A GWAS approach to find SNPs associated with salt removal in rice leaf sheath.

BACKGROUND AND AIMS: The ability for salt removal at the leaf sheath level is considered to be one of the major mechanisms associated with salt tolerance in rice. Thus, understanding the genetic control of the salt removal capacity in leaf sheaths will help improve the molecular breeding of salt-tolerant rice varieties and speed up future varietal development to increase productivity in salt-affected areas. We report a genome-wide association study (GWAS) conducted to find single nucleotide polymorphisms (SNPs) associated with salt removal in leaf sheaths of rice. METHODS: In this study, 296 accessions of a rice (Oryza sativa) diversity panel were used to identify salt removal-related traits and conduct GWAS using 36 901 SNPs. The sheath:blade ratio of Na+ and Cl- concentrations was used to determine the salt removal ability in leaf sheaths. Candidate genes were further narrowed via Gene Ontology and RNA-seq analysis to those whose putative function was likely to be associated with salt transport and were up-regulated in response to salt stress. KEY RESULTS: For the association signals of the Na+ sheath:blade ratio, significant SNPs were found only in the indica sub-population on chromosome 5. Within candidate genes found in the GWAS study, five genes were upregulated and eight genes were downregulated in the internal leaf sheath tissues in the presence of salt stress. CONCLUSIONS: These GWAS data imply that rice accessions in the indica variety group are the main source of genes and alleles associated with Na+ removal in leaf sheaths of rice under salt stress.

Exploring novel genetic sources of salinity tolerance in rice through molecular and physiological characterization.

BACKGROUND AND AIMS: Agricultural productivity is increasingly being affected by the build-up of salinity in soils and water worldwide. The genetic base of salt-tolerant rice donors being used in breeding is relatively narrow and needs broadening to breed varieties with wider adaptation to salt-affected areas. This study evaluated a large set of rice accessions of diverse origins to identify and characterize novel sources of salt tolerance. METHODS: Diversity analysis was performed on 107 germplasm accessions using a genome-wide set of 376 single-nucleotide polymorphism (SNP) markers, along with characterization of allelic diversity at the major quantitative trait locus Saltol Sixty-nine accessions were further evaluated for physiological traits likely associated with responses to salt stress during the seedling stage. KEY RESULTS: Three major clusters corresponding to the indica, aus and aromatic subgroups were identified. The largest group was indica, with the salt-tolerant Pokkali accessions in one sub-cluster, while a set of Bangladeshi landraces, including Akundi, Ashfal, Capsule, Chikirampatnai and Kutipatnai, were in a different sub-cluster. A distinct aus group close to indica contained the salt-tolerant landrace Kalarata, while a separate aromatic group closer to japonica rice contained a number of traditional, but salt-sensitive Bangladeshi landraces. These accessions have different alleles at the Saltol locus. Seven landraces - Akundi, Ashfal, Capsule, Chikirampatnai, Jatai Balam, Kalarata and Kutipatnai - accumulated less Na and relatively more K, maintaining a lower Na/K ratio in leaves. They effectively limit sodium transport to the shoot. CONCLUSIONS: New salt-tolerant landraces were identified that are genetically and physiologically distinct from known donors. These landraces can be used to develop better salt-tolerant varieties and could provide new sources of quantitative trait loci/alleles for salt tolerance for use in molecular breeding. The diversity observed within this set and in other donors suggests multiple mechanisms that can be combined for higher salt tolerance.

Salinity tolerance, Na+ exclusion and allele mining of HKT1;5 in Oryza sativa and O. glaberrima: many sources, many genes, one mechanism?

BACKGROUND: Cultivated rice species (Oryza sativa L. and O. glaberrima Steud.) are generally considered among the crop species most sensitive to salt stress. A handful of lines are known to be tolerant, and a small number of these have been used extensively as donors in breeding programs. However, these donors use many of the same genes and physiological mechanisms to confer tolerance. Little information is available on the diversity of mechanisms used by these species to cope with salt stress, and there is a strong need to identify varieties displaying additional physiological and/or genetic mechanisms to confer higher tolerance. RESULTS: Here we present data on 103 accessions from O. sativa and 12 accessions from O. glaberrima, many of which are identified as salt tolerant for the first time, showing moderate to high tolerance of high salinity. The correlation of salinity-induced senescence (as judged by the Standard Evaluation System for Rice, or SES, score) with whole-plant and leaf blade Na+ concentrations was high across nearly all accessions, and was almost identical in both O. sativa and O. glaberrima. The association of leaf Na+ concentrations with cultivar-groups was very weak, but association with the OsHKT1;5 allele was generally strong. Seven major and three minor alleles of OsHKT1;5 were identified, and their comparisons with the leaf Na+ concentration showed that the Aromatic allele conferred the highest exclusion and the Japonica allele the least. A number of exceptions to this association with the Oryza HKT1;5 allele were identified; these probably indicate the existence of additional highly effective exclusion mechanisms. In addition, two landraces were identified, one from Thailand and the other from Senegal, that show high tissue tolerance. CONCLUSIONS: Significant variation in salinity tolerance exists within both cultivated Oryza species, and this is the first report of significant tolerance in O. glaberrima. The majority of accessions display a strong quantitative relationship between tolerance and leaf blade Na+ concentration, and thus the major tolerance mechanisms found in these species are those contributing to limiting sodium uptake and accumulation in active leaves. However, there appears to be genetic variation for several mechanisms that affect leaf Na+ concentration, and rare cases of accessions displaying different mechanisms also occur. These mechanisms show great promise for improving salt tolerance in rice over that available from current donors.

Development of a phenotyping protocol for combined drought and salinity stress at seedling stage in rice.

Introduction: The case of combined drought and salinity stress is increasingly becoming a constraint to rice production, especially in coastal areas and river deltas where low rainfall not only reduces soil moisture levels but also reduces the flow of river water, resulting in intrusion of saline sea-water. A standardized screening method is needed in order to systematically evaluate rice cultivars under combined drought+salinity at the same time because sequential stress of salinity followed by drought or vice-versa is not similar to simultaneous stress effects. Therefore, we aimed to develop a screening protocol for combined drought+salinity stress applied to soil-grown plants at seedling stage. Methods: The study system used 30-L soil-filled boxes, which allowed a comparison of plant growth under control conditions, individual drought and salinity stress, as well as combined drought+salinity. A set of salinity tolerant and drought tolerant cultivars were tested, together with several popular but salinity and drought-susceptible varieties that are grown in regions prone to combined drought+salinity. A range of treatments were tested including different timings of the drought and salinity application, and different severities of stress, in order to determine the most effective that resulted in visible distinction among cultivars. The challenges related to determining a protocol with repeatable seedling stage stress treatment effects while achieving a uniform plant stand are described here. Results: The optimized protocol simultaneously applied both stresses by planting into saline soil at 75% of field capacity which was then allowed to undergo progressive drydown. Meanwhile, physiological characterization revealed that chlorophyll fluorescence at seedling stage correlated well with grain yield when drought stress was applied to vegetative stage only. Discussion: The drought+salinity protocol developed here can be used for screening rice breeding populations as part of a pipeline to develop new rice varieties with improved adaptation to combined stresses.

Mechanistic understanding of iron toxicity tolerance in contrasting rice varieties from Africa: 2. Root oxidation ability and oxidative stress control.

To enhance breeding efficiency for iron (Fe) toxicity tolerance and boost lowland rice production in sub-Saharan Africa, we have characterised the morphological, physiological and biochemical responses of contrasting rice varieties to excess iron. Here, we report the capacity of four varieties (CK801 and Suakoko8 (tolerant), Supa and IR64 (sensitive)) to oxidise iron in the rhizosphere and control iron-induced oxidative stress. The experiments were conducted in hydroponic conditions using modified Magnavaca nutrient solution and 300 ppm of ferrous iron (Fe2+) supplied in the form of FeSO4. Severe oxidative stress was observed in sensitive varieties as revealed by their high levels of lipid peroxidation. Histochemical and biochemical analyses showed that tolerant varieties exhibited a better development of the aerenchyma and greater oxygen release than the sensitive varieties in response to excess Fe. Both suberin and lignin deposits were observed in the root, stem and leaf tissues but with varying intensities depending on the variety. Under iron toxic conditions, tolerant varieties displayed increased superoxide dismutase (SOD), glutathione reductase (GR), peroxidase (POX) and ascorbate peroxidase (APX) activities in both the roots and shoots, whereas sensitive varieties showed increased APX and catalase (CAT) activities in the roots. This study had revealed also that Suakoko8 mainly uses root oxidation to exclude Fe2+ from its rhizosphere, and CK801 possesses a strong reactive oxygen species scavenging system, in addition to root oxidation ability. Key traits associated with these tolerance mechanisms such as a well-developed aerenchyma, radial oxygen loss restricted to the root cap as well as strong activation of antioxidative enzymes (SOD, GR, POX and APX) could be useful selection criteria in rice varietal improvement programs for enhanced Fe toxicity tolerance.

Assessing trait contribution and mapping novel QTL for salinity tolerance using the Bangladeshi rice landrace Capsule.

BACKGROUND: Salinity is one of the most widespread abiotic stresses affecting rice productivity worldwide. The purpose of this study was to establish the relative importance of different traits associated with salinity tolerance in rice and to identify new quantitative trait loci (QTL) conferring tolerance to salinity at seedling stage. A total of 231 F2:3 plants derived from a cross between a sensitive variety BRRI dhan29 (BR29 hereafter) and Capsule, a salt tolerant Bangladeshi indica landrace, were evaluated under salt stress in a phytotron. RESULTS: Out of the 231 F2 plants, 47 highly tolerant and 47 most sensitive lines were selected, representing the two extreme tails of the phenotypic distribution. These 94 plants were genotyped for 105 simple sequence repeat (SSR) and insertion/deletion (InDel) markers. A genetic linkage map spanning approximately 1442.9 cM of the 12 linkage groups with an average marker distance of 13.7 cM was constructed. QTL were identified on the long arm of chromosome 1 for Na+ concentration, K+ concentration, Na+-K+ ratio and survival; chromosome 3 for Na+ concentration, survival and overall phenotypic evaluation using the Standard Evaluation system (SES); and chromosome 5 for SES. A total of 6 pairwise epistatic interactions were also detected between QTL-linked and QTL-unlinked regions. Graphical genotyping indicated an association between the phenotypes of the extreme families and their QTL genotypes. Path coefficient analysis revealed that Na+ concentration, survival, Na+-K+ ratio and the overall phenotypic performance (SES score) are the major traits associated with salinity tolerance of Capsule. CONCLUSIONS: Capsule provides an alternative source of salinity tolerance aside from Pokkali and Nona Bokra, the two Indian salt tolerant landraces traditionally used for breeding salt tolerant rice varieties. Pyramiding the new QTL identified in this study with previously discovered loci, such as Saltol, will facilitate breeding varieties that are highly tolerant of salt stress.

Fundamental parenchyma cells are involved in Na+ and Cl- removal ability in rice leaf sheath.

Salt sensitivity in rice plants is associated with the accumulated amount of Na+ and Cl- in shoots and, more significantly, in photosynthetic tissues. Therefore, salt removal ability at the leaf sheath level is an important mechanism of salt tolerance. In the present study we attempted to determine whether rice leaf sheaths excluded Cl- as well as Na+, and to identify the tissues that were involved in the removal ability of both ions. In two rice genotypes, salt-tolerant FL478 and -sensitive IR29, leaf sheaths excluded Na+ and Cl- under NaCl treatment as estimated using their sheath:blade ratios. The sheath:blade ratio of Na+ but not of Cl-, was increased by NaCl treatment. Under NaCl treatment, Na+ concentration was higher in the basal leaf sheath, whereas Cl- concentration was higher in the middle and tip parts. At the tissue level, fundamental parenchyma cells of leaf sheaths retained the highest amounts of Na and Cl when treated with high amount of NaCl. These results imply that the leaf sheath potentially functions to remove excess Na+ and Cl- from xylem vessels in different locations along the axis, with the fundamental parenchyma cells of leaf sheaths being involved in over-accumulation of both Na+ and Cl-.

Newly Identified Wild Rice Accessions Conferring High Salt Tolerance Might Use a Tissue Tolerance Mechanism in Leaf.

Cultivated rice (Oryza sativa L.) is very sensitive to salt stress. So far a few rice landraces have been identified as a source of salt tolerance and utilized in rice improvement. These tolerant lines primarily use Na+ exclusion mechanism in root which removes Na+ from the xylem stream by membrane Na+ and K+ transporters, and resulted in low Na+ accumulation in shoot. Identification of a new donor source conferring high salt tolerance is imperative. Wild relatives of rice having wide genetic diversity are regarded as a potential source for crop improvement. However, they have been less exploited against salt stress. Here, we simultaneously evaluated all 22 wild Oryza species along with the cultivated tolerant lines including Pokkali, Nona Bokra, and FL478, and sensitive check varieties under high salinity (240 mM NaCl). Based on the visual salt injury score, three species (O. alta, O. latifolia, and O. coarctata) and four species (O. rhizomatis, O. eichingeri, O. minuta, and O. grandiglumis) showed higher and similar level of tolerance compared to the tolerant checks, respectively. All three CCDD genome species exhibited salt tolerance, suggesting that the CCDD genome might possess the common genetic factors for salt tolerance. Physiological and biochemical experiments were conducted using the newly isolated tolerant species together with checks under 180 mM NaCl. Interestingly, all wild species showed high Na+ concentration in shoot and low concentration in root unlike the tolerant checks. In addition, the wild-tolerant accessions showed a tendency of a high tissue tolerance in leaf, low malondialdehyde level in shoot, and high retention of chlorophyll in the young leaves. These results suggest that the wild species employ tissue tolerance mechanism to manage salt stress. Gene expression analyses of the key salt tolerance-related genes suggested that high Na+ in leaf of wild species might be affected by OsHKT1;4-mediated Na+ exclusion in leaf and the following Na+ sequestration in leaf might be occurring independent of tonoplast-localized OsNHX1. The newly isolated wild rice accessions will be valuable materials for both rice improvement to salinity stress and the study of salt tolerance mechanism in plants.