH resistance at the early seedling stage. Brown planthopper (BPH; Nilaparvata lugens Stål) is considered the most important rice pest in many Asian countries. Several BPH resistance genes have previously been identified. However, there are few reports of genes specific for BPH resistance at the early seedling stage, a crucial stage for direct-seeding cultivation. In this study, we performed a QTL-seq analysis using two bulks (20 F2 lines in each bulk) of the F2 population (n = 300) derived from a cross of Rathu Heenati (RH) × HCS-1 to identify QTL/genes associated with BPH resistance at the early seedling stage. An important QTL was identified on chromosome 3 and Bph14 was identified as a potential candidate gene based on the differences in gene expression and sequence variation when compared with the two parents. All plants in the resistant bulks possessed the functional Bph14 from RH and all plants in the susceptible bulk and HCS-1 contained a large deletion (2703 bp) in Bph14. The functional Bph14 gene of RH appears to be important for BPH resistance at the early seedling stage of rice and could be used in conjunction with other BPH resistance genes in rice breeding programs that confer resistance to BPH at the early and later growth stages.

Comparative proteomic analysis of chromosome segment substitution lines of Thai jasmine rice KDML105 under short-term salinity stress.

MAIN CONCLUSIONS: Heat shock proteins, ROS detoxifying enzymes, and ion homeostasis proteins, together with proteins in carbohydrate metabolism, cell structure, brassinosteroids, and carotenoid biosynthesis pathway were up-regulated in CSSLs under salinity stress. Rice is one of the most consumed staple foods worldwide. Salinity stress is a serious global problem affecting rice productivity. Many attempts have been made to select or produce salinity-tolerant rice varieties. Genetics and biochemical approaches were used to study the salinity-responsive pathway in rice to develop salinity tolerant strains. This study investigated the proteomic profiles of chromosome segment substitution lines (CSSLs) developed from KDML105 (Khao Dawk Mali 105, a Thai jasmine rice cultivar) under salinity stress. The CSSLs showed a clear resistant phenotype in response to 150 mM NaCl treatment compared to the salinity-sensitive line, IR29. Liquid chromatography-tandem mass spectrometry using the Ultimate 3000 Nano/Capillary LC System coupled to a Hybrid Quadrupole Q-Tof Impact II™ equipped with a nano-captive spray ion source was applied for proteomic analysis. Based on our criteria, 178 proteins were identified as differentially expressed proteins under salinity stress. Protein functions in DNA replication and transcription, and stress and defense accounted for the highest proportions in response to salinity stress, followed by protein transport and trafficking, carbohydrate metabolic process, signal transduction, and cell structure. The protein interaction network among the 75 up-regulated proteins showed connections between proteins involved in cell wall synthesis, transcription, translation, and in defense responses.

riceExplorer: Uncovering the Hidden Potential of a National Genomic Resource Against a Global Database.

Agricultural crop breeding programs, particularly at the national level, typically consist of a core panel of elite breeding cultivars alongside a number of local landrace varieties (or other endemic cultivars) that provide additional sources of phenotypic and genomic variation or contribute as experimental materials (e.g., in GWAS studies). Three issues commonly arise. First, focusing primarily on core development accessions may mean that the potential contributions of landraces or other secondary accessions may be overlooked. Second, elite cultivars may accumulate deleterious alleles away from nontarget loci due to the strong effects of artificial selection. Finally, a tendency to focus solely on SNP-based methods may cause incomplete or erroneous identification of functional variants. In practice, integration of local breeding programs with findings from global database projects may be challenging. First, local GWAS experiments may only indicate useful functional variants according to the diversity of the experimental panel, while other potentially useful loci-identifiable at a global level-may remain undiscovered. Second, large-scale experiments such as GWAS may prove prohibitively costly or logistically challenging for some agencies. Here, we present a fully automated bioinformatics pipeline (riceExplorer) that can easily integrate local breeding program sequence data with international database resources, without relying on any phenotypic experimental procedure. It identifies associated functional haplotypes that may prove more robust in determining the genotypic determinants of desirable crop phenotypes. In brief, riceExplorer evaluates a global crop database (IRRI 3000 Rice Genomes) to identify haplotypes that are associated with extreme phenotypic variation at the global level and recorded in the database. It then examines which potentially useful variants are present in the local crop panel, before distinguishing between those that are already incorporated into the elite breeding accessions and those only found among secondary varieties (e.g., landraces). Results highlight the effectiveness of our pipeline, identifying potentially useful functional haplotypes across the genome that are absent from elite cultivars and found among landraces and other secondary varieties in our breeding program. riceExplorer can automatically conduct a full genome analysis and produces annotated graphical output of chromosomal maps, potential global diversity sources, and summary tables.

Photosynthetic Plasticity and Stomata Adjustment in Chromosome Segment Substitution Lines of Rice Cultivar KDML105 under Drought Stress.

The impact of increasing drought periods on crop yields as a result of global climate change is a major concern in modern agriculture. Thus, a greater understanding of crop physiological responses under drought stress can guide breeders to develop new cultivars with enhanced drought tolerance. In this study, selected chromosome segment substitution lines of KDML105 (KDML105-CSSL) were grown in the Plant Phenomics Center of Kasetsart University in Thailand under well-watered and drought-stressed conditions. Physiological traits were measured by observing gas exchange dynamics and using a high-throughput phenotyping platform. Furthermore, because of its impact on plant internal gas and water regulation, stomatal morphological trait variation was recorded. The results show that KDML105-CSS lines exhibited plasticity responses to enhance water-use efficiency which increased by 3.62%. Moreover, photosynthesis, stomatal conductance and transpiration decreased by approximately 40% and plant height was reduced by 17.69%. Stomatal density tended to decrease and was negatively correlated with stomatal size, and stomata on different sides of the leaves responded differently under drought stress. Under drought stress, top-performing KDML105-CSS lines with high net photosynthesis had shorter plant height and improved IWUE, as influenced by an increase in stomatal density on the upper leaf side and a decrease on the lower leaf side.

Combining Genome and Gene Co-expression Network Analyses for the Identification of Genes Potentially Regulating Salt Tolerance in Rice.

Salinity stress tolerance is a complex polygenic trait involving multi-molecular pathways. This study aims to demonstrate an effective transcriptomic approach for identifying genes regulating salt tolerance in rice. The chromosome segment substitution lines (CSSLs) of "Khao Dawk Mali 105 (KDML105)" rice containing various regions of DH212 between markers RM1003 and RM3362 displayed differential salt tolerance at the booting stage. CSSL16 and its nearly isogenic parent, KDML105, were used for transcriptome analysis. Differentially expressed genes in the leaves of seedlings, flag leaves, and second leaves of CSSL16 and KDML105 under normal and salt stress conditions were subjected to analyses based on gene co-expression network (GCN), on two-state co-expression with clustering coefficient (CC), and on weighted gene co-expression network (WGCN). GCN identified 57 genes, while 30 and 59 genes were identified using CC and WGCN, respectively. With the three methods, some of the identified genes overlapped, bringing the maximum number of predicted salt tolerance genes to 92. Among the 92 genes, nine genes, OsNodulin, OsBTBZ1, OsPSB28, OsERD, OsSub34, peroxidase precursor genes, and three expressed protein genes, displayed SNPs between CSSL16 and KDML105. The nine genes were differentially expressed in CSSL16 and KDML105 under normal and salt stress conditions. OsBTBZ1 and OsERD were identified by the three methods. These results suggest that the transcriptomic approach described here effectively identified the genes regulating salt tolerance in rice and support the identification of appropriate QTL for salt tolerance improvement.

Expression level of Na+ homeostasis-related genes and salt-tolerant abilities in backcross introgression lines of rice crop under salt stress at reproductive stage.

Salt stress in the rice field is one of the most common abiotic stresses, reducing crop productivity, especially at reproductive stage, which is very sensitive to salt stress. The aim of this investigation was to study mRNA-related Na+ uptake/translocation and Na+ enrichment in the cellular level, leading to physiological changes, growth characteristics, and yield attributes in FL530 [salt-tolerant genotype; carrying SKC1 (in relation to high-affinity potassium transporters controlling Na+ and K+ translocation) and qSt1b (linking to salt injury score) QTLs] and KDML105 (salt-sensitive cultivar; lacking both QTLs) parental lines and 221-48 (carrying SKC1 and qSt1b QTLs) derived from BILs (backcross introgression lines) at 50% flowering of rice, under 150-mM NaCl until harvesting process. The upregulation of OsHKT1;5 (mediating Na+ exclusion into xylem parenchyma cells) and OsNHX1 (Na+/H+ exchanger to secrete Na+ into vacuole) and downregulation of OsHKT2;1 and OsHKT2;2 (mediating Na+ restriction in the roots, leaf sheath and older leaves) in cvs. FL530 and 221-48 (+ SKC1; + qSt1b) under salt stress were observed. It restricted Na+ level in flag leaf, thereby preventing salt toxicity, as indicated by maintenance of photon yield of PSII (ΦPSII), net photosynthetic rate (Pn), transpiration rate (E) and overall growth performances. In contrast, Na+ enrichment in flag leaf of cv. KDML105 (-SKC1;-qSt1b) caused the reduction in ΦPSII by 30.5% over the control, leading to the reduction in Pn by 62.3%, in seed sterility by 88.2%, and yield loss by 85.1%. Moreover, the negative relationships between Na+ enrichment in flag leaf, physiological changes, and yield traits in rice crop grown under salt stress were demonstrated. Based on this investigation, rice genotype 221-48 was found to possess salt-tolerant traits at reproductive stage and thus could prove to be a potential candidate for future breeding programs.

Comparison between the Transcriptomes of 'KDML105' Rice and a Salt-Tolerant Chromosome Segment Substitution Line.

'KDML105' rice, known as jasmine rice, is grown in northeast Thailand. The soil there has high salinity, which leads to low productivity. Chromosome substitution lines (CSSLs) with the 'KDML105' rice genetic background were evaluated for salt tolerance. CSSL18 showed the highest salt tolerance among the four lines tested. Based on a comparison between the CSSL18 and 'KDML105' transcriptomes, more than 27,000 genes were mapped onto the rice genome. Gene ontology enrichment of the significantly differentially expressed genes (DEGs) revealed that different mechanisms were involved in the salt stress responses between these lines. Biological process and molecular function enrichment analysis of the DEGs from both lines revealed differences in the two-component signal transduction system, involving LOC_Os04g23890, which encodes phototropin 2 (PHOT2), and LOC_Os07g44330, which encodes pyruvate dehydrogenase kinase (PDK), the enzyme that inhibits pyruvate dehydrogenase in respiration. OsPHOT2 expression was maintained in CSSL18 under salt stress, whereas it was significantly decreased in 'KDML105', suggesting OsPHOT2 signaling may be involved in salt tolerance in CSSL18. PDK expression was induced only in 'KDML105'. These results suggested respiration was more inhibited in 'KDML105' than in CSSL18, and this may contribute to the higher salt susceptibility of 'KDML105' rice. Moreover, the DEGs between 'KDML105' and CSSL18 revealed the enrichment in transcription factors and signaling proteins located on salt-tolerant quantitative trait loci (QTLs) on chromosome 1. Two of them, OsIRO2 and OsMSR2, showed the potential to be involved in salt stress response, especially, OsMSR2, whose orthologous genes in Arabidopsis had the potential role in photosynthesis adaptation under salt stress.

Data in support of photosynthetic responses in a chromosome segment substitution line of 'Khao Dawk Mali 105' rice at seedling stage.

The rice chromosome segment substitution lines (CSSLs) of 'Khao Dawk Mali 105' ('KDML105') genetic background were developed by backcrossing with 'KDML105' rice and transferring the region from chromosome 1 of DH212 which was expected to contain the full putative salt tolerance genetic region. Line of CSSL11, CSSL12, and CSSL16 contained the full putative salt tolerance genetic region were evaluated with the parental lines, 'KDML105' and DH212 at seedling stage of rice. The physiological responses in rice plants were grown under normal condition and 75 mM of NaCl, and then comparative photosynthetic parameters, chlorophyll fluorescence parameters, PhiPS2, ETR, NPQ, as well as growth analysis. In this article, the data of physiological response evaluation in rice at seedling stage after salt stress treatment can be found. This can be useful as the information of the photosynthesis response to salt stress to other rice cultivars and related species.

Thai Hom Mali Rice: Origin and Breeding for Subsistence Rainfed Lowland Rice System.

The world-renowned Thai Hom Mali Rice has been the most important aromatic rice originating in Thailand. The aromatic variety was collected from Chachoengsao, a central province, and after pure-line selection, it was officially named as Khao Dawk Mali 105, (KDML105). Because of its superb fragrance and cooking quality, KDML105 has been a model variety for studying genes controlling grain quality and aroma. The aromatic gene was cloned in KDML105, as an amino aldehyde dehydrogenase (AMADH) or better known as BADH2 located on chromosome 8. Later on, all other aromatic rice genes were discovered as allelic to the AMADH. As a selection of local landrace variety found in rainfed areas, the Thai Jasmine rice showed adaptive advantages over improved irrigated rice in less fertile lowland rainfed conditions. Because KDML105 was susceptible to most diseases and insect pests, marker-assisted backcross selection (MABC) was used for the genetic improvement since 2000. After nearly 17 years of MABC for integrating new traits into KDML105, a new generation of KDML105, designated HM84, was developed which maintains the cooking quality and fragrance, and has gained advantages during flash flooding, disease, and insect outbreak.

Contribution of Ebullition to Methane and Carbon Dioxide Emission from Water between Plant Rows in a Tropical Rice Paddy Field.

Although bubble ebullition through water in rice paddy fields dominates direct methane (CH4) emissions from paddy soil to the atmosphere in tropical regions, the temporal changes and regulating factors of this ebullition are poorly understood. Bubbles in a submerged paddy soil also contain high concentrations of carbon dioxide (CO2), implying that CO2 ebullition may occur in addition to CH4 ebullition. We investigated the dynamics of CH4 and CO2 ebullition in tropical rice paddy fields using an automated closed chamber installed between rice plants. Abrupt increases in CH4 concentrations occurred by bubble ebullition. The CO2 concentration in the chamber air suddenly increased at the same time, which indicated that CO2 ebullition was also occurring. The CH4 and CO2 emissions by bubble ebullition were correlated with falling atmospheric pressure and increasing soil surface temperature. The relative contribution of CH4 and CO2 ebullitions to the daily total emissions was 95-97% and 13-35%, respectively.